Publications by year
In Press
Costello J, Islinger M, Schrader M (In Press). Editorial: “Molecular mechanisms and physiological significance of organelle interactions and cooperation—Volume II”. Frontiers in Cell and Developmental Biology
Kamoshita M, Kumar R, Anteghini M, Kunze M, Islinger M, Martins dos Santos V, Schrader M (In Press). Insights into the peroxisomal protein inventory of zebrafish. Frontiers in Physiology
Kors S, Schrader M, Costello J (In Press). Multiple ways to keep FFAT under control!. Contact
Carmichael R, Richards D, Fahimi HD, Schrader M (In Press). Organelle Membrane Extensions in Mammalian Cells. Biology
Aroso M, Agricola B, Hacker C, Schrader M (In Press). Proteoglycans support proper granule formation in pancreatic acinar cells.
2023
Kors S, Schrader M (2023). Assessing Peroxisomal Protein Interaction by Immunoprecipitation.
Methods Mol Biol,
2643, 345-357.
Abstract:
Assessing Peroxisomal Protein Interaction by Immunoprecipitation.
Organelles physically interact with each other via protein tethering complexes that bridge the opposing membranes. Organelle membrane contacts are highly dynamic, implying dynamism of the tethering complexes. Alterations in the binding of the tethering proteins can be assessed by immunoprecipitation. Antibody-conjugated beads allow for purification of the target protein with its binding partners, which can subsequently be examined by western blot analysis. We present immunoprecipitation methods and strategies to examine protein interaction domains, and for the identification of residues important for the regulation of the interaction, here focusing on phosphorylation. We use the peroxisomal membrane protein ACBD5 and its paralog ACBD4, which both bind ER membrane protein VAPB to mediate peroxisome-ER contacts, as example. However, this method can be applied to other peroxisomal and non-peroxisomal (membrane) proteins.
Abstract.
Author URL.
Costello JL, Koster J, Silva BSC, Worthy HL, Schrader TA, Hacker C, Passmore J, Kuypers FA, Waterham HR, Schrader M, et al (2023). Differential roles for ACBD4 and ACBD5 in peroxisome–ER interactions and lipid metabolism. Journal of Biological Chemistry, 299(8), 105013-105013.
Silva B, Schrader TA, Schrader M, Carmichael R (2023). Generation of reporter cell lines for endogenous expression analysis of peroxisomal proteins. Methods in Molecular Biology
Schrader M (2023). Immunolabelling for detection of endogenous and overexpressed peroxisomal proteins in mammalian cells. In (Ed) , Springer.
Schrader TA, Carmichael R, Schrader M (2023). Immunolabelling for detection of endogenous and overexpressed peroxisomal proteins in mammalian cells., Springer.
Schrader T, Carmichael R, Schrader M (2023). Immunolabelling for detection of endogenous and overexpressed peroxisomal proteins in mammalian cells. Methods in Molecular Biology
Schrader M (2023). Preface.
Kamoshita M, Schrader M (2023). Proximity-Ligation Assay to Detect Peroxisome-Organelle Interaction.
Methods Mol Biol,
2643, 135-148.
Abstract:
Proximity-Ligation Assay to Detect Peroxisome-Organelle Interaction.
Peroxisomes are essential organelles in mammals, which contribute to cellular lipid metabolism and redox homeostasis. They do not function as isolated entities but cooperate and interact with other subcellular organelles, in particular the endoplasmic reticulum, mitochondria, and lipid droplets. Those interactions are often mediated by membrane contact sites. Tether proteins at those sites bring the organelles in close proximity to facilitate metabolite and lipid transfer as well as organelle communication. There is great interest in the investigation of the physiological functions of peroxisome-organelle contacts and how they are regulated. Here, we present an antibody- and fluorescence-based proximity ligation approach used successfully in our laboratory for the detection and quantification of peroxisome-organelle interactions in cultured mammalian cells.
Abstract.
Author URL.
Hacker C, Schrader TA, Schrader M (2023). Ultrastructural Analysis and Quantification of Peroxisome-Organelle Contacts.
Methods Mol Biol,
2643, 105-122.
Abstract:
Ultrastructural Analysis and Quantification of Peroxisome-Organelle Contacts.
Transmission electron microscopy (TEM) has long been a vital technology to visualize the interaction of cellular compartments at the highest possible resolution. While this paved the way to describing organelles within the cellular context in detail, TEM has long been underused to generate quantitative data, analyzing those interactions as well as underlying mechanisms leading to their formation and modification. Here we describe a simple stereological method to unbiasedly assess the extent of organelle-organelle membrane contact sites, able to efficiently generate accurate and reproducible quantitative data from cultured mammalian cells prepared for TEM.
Abstract.
Author URL.
2022
Schrader M, Islinger M, Costello J (eds)(2022). 978-2-83250-838-1. Lausanne, Frontiers Media SA.
Kors S, Kurian SM, Costello JL, Schrader M (2022). Controlling contacts-Molecular mechanisms to regulate organelle membrane tethering.
Bioessays,
44(11).
Abstract:
Controlling contacts-Molecular mechanisms to regulate organelle membrane tethering.
In recent years, membrane contact sites (MCS), which mediate interactions between virtually all subcellular organelles, have been extensively characterized and shown to be essential for intracellular communication. In this review essay, we focus on an emerging topic: the regulation of MCS. Focusing on the tether proteins themselves, we discuss some of the known mechanisms which can control organelle tethering events and identify apparent common regulatory hubs, such as the VAP interface at the endoplasmic reticulum (ER). We also highlight several currently hypothetical concepts, including the idea of tether oligomerization and redox regulation playing a role in MCS formation. We identify gaps in our current understanding, such as the identity of the majority of kinases/phosphatases involved in tether modification and conclude that a holistic approach-incorporating the formation of multiple MCS, regulated by interconnected regulatory modulators-may be required to fully appreciate the true complexity of these fascinating intracellular communication systems.
Abstract.
Author URL.
Kors S (2022). Controlling peroxisome-ER contacts – Molecular mechanisms to regulate peroxisome tethering and distribution.
Abstract:
Controlling peroxisome-ER contacts – Molecular mechanisms to regulate peroxisome tethering and distribution
Peroxisomes are small, single membrane bound organelles with key roles in cellular lipid and hydrogen peroxide metabolism. They contribute to a wide range of metabolic processes including the β-oxidation of fatty acids and the synthesis of ether-phospholipids. To fulfil those functions, peroxisomes cooperate with other organelles, such as the endoplasmic reticulum (ER) and mitochondria. This collaboration requires close proximity of the organelles, which is mediated by protein tethering complexes that physically bridge apposing organelles.
This thesis focuses on the interaction of peroxisomes with the ER that are mediated through interaction of the peroxisomal membrane protein ACBD5 and the ER-resident protein VAPB. These peroxisome-ER contacts are important for peroxisome motility, and the transfer of membrane lipids and ether-phospholipid intermediates. ACBD5 binds to VAPB via its FFAT motif. However, molecular mechanisms, which regulate formation of these contact sites, are unknown. Additionally, ACBD5 deficient patients present with neurological problems, but the pathological mechanisms are not well understood. Here, I explore the regulation of peroxisome-ER contacts and study them in model organisms.
My findings reveal that peroxisome-ER associations via the ACBD5-VAPB tether are regulated by phosphorylation. I show that ACBD5-VAPB binding is phosphatase-sensitive and identify phosphorylation sites in the flanking regions and core of the FFAT motif, which alter interaction with VAPB – and thus peroxisome-ER contact sites – differently. Moreover, I demonstrate that the kinase GSK3β regulates this interaction. These results reveal for the first time a molecular mechanism for the regulation of peroxisome-ER contacts in mammalian cells and expand the current model of the FFAT-VAP interaction.
In addition, I used two model systems, the fruit fly Drosophila melanogaster and the filamentous fungus Ustilago maydis, to study the impact of ACBD5 depletion on peroxisome dynamics in vivo in long polarized neurites and hyphae, respectively. I discovered that the D. melanogaster ACBD5 and VAP homologs interact, while the U. maydis proteins do not, but depletion of ACBD5 in both species leads to redistribution of peroxisomes. Overall, this provides valuable findings to increase our understanding of the pathophysiologic processes in ACBD5 deficient patients.
Abstract.
Carmichael RE, Schrader M (2022). Determinants of Peroxisome Membrane Dynamics.
Front Physiol,
13Abstract:
Determinants of Peroxisome Membrane Dynamics.
Organelles within the cell are highly dynamic entities, requiring dramatic morphological changes to support their function and maintenance. As a result, organelle membranes are also highly dynamic, adapting to a range of topologies as the organelle changes shape. In particular, peroxisomes-small, ubiquitous organelles involved in lipid metabolism and reactive oxygen species homeostasis-display a striking plasticity, for example, during the growth and division process by which they proliferate. During this process, the membrane of an existing peroxisome elongates to form a tubule, which then constricts and ultimately undergoes scission to generate new peroxisomes. Dysfunction of this plasticity leads to diseases with developmental and neurological phenotypes, highlighting the importance of peroxisome dynamics for healthy cell function. What controls the dynamics of peroxisomal membranes, and how this influences the dynamics of the peroxisomes themselves, is just beginning to be understood. In this review, we consider how the composition, biophysical properties, and protein-lipid interactions of peroxisomal membranes impacts on their dynamics, and in turn on the biogenesis and function of peroxisomes. In particular, we focus on the effect of the peroxin PEX11 on the peroxisome membrane, and its function as a major regulator of growth and division. Understanding the roles and regulation of peroxisomal membrane dynamics necessitates a multidisciplinary approach, encompassing knowledge across a range of model species and a number of fields including lipid biochemistry, biophysics and computational biology. Here, we present an integrated overview of our current understanding of the determinants of peroxisome membrane dynamics, and reflect on the outstanding questions still remaining to be solved.
Abstract.
Author URL.
Carmichael RE, Islinger M, Schrader M (2022). Fission Impossible (?)-New Insights into Disorders of Peroxisome Dynamics.
Cells,
11(12).
Abstract:
Fission Impossible (?)-New Insights into Disorders of Peroxisome Dynamics.
Peroxisomes are highly dynamic and responsive organelles, which can adjust their morphology, number, intracellular position, and metabolic functions according to cellular needs. Peroxisome multiplication in mammalian cells involves the concerted action of the membrane-shaping protein PEX11β and division proteins, such as the membrane adaptors FIS1 and MFF, which recruit the fission GTPase DRP1 to the peroxisomal membrane. The latter proteins are also involved in mitochondrial division. Patients with loss of DRP1, MFF or PEX11β function have been identified, showing abnormalities in peroxisomal (and, for the shared proteins, mitochondrial) dynamics as well as developmental and neurological defects, whereas the metabolic functions of the organelles are often unaffected. Here, we provide a timely update on peroxisomal membrane dynamics with a particular focus on peroxisome formation by membrane growth and division. We address the function of PEX11β in these processes, as well as the role of peroxisome-ER contacts in lipid transfer for peroxisomal membrane expansion. Furthermore, we summarize the clinical phenotypes and pathophysiology of patients with defects in the key division proteins DRP1, MFF, and PEX11β as well as in the peroxisome-ER tether ACBD5. Potential therapeutic strategies for these rare disorders with limited treatment options are discussed.
Abstract.
Author URL.
Schrader TA, Carmichael RE, Islinger M, Costello JL, Hacker C, Bonekamp NA, Weishaupt JH, Andersen PM, Schrader M (2022). PEX11β and FIS1 cooperate in peroxisome division independently of mitochondrial fission factor.
J Cell Sci,
135(13).
Abstract:
PEX11β and FIS1 cooperate in peroxisome division independently of mitochondrial fission factor.
Peroxisome membrane dynamics and division are essential to adapt the peroxisomal compartment to cellular needs. The peroxisomal membrane protein PEX11β (also known as PEX11B) and the tail-anchored adaptor proteins FIS1 (mitochondrial fission protein 1) and MFF (mitochondrial fission factor), which recruit the fission GTPase DRP1 (dynamin-related protein 1, also known as DNML1) to both peroxisomes and mitochondria, are key factors of peroxisomal division. The current model suggests that MFF is essential for peroxisome division, whereas the role of FIS1 is unclear. Here, we reveal that PEX11β can promote peroxisome division in the absence of MFF in a DRP1- and FIS1-dependent manner. We also demonstrate that MFF permits peroxisome division independently of PEX11β and restores peroxisome morphology in PEX11β-deficient patient cells. Moreover, targeting of PEX11β to mitochondria induces mitochondrial division, indicating the potential for PEX11β to modulate mitochondrial dynamics. Our findings suggest the existence of an alternative, MFF-independent pathway in peroxisome division and report a function for FIS1 in the division of peroxisomes. This article has an associated First Person interview with the first authors of the paper.
Abstract.
Author URL.
Kors S, Hacker C, Bolton C, Maier R, Reimann L, Kitchener EJA, Warscheid B, Costello JL, Schrader M (2022). Regulating peroxisome–ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3β.
Journal of Cell Biology,
221(3).
Abstract:
Regulating peroxisome–ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3β
Peroxisomes and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism. They form membrane contacts through interaction of the peroxisomal membrane protein ACBD5 (acyl-coenzyme A–binding domain protein 5) and the ER-resident protein VAPB (vesicle-associated membrane protein–associated protein B). ACBD5 binds to the major sperm protein domain of VAPB via its FFAT-like (two phenylalanines [FF] in an acidic tract) motif. However, molecular mechanisms, which regulate formation of these membrane contact sites, are unknown. Here, we reveal that peroxisome–ER associations via the ACBD5-VAPB tether are regulated by phosphorylation. We show that ACBD5-VAPB binding is phosphatase-sensitive and identify phosphorylation sites in the flanking regions and core of the FFAT-like motif, which alter interaction with VAPB—and thus peroxisome–ER contact sites—differently. Moreover, we demonstrate that GSK3β (glycogen synthase kinase-3 β) regulates this interaction. Our findings reveal for the first time a molecular mechanism for the regulation of peroxisome–ER contacts in mammalian cells and expand the current model of FFAT motifs and VAP interaction.
Abstract.
Soares Carneiro Da Silva B (2022). Unravelling the molecular mechanisms of peroxisome-organelle communication and interplay.
Abstract:
Unravelling the molecular mechanisms of peroxisome-organelle communication and interplay
Peroxisomes are essential metabolic organelles which must communicate and interact extensively with their environment to exchange metabolites and coordinate cellular responses. This communication is often established through membrane contact sites (MCSs), where membranes of two organelles are physically tethered to enable rapid transfer of small molecules, which are crucial for coordinating cellular functions and hence human health. Identifying, understanding and characterising these MCSs and their regulation has been a challenging research topic in the current cell biology field.
In this thesis, I characterised two isoforms of the Acyl-CoA Binding Domain Protein 4 (ACBD4) in order to understand the physiological role of ACBD4 in the cell. Our group previously identified ACBD4 isoform 2 (ACBD4.2) as a tail-anchored peroxisomal
membrane protein. This protein belongs to the ACBD family, which binds acyl-CoA fatty acids via their acyl-CoA binding (ACB) domain. This isoform was described to be involved in the formation of a MCS by tethering with the ER protein, VAPB (Vesicle-Associated membrane Protein B). Looking at the two other uncharacterised isoforms of ACBD4, I found that ACBD4 isoform 1 (ACBD4.1) is localised only in the nucleus, and ACBD4 isoform 3 (ACBD4.3) is predominantly nuclear but also located at
peroxisomes. I explored their physiological role by analysing a BioID, which revealed candidate proximal interaction partners in the nucleus. Notably, the peroxisomal protein ACBD5 (ACBD Protein 5) was identified as a binding partner of ACBD4.3 but
not ACBD4.1. Similar to ACBD4.2, ACBD5 is also described as a tail-anchored peroxisomal protein involved in the interaction with VAPB to enable the exchange of lipids and metabolites between peroxisomes and the ER. We have confirmed that
ACBD5 interacts with ACBD4.3, supporting the idea that there is communication between peroxisomes and the nucleus, although the function of this, and the role of ACBD4.3 in this process, are not yet understood. Notably, the peroxisomal localisation
of ACBD4.3 seems to be dependent on its acyl-CoA fatty acid binding state, as ACB domain mutants decreased localisation at peroxisomes. Additionally, more prolonged expression of ACBD4.3 led to a rearrangement of the ER around peroxisomes,
suggesting a role of this protein in the peroxisome-ER hub. Further studies intend to investigate the impact of ACBD4.3 on peroxisome and nuclear function as well as on peroxisome-ER communication.
In parallel, I investigated the regulation of ACBD5 as an important mediator of peroxisome-ER contacts, which have a huge impact on maintaining cellular well-being. Therefore, I generated an endogenously tagged ACBD5 reporter cell line by CRIS PITCh to study the response of ACBD5 protein levels to different compounds. A small compound screen using a luminescence read-out of endogenous ACBD5 levels revealed that ACBD5 may be modulated by different compounds which activate PPARα signalling, although nothing significant was revealed. Further studies will explore other compounds that could impact lipid metabolism and extend to an
extensive compound screen in cooperation with Novartis. The identified compounds may prove useful to modulate organelle contacts and consequently improve cell performance in pathophysiological conditions.
Abstract.
Kors S, Costello JL, Schrader M (2022). VAP Proteins - from Organelle Tethers to Pathogenic Host Interactors and Their Role in Neuronal Disease.
Front Cell Dev Biol,
10Abstract:
VAP Proteins - from Organelle Tethers to Pathogenic Host Interactors and Their Role in Neuronal Disease.
Vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) are ubiquitous ER-resident tail-anchored membrane proteins in eukaryotic cells. Their N-terminal major sperm protein (MSP) domain faces the cytosol and allows them to interact with a wide variety of cellular proteins. Therefore, VAP proteins are vital to many cellular processes, including organelle membrane tethering, lipid transfer, autophagy, ion homeostasis and viral defence. Here, we provide a timely overview of the increasing number of VAPA/B binding partners and discuss the role of VAPA/B in maintaining organelle-ER interactions and cooperation. Furthermore, we address how viruses and intracellular bacteria hijack VAPs and their binding partners to induce interactions between the host ER and pathogen-containing compartments and support pathogen replication. Finally, we focus on the role of VAP in human disease and discuss how mutated VAPB leads to the disruption of cellular homeostasis and causes amyotrophic lateral sclerosis.
Abstract.
Author URL.
2021
Kors S, Hacker C, Bolton C, Maier R, Reimann L, Kitchener EJA, Warscheid B, Costello JL, Schrader M (2021). Regulating peroxisome-ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3β.
2020
Azadi AS, Carmichael RE, Kovacs WJ, Koster J, Kors S, Waterham HR, Schrader M (2020). A Functional SMAD2/3 Binding Site in the PEX11β Promoter Identifies a Role for TGFβ in Peroxisome Proliferation in Humans.
Frontiers in Cell and Developmental Biology,
8Abstract:
A Functional SMAD2/3 Binding Site in the PEX11β Promoter Identifies a Role for TGFβ in Peroxisome Proliferation in Humans
In mammals, peroxisomes perform crucial functions in cellular metabolism, signaling and viral defense which are essential to the viability of the organism. Molecular cues triggered by changes in the cellular environment induce a dynamic response in peroxisomes, which manifests itself as a change in peroxisome number, altered enzyme levels and adaptations to the peroxisomal morphology. How the regulation of this process is integrated into the cell’s response to different stimuli, including the signaling pathways and factors involved, remains unclear. Here, a cell-based peroxisome proliferation assay has been applied to investigate the ability of different stimuli to induce peroxisome proliferation. We determined that serum stimulation, long-chain fatty acid supplementation and TGFβ application all increase peroxisome elongation, a prerequisite for proliferation. Time-resolved mRNA expression during the peroxisome proliferation cycle revealed a number of peroxins whose expression correlated with peroxisome elongation, including the β isoform of PEX11, but not the α or γ isoforms. An initial map of putative regulatory motif sites in the respective promoters showed a difference between binding sites in PEX11α and PEX11β, suggesting that these genes may be regulated by distinct pathways. A functional SMAD2/3 binding site in PEX11β points to the involvement of the TGFβ signaling pathway in expression of this gene and thus peroxisome proliferation/dynamics in humans.
Abstract.
Steinberg G, Schuster M, Gurr SJ, Schrader TA, Schrader M, Wood M, Early A, Kilaru S (2020). A lipophilic cation protects crops against fungal pathogens by multiple modes of action.
Nat Commun,
11(1).
Abstract:
A lipophilic cation protects crops against fungal pathogens by multiple modes of action.
The emerging resistance of crop pathogens to fungicides poses a challenge to food security and compels discovery of new antifungal compounds. Here, we show that mono-alkyl lipophilic cations (MALCs) inhibit oxidative phosphorylation by affecting NADH oxidation in the plant pathogens Zymoseptoria tritici, Ustilago maydis and Magnaporthe oryzae. One of these MALCs, consisting of a dimethylsulfonium moiety and a long alkyl chain (C18-SMe2+), also induces production of reactive oxygen species at the level of respiratory complex I, thus triggering fungal apoptosis. In addition, C18-SMe2+ activates innate plant defense. This multiple activity effectively protects cereals against Septoria tritici blotch and rice blast disease. C18-SMe2+ has low toxicity in Daphnia magna, and is not mutagenic or phytotoxic. Thus, MALCs hold potential as effective and non-toxic crop fungicides.
Abstract.
Author URL.
Silva BSC, DiGiovanni L, Kumar R, Carmichael RE, Kim PK, Schrader M (2020). Maintaining social contacts: the physiological relevance of organelle interactions.
Biochimica et Biophysica Acta - Molecular Cell Research,
1867(11).
Abstract:
Maintaining social contacts: the physiological relevance of organelle interactions
Membrane-bound organelles in eukaryotic cells form an interactive network to coordinate and facilitate cellular functions. The formation of close contacts, termed “membrane contact sites” (MCSs), represents an intriguing strategy for organelle interaction and coordinated interplay. Emerging research is rapidly revealing new details of MCSs. They represent ubiquitous and diverse structures, which are important for many aspects of cell physiology and homeostasis. Here, we provide a comprehensive overview of the physiological relevance of organelle contacts. We focus on mitochondria, peroxisomes, the Golgi complex and the plasma membrane, and discuss the most recent findings on their interactions with other subcellular organelles and their multiple functions, including membrane contacts with the ER, lipid droplets and the endosomal/lysosomal compartment.
Abstract.
Passmore JB, Carmichael RE, Schrader TA, Godinho LF, Ferdinandusse S, Lismont C, Wang Y, Hacker C, Islinger M, Fransen M, et al (2020). Mitochondrial fission factor (MFF) is a critical regulator of peroxisome maturation.
Biochimica et Biophysica Acta - Molecular Cell Research,
1867(7).
Abstract:
Mitochondrial fission factor (MFF) is a critical regulator of peroxisome maturation
Peroxisomes are highly dynamic subcellular compartments with important functions in lipid and ROS metabolism. Impaired peroxisomal function can lead to severe metabolic disorders with developmental defects and neurological abnormalities. Recently, a new group of disorders has been identified, characterised by defects in the membrane dynamics and division of peroxisomes rather than by loss of metabolic functions. However, the contribution of impaired peroxisome plasticity to the pathophysiology of those disorders is not well understood. Mitochondrial fission factor (MFF) is a key component of both the peroxisomal and mitochondrial division machinery. Patients with MFF deficiency present with developmental and neurological abnormalities. Peroxisomes (and mitochondria) in patient fibroblasts are highly elongated as a result of impaired organelle division. The majority of studies into MFF-deficiency have focused on mitochondrial dysfunction, but the contribution of peroxisomal alterations to the pathophysiology is largely unknown. Here, we show that MFF deficiency does not cause alterations to overall peroxisomal biochemical function. However, loss of MFF results in reduced import-competency of the peroxisomal compartment and leads to the accumulation of pre-peroxisomal membrane structures. We show that peroxisomes in MFF-deficient cells display alterations in peroxisomal redox state and intra-peroxisomal pH. Removal of elongated peroxisomes through induction of autophagic processes is not impaired. A mathematical model describing key processes involved in peroxisome dynamics sheds further light into the physical processes disturbed in MFF-deficient cells. The consequences of our findings for the pathophysiology of MFF-deficiency and related disorders with impaired peroxisome plasticity are discussed.
Abstract.
Passmore JB, Godinho LF, Ferdinandusse S, Lismont C, Wang Y, Hacker C, Islinger M, Fransen M, Richards DM, Freisinger P, et al (2020). Mitochondrial fission factor (MFF) is a critical regulator of peroxisome maturation.
bioRxiv, 2020.01.08.898486-2020.01.08.898486.
Abstract:
Mitochondrial fission factor (MFF) is a critical regulator of peroxisome maturation
Peroxisomes are highly dynamic subcellular compartments with important functions in lipid and ROS metabolism. Impaired peroxisomal function can lead to severe metabolic disorders with developmental defects and neurological abnormalities. Recently, a new group of disorders has been identified, characterised by defects in the membrane dynamics and division of peroxisomes rather than by loss of metabolic functions. However, the contribution of impaired peroxisome plasticity to the pathophysiology of those disorders is not well understood. Mitochondrial fission factor (MFF) is a key component of both the peroxisomal and mitochondrial division machinery. Patients with MFF deficiency present with developmental and neurological abnormalities. Peroxisomes (and mitochondria) in patient fibroblasts are highly elongated as a result of impaired organelle division. The majority of studies into MFF-deficiency have focused on mitochondrial dysfunction, but the contribution of peroxisomal alterations to the pathophysiology is largely unknown. Here, we show that MFF deficiency does not cause alterations to overall peroxisomal biochemical function. However, loss of MFF results in reduced import-competency of the peroxisomal compartment and leads to the accumulation of pre-peroxisomal membrane structures. We show that peroxisomes in MFF-deficient cells display alterations in peroxisomal redox state and intra-peroxisomal pH as well as altered distribution in neuronal cells. Removal of elongated peroxisomes through induction of autophagic processes is not impaired. A mathematical model describing key processes involved in peroxisome dynamics sheds further light into the physical processes disturbed in MFF-deficient cells. The consequences of our findings for the pathophysiology of MFF-deficiency and related disorders with impaired peroxisome plasticity are discussed. ACOX1 :. acyl-CoA oxidase 1; PBD :. peroxisome biogenesis disorder; PED :. single peroxisomal enzyme deficiency; DRP1 :. dynamin-related protein 1; ER :. endoplasmic reticulum; FIS1 :. mitochondrial fission 1 protein; MFF :. Mitochondrial fission factor; ROS :. reactive oxygen species; PTS :. peroxisome targeting signal; VLCFA :. very-long-chain fatty acid.
Abstract.
Schrader M, Kamoshita M, Islinger M (2020). Organelle interplay—peroxisome interactions in health and disease.
Journal of Inherited Metabolic Disease,
43(1), 71-89.
Abstract:
Organelle interplay—peroxisome interactions in health and disease
Peroxisomes are multifunctional, dynamic, membrane-bound organelles with important functions in cellular lipid metabolism, rendering them essential for human health and development. Important roles for peroxisomes in signaling and the fine-tuning of cellular processes are emerging, which integrate them in a complex network of interacting cellular compartments. Like many other organelles, peroxisomes communicate through membrane contact sites. For example, peroxisomal growth, positioning, and lipid metabolism involves contacts with the endoplasmic reticulum (ER). Here, we discuss the most recent findings on peroxisome-organelle interactions including peroxisome-ER interplay at membrane contacts sites, and functional interplay with mitochondria, lysosomes, and lipid droplets in mammalian cells. We address tether proteins, metabolic cooperation, and the impact of peroxisome interactions on human health and disease.
Abstract.
Passmore J (2020). Peroxisomal membrane dynamics in health and disease.
Abstract:
Peroxisomal membrane dynamics in health and disease
Peroxisomes are oxidative subcellular organelles present in the majority of eukaryotic cells, with key functions in lipid metabolism, reactive oxygen species (ROS) homeostasis, and production of ether phospholipids (myelin lipids). Modulation of peroxisome number in the cell through coordinated proliferation and degradation is crucial for supporting these functions, with peroxisomes rapidly responding to environmental changes in order to maintain cell vitality. As a result, patients with defects in peroxisome proliferation present with a range of symptoms and severity, with vision/hearing loss, neurological defects, seizures, and organ dysfunction being common in peroxisome biogenesis disorders.
To proliferate, peroxisomes follow a multi-step process of growth and division, whereby (i) the peroxisome membrane is elongated, (ii) this elongation is constricted at several points to produce a ‘beads on a string’ morphology, and (iii) the membrane undergoes scission at these constriction points to produce new peroxisomes. This process requires extensive remodelling of the peroxisomal membrane, through the action of dedicated division machinery. Our knowledge of the key processes underlying the regulation of this membrane remodelling and the contribution of alterations to membrane dynamics to health and disease are still not well understood.
In this thesis, a literature review is presented describing knowns and unknowns in the peroxisome field, with particular focus on the role peroxisome membrane dynamics play in maintaining peroxisome function, and the role of these dynamics in human health and disease.
Results chapters begin first by investigating alterations to peroxisomal membrane dynamics in response to the mitochondrial respiratory chain inhibitor rotenone, to understand the resistance of peroxisomes to mitochondria-derived ROS. Surprisingly, alterations to the peroxisome compartment are due to the microtubule-destabilising ability of rotenone, and peroxisomes are unaffected by mitochondrial ROS, despite this not being the case vice versa. The implication of these findings in understanding the peroxisome-mitochondria redox relationship are discussed, in addition to highlighting the importance of investigating peroxisome membrane dynamics in in cellulo models of disease. Second, alterations to peroxisomal membrane dynamics in response to lack of division protein MFF are investigated. It is revealed that despite completely normal biochemical parameters, there are more underlying alterations to the peroxisomes than hyper-elongation, and that MFF has a crucial role in the maturation of peroxisomes. In addition, these findings suggest that peroxisomal import complex protein PEX14 may play a role in peroxisomal membrane dynamics, by stabilising elongated peroxisomal tubules through interaction with the cytoskeleton. Third, an interdisciplinary, combined experimental-modelling approach is used to characterise alterations to peroxisomal membrane dynamics, using peroxisomes with a block in division (loss of function of MFF), and peroxisomes with altered membrane dynamics (elongation through expression of Rho GTPase MIRO1), as case studies. The key cellular processes underlying the regulation of peroxisome growth and division are highlighted, and findings from this chapter provide a proof-of-concept that this interdisciplinary approach is useful not only for characterisation and understanding mechanisms of membrane dynamics, but as a prediction tool to help suggest future therapeutics.
Finally, a discussion of the key findings of this thesis and their implications is presented, highlighting the real importance of studying peroxisome membrane dynamics through interdisciplinary approaches such as the ones employed here.
Abstract.
Islinger M, Costello JL, Kors S, Soupene E, Levine TP, Kuypers FA, Schrader M (2020). The diversity of ACBD proteins – from lipid binding to protein modulators and organelle tethers.
Biochimica et Biophysica Acta - Molecular Cell Research,
1867(5).
Abstract:
The diversity of ACBD proteins – from lipid binding to protein modulators and organelle tethers
Members of the large multigene family of acyl-CoA binding domain containing proteins (ACBDs) share a conserved motif required for binding of Coenzyme a esterified fatty acids of various chain length. These proteins are present in the three kingdoms of life, and despite their predicted roles in cellular lipid metabolism, knowledge about the precise functions of many ACBD proteins remains scarce. Interestingly, several ACBD proteins are now suggested to function at organelle contact sites, and are recognized as host interaction proteins for different pathogens including viruses and bacteria. Here, we present a thorough phylogenetic analysis of the ACBD family and discuss their structure and evolution. We summarize recent findings on the various functions of animal and fungal ACBDs with particular focus on peroxisomes, the role of ACBD proteins at organelle membranes, and their increasing recognition as targets for pathogens.
Abstract.
Porter L (2020). Wnt signalling and peroxisome dynamics in the zebrafish (Danio rerio).
Abstract:
Wnt signalling and peroxisome dynamics in the zebrafish (Danio rerio)
Cell-cell or paracrine signalling is a form of cellular communication in which a cell produces a signal that influences the behaviour of neighbouring cells, which is important because it allows for the local coordination of the activities of groups of cells. This coordination is indispensable during development; for example, paracrine Wnt signalling is fundamental to body patterning in all metazoans where it helps to determine cell fate in a developing embryo. Wnt can regulate the transcription of target genes including cyclin and peroxisome-proliferator activated receptor-ẟ. The importance of Wnt signalling is not temporally limited, and Wnt has roles throughout the life of an organism such as the management of stem cells and the cellular abundance of mitochondria, the ‘sister organelle’ of the peroxisome. The peroxisome is a single membrane-bound organelle with diverse roles in healthy development and life, inclusive of the breakdown of very long chain fatty acids (VLCFAs) and the production of plasmalogens for efficient nervous conduction. The relationship between the Wnt signalling pathway and peroxisomes is unknown. Here I investigate the influence of Wnt signalling on peroxisome dynamics in zebrafish. To do so, canonical wnt8a was knocked out and knocked down using the genomic engineering tool CRISPR and Morpholino oligomers. The number and morphology of peroxisomes was observed in Wnt8a-deficient zebrafish embryos and appeared to be aggregated and less numerate than in wild type zebrafish. Consistently, in zebrafish embryos overexpressing wnt8a, peroxisomes were visualised as highly numerate singular puncta. I hypothesise that - in addition to a set of functions in development and tissue homeostasis - Wnt signalling has a novel role in regulating peroxisome proliferation in zebrafish.
Abstract.
2019
Kustatscher G, Grabowski P, Schrader TA, Passmore JB, Schrader M, Rappsilber J (2019). Co-regulation map of the human proteome enables identification of protein functions.
Nature Biotechnology,
37(11), 1361-1371.
Abstract:
Co-regulation map of the human proteome enables identification of protein functions
Assigning functions to the vast array of proteins present in eukaryotic cells remains challenging. To identify relationships between proteins, and thereby enable functional annotation of proteins, we determined changes in abundance of 10,323 human proteins in response to 294 biological perturbations using isotope-labeling mass spectrometry. We applied the machine learning algorithm treeClust to reveal functional associations between co-regulated human proteins from ProteomeHD, a compilation of our own data and datasets from the Proteomics Identifications database. This produced a co-regulation map of the human proteome. Co-regulation was able to capture relationships between proteins that do not physically interact or colocalize. For example, co-regulation of the peroxisomal membrane protein PEX11β with mitochondrial respiration factors led us to discover an organelle interface between peroxisomes and mitochondria in mammalian cells. We also predicted the functions of microproteins that are difficult to study with traditional methods. The co-regulation map can be explored at www.proteomeHD.net.
Abstract.
Bishop A, Kamoshita M, Passmore J, Hacker C, Schrader T, Waterham H, Costello J, Schrader M (2019). Fluorescent tools to analyse peroxisome-ER interactions in mammalian cells. Contact, 2
Taatjes DJ, Schrader M, Koji T, Roth J (2019). Histochemistry and Cell Biology: 61 years and not tired at all. Histochemistry and Cell Biology, 152(1).
Harrison C (2019). Super-resolution imaging of cardiac immuno-markers: Defining quality criteria for use in dual colour STORM and. DNA PAINT single molecule localisation microscopy.
Abstract:
Super-resolution imaging of cardiac immuno-markers: Defining quality criteria for use in dual colour STORM and. DNA PAINT single molecule localisation microscopy
Aberrant Ryanodine receptor behaviour is highly implicated in cardiovascular disease. Post-translational modifications are used widely in the body to control the dynamics of proteins to respond to acute and chronic demands. Phosphorylation is a key, highly tuneable modification used by cells by the reversible enzymatic addition of a phosphate group to single amino acids within protein structures. Common cardiac diseases such as arrhythmia, hypertension, and heart failure have been linked to excessive ryanodine receptor phosphorylation and transgenic constitutive phosphorylation has shown disease aetiology in disease models.
Phosphorylation is typically measured en masse by use of Western blots, or more recently phosphoproteomics, however the spatial distribution has remained a mystery. Ryanodine receptors are found in clusters and their influence tightly controlled spatially. As phosphorylation increases their range of influence, it is of great interest to observe the pattern of phosphorylation within and between ryanodine receptor clusters.
Ryanodine receptor clusters have been well characterised in electron microscopy and the fluorescence based super resolution microscopy, achieving single receptor resolution. This thesis details the validation pipeline for translation phosphorylation-state specific antibodies from Western blot through to super resolution microscopy. The phosphorylation distribution was compared between isolated ventricular cardiomyocytes and ventricular tissue sections for Ser 2808 and Ser 2814 phosphorylation. Strong reductions in basal phosphorylation caused by the isolation procedure were observed for Ser 2808 but not Ser 2814. These differences in Ser¬ 2808 phosphorylation were then investigated in dual channel STORM super resolution microscopy, highlighting stark contracts in colocalisation between the confocal and STORM techniques.
This population and sub population experiment was then translated into the new DNA PAINT technology and a direct comparison of performance between STORM and DNA PAINT was discussed. The data described in this thesis shows a methodological approach to enabling other biophysicists to perform quantitative super resolution microscopy to determine the extent and spatial distribution pattern of phosphorylation of a protein of interest at the nanoscale. Important differences were observed in the phosphorylation state due to cardiomyocyte isolation procedures that are of interest to a wide audience of cardiovascular researchers.
DNA-PAINT is emerging as the progression of SMLM from STORM microscopy due to the greater control of imaging parameters it affords. Parallel experiments of Ryanodine receptor Ser-2808 phosphorylation were performed in tissue sections. Comparisons between dual channel STORM and DNA-PAINT were evaluated. Open questions about DNA PAINT are also highlighted and discussed.
Abstract.
Harrison C (2019). Super-resolution imaging of cardiac immuno-markers: Defining quality criteria for use in dual colour STORM and. DNA-PAINT single molecule localisation microscopy.
Abstract:
Super-resolution imaging of cardiac immuno-markers: Defining quality criteria for use in dual colour STORM and. DNA-PAINT single molecule localisation microscopy
Aberrant Ryanodine receptor behaviour is highly implicated in cardiovascular disease. Post-translational modifications are used widely in the body to control the dynamics of proteins to respond to acute and chronic demands. Phosphorylation is a key, highly tuneable modification used by cells by the reversible enzymatic addition of a phosphate group to single amino acids within protein structures. Common cardiac diseases such as arrhythmia, hypertension, and heart failure have been linked to excessive ryanodine receptor phosphorylation and transgenic constitutive phosphorylation has shown disease aetiology in disease models.
Phosphorylation is typically measured en masse by use of Western blots, or more recently phosphoproteomics, however the spatial distribution has
remained a mystery. Ryanodine receptors are found in clusters and their
influence tightly controlled spatially. As phosphorylation increases their range of influence, it is of great interest to observe the pattern of phosphorylation within and between ryanodine receptor clusters.
Ryanodine receptor clusters have been well characterised in electron
microscopy and the fluorescence based super-resolution microscopy,
achieving single receptor resolution. This thesis details the validation pipeline for translation phosphorylation-state specific antibodies from Western blot through to super-resolution microscopy. The phosphorylation distribution was compared between isolated ventricular cardiomyocytes and ventricular tissue sections for Ser-2808 and Ser-2814 phosphorylation. Strong reductions in basal phosphorylation caused by the isolation procedure were observed for Ser-2808 but not Ser-2814. These differences in Ser-2808 phosphorylation were then investigated in dual channel STORM super-resolution microscopy, highlighting stark contracts in colocalisation between the confocal and STORM techniques.
This population and sub-population experiment was then translated into the new DNA-PAINT technology and a direct comparison of performance between STORM and DNA-PAINT was discussed. The data described in this thesis shows a methodological approach to enabling other biophysicists to perform quantitative super-resolution microscopy to determine the extent and spatial distribution pattern of phosphorylation of a protein of interest at the nanoscale. Important differences were observed in the phosphorylation state due to cardiomyocyte isolation procedures that are of interest to a wide audience of cardiovascular researchers.
DNA-PAINT is emerging as the progression of SMLM from STORM
microscopy due to the greater control of imaging parameters it affords.
Parallel experiments of Ryanodine receptor Ser-2808 phosphorylation were performed in tissue sections. Comparisons between dual channel STORM and DNA-PAINT were evaluated. Open questions about DNA-PAINT are also highlighted and discussed.
Abstract.
Harrison C (2019). Super-resolution imaging of cardiac immuno-markers: Defining quality criteria for use in dual colour STORM and DNA PAINT single molecule localisation microscopy.
Abstract:
Super-resolution imaging of cardiac immuno-markers: Defining quality criteria for use in dual colour STORM and DNA PAINT single molecule localisation microscopy
Aberrant Ryanodine receptor behaviour is highly implicated in cardiovascular disease. Post-translational modifications are used widely in the body to control the dynamics of proteins to respond to acute and chronic demands. Phosphorylation is a key, highly tuneable modification used by cells by the reversible enzymatic addition of a phosphate group to single amino acids within protein structures. Common cardiac diseases such as arrhythmia, hypertension, and heart failure have been linked to excessive ryanodine receptor phosphorylation and transgenic constitutive phosphorylation has shown disease aetiology in disease models.
Phosphorylation is typically measured en masse by use of Western blots, or more recently phosphoproteomics, however the spatial distribution has remained a mystery. Ryanodine receptors are found in clusters and their influence tightly controlled spatially. As phosphorylation increases their range of influence, it is of great interest to observe the pattern of phosphorylation within and between ryanodine receptor clusters.
Ryanodine receptor clusters have been well characterised in electron microscopy and the fluorescence based super resolution microscopy, achieving single receptor resolution. This thesis details the validation pipeline for translation phosphorylation-state specific antibodies from Western blot through to super resolution microscopy. The phosphorylation distribution was compared between isolated ventricular cardiomyocytes and ventricular tissue sections for Ser 2808 and Ser 2814 phosphorylation. Strong reductions in basal phosphorylation caused by the isolation procedure were observed for Ser 2808 but not Ser 2814. These differences in Ser¬ 2808 phosphorylation were then investigated in dual channel STORM super resolution microscopy, highlighting stark contracts in colocalisation between the confocal and STORM techniques.
This population and sub population experiment was then translated into the new DNA PAINT technology and a direct comparison of performance between STORM and DNA PAINT was discussed. The data described in this thesis shows a methodological approach to enabling other biophysicists to perform quantitative super resolution microscopy to determine the extent and spatial distribution pattern of phosphorylation of a protein of interest at the nanoscale. Important differences were observed in the phosphorylation state due to cardiomyocyte isolation procedures that are of interest to a wide audience of cardiovascular researchers.
DNA-PAINT is emerging as the progression of SMLM from STORM microscopy due to the greater control of imaging parameters it affords. Parallel experiments of Ryanodine receptor Ser-2808 phosphorylation were performed in tissue sections. Comparisons between dual channel STORM and DNA-PAINT were evaluated. Open questions about DNA PAINT are also highlighted and discussed.
Abstract.
Kustatscher G, Grabowski P, Schrader TA, Passmore JB, Schrader M, Rappsilber J (2019). The human proteome co-regulation map reveals functional relationships between proteins.
Turfah M (2019). Understanding the role of unusual dynamin- related proteins in the lizard pathogen Entamoeba invadens.
Abstract:
Understanding the role of unusual dynamin- related proteins in the lizard pathogen Entamoeba invadens
Amoebiasis is the third most common cause of death due to parasitic diseases in the world after malaria and schistosomiasis. In developing countries, it infects more than 50 million individuals annually causing 50,000 -100,000 deaths. Entamoeba. histolytica, an intestinal protozoan parasite, is the causative agent of amoebiasis in humans. The cyst is responsible for the transmission of the disease, in which the host gets infected through the ingestion of cyst-contaminated foods and water. About 90% of infections are asymptomatic cyst carriers and spreaders, in which each individual shed up to 45 million cysts per day. The process of conversion from motile trophozoite to dormant cyst is called encystation. However, little known about the molecular and cellular events that trigger this encystation. Numerous studies have attempted to investigate the molecules and genes involved in encystation. Dynamin, which is a conserved family of large GTPase, were suggested to play a role during encystation in Entamoeba. The unusual dynamin-related proteins Drp3 and Drp4 were revealed to be upregulated during stage transition. Despite the fact that all eukaryotes contain at least one dynamin protein, no homologues of Drp3 and Drp4 were identified in mammals. In this study, I attempted to study potential role of Drp3 and Drp4 during encystation in the laboratory model Entamoeba invadens. Furthermore, study the effect of the overexpression of these dynamins in mammalian cells. Using molecular biology techniques, immunofluorescence microscopy, transmission electron microscopy, and immunoelectron microscopy. In this study, we conducted stage conversion experiments to understand the importance of dynamins during cyst formation. Localization experiments showed that Drp3 and Drp4 have dual localization to both nucleus and cytoplasm. Drp3 existed as punctate structures localized to the cytoplasm and the nucleus in trophozoites and elongated structures localized to the nucleus in the cyst. The localization of the mutated form of Drp3 lacking the key residues in the GTPase domain (Drp3-mutant) was mainly nuclear and to a lesser extent cytoplasmic. The nuclear localization revealed the presence of Drp3 on the nucleus, the nuclear vesicles, and the nuclear envelope. Interestingly, cells overexpressing Drp3-mutant exhibited cytokinesis failure and multinucleation, suggesting a possible role in cytokinesis. In the mammalian cells, both Drp3 and Drp4 displayed the negative dominant effect on COS-7 cells. Drp3 localized to the nucleus and Drp4 localized to the mitochondria. However, both dynamins induced membranous tubulation in COS-7 cells. The work presented in this study demonstrated that Drp3 and Drp4 have nuclear and cytoplasmic functions. Drp3 is associated with the nucleus in mammalian cells and with the nucleus and the cytoplasm in E. invadens. The effect of Drp3 mutant on E. invadens cells indicates a potential role in cytokinesis and nuclear division. Drp4 is associated with Mitochondria in mammalian cells and some cytoplasmic compartments in E. invadens might involve the mitosomes.
Abstract.
2018
Castro IG, Richards DM, Metz J, Costello JL, Passmore JB, Schrader TA, Gouveia A, Ribeiro D, Schrader M (2018). A role for Mitochondrial Rho GTPase 1 (MIRO1) in motility and membrane dynamics of peroxisomes.
Traffic,
19(3), 229-242.
Abstract:
A role for Mitochondrial Rho GTPase 1 (MIRO1) in motility and membrane dynamics of peroxisomes
Peroxisomes are dynamic organelles which fulfil essential roles in lipid and ROS metabolism. Peroxisome movement and positioning allows interaction with other organelles and is crucial for their cellular function. In mammalian cells, such movement is microtubule-dependent and mediated by kinesin and dynein motors. The mechanisms of motor recruitment to peroxisomes are largely unknown, as well as the role this plays in peroxisome membrane dynamics and proliferation. Here, using a combination of microscopy, live-cell imaging analysis and mathematical modelling, we identify a role for Mitochondrial Rho GTPase 1 (MIRO1) as an adaptor for microtubule-dependent peroxisome motility in mammalian cells. We show that MIRO1 is targeted to peroxisomes and alters their distribution and motility. Using a peroxisome-targeted MIRO1 fusion protein, we demonstrate that MIRO1-mediated pulling forces contribute to peroxisome membrane elongation and proliferation in cellular models of peroxisome disease. Our findings reveal a molecular mechanism for establishing peroxisome-motor protein associations in mammalian cells and provide new insights into peroxisome membrane dynamics in health and disease.
Abstract.
Bishop A (2018). Characterisation of peroxisome-organelle contacts and cooperation.
Abstract:
Characterisation of peroxisome-organelle contacts and cooperation
Peroxisomes are organelles which are vital for human health and development. They represent dynamic subcellular compartments which play cooperative roles in essential cellular metabolic processes such as lipid metabolism and redox balance. For example, cooperation between peroxisomes and the endoplasmic reticulum (ER) is essential for the production of myelin lipids which are required for normal neurological function. We recently discovered that peroxisome-ER interaction is mediated by physical linkages in the form of membrane contact sites. These contact sites are mediated by the interaction of peroxisomal ACBD5 and ER-resident VAPB proteins. ACBD5-deficient patients have recently been identified who display retinal dystrophy, white matter disease and accumulation of very-long-chain fatty acids, which can only be degraded in peroxisomes. There is currently a need to develop simple and robust tools to allow efficient visualisation and quantification of these membrane contact sites to further their characterisation and investigate their function. Moreover, these should allow the dynamics of membrane contact sites under physiological conditions to be assessed. This study presents the optimisation of two systems to investigate peroxisome-ER interactions, the proximity ligation assay, Duolink® and a split fluorescent reporter system, split superfolder green fluorescent protein. These allow peroxisome-ER interactions to be visualised and measured in situ with a fluorescence-based readout when the organelles are in close proximity. These systems are powerful and modifiable and will help further characterise peroxisome-ER (or other organelle) membrane contacts and shed light on the interplay between peroxisomes and the ER.
Abstract.
Barel O, Malicdan MCV, Ben-Zeev B, Kandel J, Pri-Chen H, Stephen J, Castro IG, Metz J, Atawa O, Moshkovitz S, et al (2018). Deleterious variants in TRAK1 disrupt mitochondrial movement and cause fatal encephalopathy (vol 140, pg 568, 2017).
BRAIN,
141 Author URL.
Wang Y, Metz J, Costello JL, Passmore J, Schrader M, Schultz C, Islinger M (2018). Intracellular redistribution of neuronal peroxisomes in response to ACBD5 expression.
PLoS ONE,
13(12).
Abstract:
Intracellular redistribution of neuronal peroxisomes in response to ACBD5 expression
Peroxisomes can be frequently found in proximity to other subcellular organelles such as the endoplasmic reticulum (ER), mitochondria or lysosomes. The tail-anchored protein ACBD5 was recently identified as part of a tethering complex at peroxisome–ER contact sites, interacting with the ER resident protein VAPB. Contact site disruption was found to significantly increase peroxisome motility, apparently interfering with intracellular positioning systems. Unlike other somatic cells, neurons have to distribute organelles across relatively long distances in order to maintain their extraordinary cellular polarity. Using confocal live imaging microscopy in cultured hippocampal neurons we observed that peroxisomes and mitochondria show a strikingly similar motility with approximately 10% performing microtubule-driven long range movements. In order to investigate if ER contacts influence overall peroxisome motility and cellular distribution patterns, hippocampal neurons were transfected with plasmids encoding ACBD5 to stimulate peroxisome–ER interactions. Overex-pression of ACBD5 reduced peroxisomal long range movements in the neurites of the hippocampal cells by 70%, implying that ER attachment counteracts microtubule-driven peroxisome transport, while mitochondrial motility was unaffected. Moreover, the analyses of peroxisome distribution in fixed neurons unveiled a significant redistribution of peroxisomes towards the periphery of the perikaryon underneath the plasma membrane and into neurites, where peroxisomes are frequently found in close proximity to mitochondria. Surprisingly, further analysis of peroxisome and VAPB distribution upon ACBD5 expression did not reveal a substantial colocalization, implying this effect may be independent of VAPB. In line with these findings, expression of an ACBD5 variant unable to bind to VAPB still altered the localization of peroxisomes in the same way as the wild-type ACBD5. Thus, we conclude, that the VAPB-ACBD5 facilitated peroxisome-ER interaction is not responsible for the observed organelle redistribution in neurons. Rather, we suggest that additional ACBD5-binding proteins in neurons may tether peroxisomes to contact sites at or near the plasma membrane of neurons.
Abstract.
Castro IG, Schrader M (2018). Miro1–the missing link to peroxisome motility.
Communicative and Integrative Biology,
11(4).
Abstract:
Miro1–the missing link to peroxisome motility
Peroxisomes are ubiquitous, highly dynamic, multifunctional compartments in eukaryotic cells, which perform key roles in cellular lipid metabolism and redox balance. Like other membrane-bound organelles, peroxisomes must move in the cellular landscape to perform localized functions, interact with other organelles and to properly distribute during cell division. However, our current knowledge of peroxisome motility in mammalian cells is still very limited. Recently, three independent studies have identified Miro1 as a regulator of peroxisome motility in mammalian cells. In these studies, the authors show that Miro1 is targeted to peroxisomes in several cell lines, in a process that relies on its interaction with the peroxisomal chaperone Pex19. Interestingly, however, different conclusions are drawn about which Miro1 isoforms are targeted to peroxisomes, how it interacts with Pex19 and most importantly, the type of motility Miro1 is regulating.
Abstract.
Costello J, Passmore J, Islinger M, Schrader M (2018). Multi-localized proteins: the peroxisome-mitochondria connection. In del Rio L, Schrader M (Eds.) Proteomics of Peroxisomes. Identifying Novel Functions and Regulatory Networks, Springer.
Del Río LA, Schrader M (2018). Preface.
Schrader M (eds)(2018). Proteomics of Peroxisomes -
Identifying Novel Functions and Regulatory Networks
978-981-13-2232-7. Dordrecht, Springer Science + Business Media.
Islinger M, Voelkl A, Fahimi HD, Schrader M (2018). The peroxisome: an update on mysteries 2.0.
Histochemistry and Cell Biology,
150(5), 443-471.
Abstract:
The peroxisome: an update on mysteries 2.0
Peroxisomes are key metabolic organelles, which contribute to cellular lipid metabolism, e.g. the β-oxidation of fatty acids and the synthesis of myelin sheath lipids, as well as cellular redox balance. Peroxisomal dysfunction has been linked to severe metabolic disorders in man, but peroxisomes are now also recognized as protective organelles with a wider significance in human health and potential impact on a large number of globally important human diseases such as neurodegeneration, obesity, cancer, and age-related disorders. Therefore, the interest in peroxisomes and their physiological functions has significantly increased in recent years. In this review, we intend to highlight recent discoveries, advancements and trends in peroxisome research, and present an update as well as a continuation of two former review articles addressing the unsolved mysteries of this astonishing organelle. We summarize novel findings on the biological functions of peroxisomes, their biogenesis, formation, membrane dynamics and division, as well as on peroxisome–organelle contacts and cooperation. Furthermore, novel peroxisomal proteins and machineries at the peroxisomal membrane are discussed. Finally, we address recent findings on the role of peroxisomes in the brain, in neurological disorders, and in the development of cancer.
Abstract.
Costello JL, Schrader M (2018). Unloosing the Gordian knot of peroxisome formation.
Current Opinion in Cell Biology,
50, 50-56.
Abstract:
Unloosing the Gordian knot of peroxisome formation
Peroxisome biogenesis is governed by molecular machineries, which are either unique to peroxisomes or are partially shared with mitochondria. As peroxisomes have important protective functions in the cell, modulation of their number is important for human health and disease. Significant progress has been made towards our understanding of the mechanisms of peroxisome formation, revealing a remarkable plasticity of the peroxisome biogenesis pathway. Here we discuss most recent findings with particular focus on peroxisome formation in mammalian cells.
Abstract.
2017
Costello JL, Castro IG, Hacker C, Schrader TA, Metz J, Zeuschner D, Azadi AS, Godinho LF, Costina V, Findeisen P, et al (2017). ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER.
Journal of Cell Biology,
216(2), 331-342.
Abstract:
ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER
Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form tight structural associations, which were first observed in ultrastructural studies decades ago. PO-ER associations have been suggested to impact on a diverse number of physiological processes, including lipid metabolism, phospholipid exchange, metabolite transport, signaling, and PO biogenesis. Despite their fundamental importance to cell metabolism, the mechanisms by which regions of the ER become tethered to POs are unknown, in particular in mammalian cells. Here, we identify the PO membrane protein acyl-coenzyme A-binding domain protein 5 (ACBD5) as a binding partner for the resident ER protein vesicle-associated membrane protein-associated protein B (VAPB). We show that ACBD5-VAPB interaction regulates PO-ER associations. Moreover, we demonstrate that loss of PO-ER association perturbs PO membrane expansion and increases PO movement. Our findings reveal the first molecular mechanism for establishing PO-ER associations in mammalian cells and report a new function for ACBD5 in PO-ER tethering.
Abstract.
Costello JL, Castro IG, Hacker C, Schrader TA, Metz J, Zeuschner D, Azadi AS, Godinho LF, Costina V, Findeisen P, et al (2017). ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER. JOURNAL OF CELL BIOLOGY, 216(2), 331-342.
Ferdinandusse S, Falkenberg KD, Koster J, Mooyer PA, Jones R, van Roermund CWT, Pizzino A, Schrader M, Wanders RJA, Vanderver A, et al (2017). ACBD5 deficiency causes a defect in peroxisomal very long-chain fatty acid metabolism.
Journal of Medical Genetics,
54(5), 330-337.
Abstract:
ACBD5 deficiency causes a defect in peroxisomal very long-chain fatty acid metabolism
Background: Acyl-CoA binding domain containing protein 5 (ACBD5) is a peroxisomal membrane protein with a cytosolic acyl-CoA binding domain. Because of its acyl-CoA binding domain, ACBD5 has been assumed to function as an intracellular carrier of acyl-CoA esters. In addition, a role for ACBD5 in pexophagy has been suggested. However, the precise role of ACBD5 in peroxisomal metabolism and/or functioning has not yet been established. Previously, a genetic ACBD5 deficiency was identified in three siblings with retinal dystrophy and white matter disease. We identified a pathogenic mutation in ACBD5 in another patient and studied the consequences of the ACBD5 defect in patient material and in ACBD5-deficient HeLa cells to uncover this role. Methods: We studied a girl who presented with progressive leukodystrophy, syndromic cleft palate, ataxia and retinal dystrophy. We performed biochemical, cell biological and molecular studies in patient material and in ACBD5-deficient HeLa cells generated by CRISPR-Cas9 genome editing. Results: We identified a homozygous deleterious indel mutation in ACBD5, leading to complete loss of ACBD5 protein in the patient. Our studies showed that ACBD5 deficiency leads to accumulation of very long-chain fatty acids (VLCFAs) due to impaired peroxisomal β-oxidation. No effect on pexophagy was found. Conclusions: Our investigations strongly suggest that ACBD5 plays an important role in sequestering C26-CoA in the cytosol and thereby facilitates transport into the peroxisome and subsequent β-oxidation. Accordingly, ACBD5 deficiency is a novel single peroxisomal enzyme deficiency caused by impaired VLCFA metabolism, leading to retinal dystrophy and white matter disease.
Abstract.
Barel O, Malicdan MCV, Ben-Zeev B, Kandel J, Pri-Chen H, Stephen J, Castro IG, Metz J, Atawa O, Moshkovitz S, et al (2017). Deleterious variants in TRAK1 disrupt mitochondrial movement and cause fatal encephalopathy. Brain, 140, 568-581.
Schrader TA, Islinger M, Schrader M (2017). Detection and immunolabeling of peroxisomal proteins.
,
1595, 113-130.
Abstract:
Detection and immunolabeling of peroxisomal proteins
Peroxisomes are essential organelles in mammals which contribute to cellular lipid metabolism and redox homeostasis. The spectrum of their functions in human health and disease is far from being complete, and unexpected and novel roles of peroxisomes are being discovered. To date, those include novel biological roles in antiviral defence, as intracellular signaling platforms and as protective organelles in sensory cells. Furthermore, peroxisomes are part of a complex network of interacting subcellular compartments which involves metabolic cooperation, cross-talk and membrane contacts. As potentially novel peroxisomal proteins are continuously discovered, there is great interest in the verification of their peroxisomal localization. Here, we present protocols used successfully in our laboratory for the detection and immunolabeling of peroxisomal proteins in cultured mammalian cells. We present immunofluorescence and fluorescence-based techniques as well as reagents to determine peroxisome-specific targeting and localization of candidate proteins.
Abstract.
Godinho LF, Schrader M (2017). Determination of peroxisomal pH in living mammalian cells using pHRed.
,
1595, 181-189.
Abstract:
Determination of peroxisomal pH in living mammalian cells using pHRed
Organelle pH homeostasis is crucial for maintaining proper cellular function. The nature of the peroxisomal pH remains somewhat controversial, with several studies reporting conflicting results. Here, we describe in detail a rapid and accurate method for the measurement of peroxisomal pH, using the pHRed sensor protein and confocal microscopy of living mammalian cells. pHRed, a ratiometric sensor of pH, is targeted to the peroxisomes by virtue of a C-terminal targeting sequence. The probe has a maximum fluorescence emission at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm, allowing for ratiometric imaging and determination of intracellular pH in live cell microscopy.
Abstract.
Guimarães SC, Kilaru S, Schrader M, Schuster M (2017). Labeling of peroxisomes for live cell imaging in the filamentous fungus Ustilago maydis. In (Ed)
Methods in Molecular Biology, 131-150.
Abstract:
Labeling of peroxisomes for live cell imaging in the filamentous fungus Ustilago maydis
Abstract.
Schrader M, Islinger M (eds)(2017). MOLECULAR MECHANISMS AND PHYSIOLOGICAL SIGNIFICANCE OF
ORGANELLE INTERACTIONS AND COOPERATION., Lausanne: Frontiers Media.
Costello JL, Castro IG, Schrader TA, Islinger M, Schrader M (2017). Peroxisomal ACBD4 interacts with VAPB and promotes ER-peroxisome associations.
Cell Cycle,
16(11), 1039-1045.
Abstract:
Peroxisomal ACBD4 interacts with VAPB and promotes ER-peroxisome associations
Cooperation between cellular organelles such as mitochondria, peroxisomes and the ER is essential for a variety of important and diverse metabolic processes. Effective communication and metabolite exchange requires physical linkages between the organelles, predominantly in the form of organelle contact sites. At such contact sites organelle membranes are brought into close proximity by the action of molecular tethers, which often consist of specific protein pairs anchored in the membrane of the opposing organelles. Currently numerous tethering components have been identified which link the ER with multiple other organelles but knowledge of the factors linking the ER with peroxisomes is limited. Peroxisome-ER interplay is important because it is required for the biosynthesis of unsaturated fatty acids, ether-phospholipids and sterols with defects in these functions leading to severe diseases. Here, we characterize acyl-CoA binding domain protein 4 (ACBD4) as a tail-anchored peroxisomal membrane protein which interacts with the ER protein, vesicle-associated membrane protein-associated protein–B (VAPB) to promote peroxisome-ER associations.
Abstract.
Metz J, Castro I, Schrader M (2017). Peroxisome Motility Measurement and Quantification Assay.
BIO-PROTOCOL,
7Abstract:
Peroxisome Motility Measurement and Quantification Assay
Organelle movement, distribution and interaction contribute to the organisation of the eukaryotic cell. Peroxisomes are multifunctional organelles which contribute to cellular lipid metabolism and ROS homeostasis. They distribute uniformly in mammalian cells and move along microtubules via kinesin and dynein motors. Their metabolic cooperation with mitochondria and the endoplasmic reticulum (ER) is essential for the β-oxidation of fatty acids and the synthesis of myelin lipids and polyunsaturated fatty acids. A key assay to assess peroxisome motility in mammalian cells is the expression of a fluorescent fusion protein with a peroxisomal targeting signal (e.g. GFP-PTS1), which targets the peroxisomal matrix and allows live-cell imaging of peroxisomes. Here, we first present a protocol for the transfection of cultured mammalian cells with the peroxisomal marker EGFP-SKL to observe peroxisomes in living cells. This approach has revealed different motile behaviour of peroxisomes and novel insight into peroxisomal membrane dynamics (Rapp et al. 1996; Wiemer et al. 1997; Schrader et al. 2000). We then present a protocol which combines the live-cell approach with peroxisome motility measurements and quantification of peroxisome dynamics in mammalian cells. More recently, we used this approach to demonstrate that peroxisome motility and displacement is increased when a molecular tether, which associates peroxisomes with the ER, is lost (Costello et al. 2017b). Silencing of the peroxisomal acyl-CoA binding domain protein ACBD5, which interacts with ER-localised VAPB, increased peroxisome movement in skin fibroblasts, indicating that membrane contact sites can modulate organelle distribution and motility. The protocols described can be adapted to other cell types and organelles to measure and quantify organelle movement under different experimental conditions.
Abstract.
Costello JL, Castro IG, Camões F, Schrader TA, McNeall D, Yang J, Giannopoulou EA, Gomes S, Pogenberg V, Bonekamp NA, et al (2017). Predicting the targeting of tail-anchored proteins to subcellular compartments in mammalian cells.
Journal of Cell Science,
130(9), 1675-1687.
Abstract:
Predicting the targeting of tail-anchored proteins to subcellular compartments in mammalian cells
Tail-anchored (TA) proteins contain a single transmembrane domain (TMD) at the C-terminus that anchors them to the membranes of organelles where they mediate critical cellular processes. Accordingly, mutations in genes encoding TA proteins have been identified in a number of severe inherited disorders. Despite the importance of correctly targeting a TA protein to its appropriate membrane, the mechanisms and signals involved are not fully understood. In this study, we identify additional peroxisomal TA proteins, discover more proteins that are present on multiple organelles, and reveal that a combination of TMD hydrophobicity and tail charge determines targeting to distinct organelle locations in mammals. Specifically, an increase in tail charge can override a hydrophobic TMD signal and re-direct a protein from the ER to peroxisomes or mitochondria and vice versa. We show that subtle changes in those parameters can shift TA proteins between organelles, explaining why peroxisomes and mitochondria have many of the same TA proteins. This enabled us to associate characteristic physicochemical parameters in TA proteins with particular organelle groups. Using this classification allowed successful prediction of the location of uncharacterized TA proteins for the first time.
Abstract.
Schrader M, Pellegrini L (2017). The making of a mammalian peroxisome, version 2.0: Mitochondria get into the mix.
Cell Death and Differentiation,
24(7), 1148-1152.
Abstract:
The making of a mammalian peroxisome, version 2.0: Mitochondria get into the mix
A recent report from the Laboratory of Heidi McBride (McGill University) presents a role for mitochondria in the de novo biogenesis of peroxisomes in mammalian cells. Peroxisomes are essential organelles responsible for a wide variety of biochemical functions, from the generation of bile to plasmalogen synthesis, reduction of peroxides, and the oxidation of very-long-chain fatty acids. Like mitochondria, peroxisomes proliferate primarily through growth and division of pre-existing peroxisomes. However, unlike mitochondria, peroxisomes do not fuse; further, and perhaps most importantly, they can also be born de novo, a process thought to occur through the generation of pre-peroxisomal vesicles that originate from the endoplasmic reticulum. De novo peroxisome biogenesis has been extensively studied in yeast, with a major focus on the role of the ER in this process; however, in the mammalian system this field is much less explored. By exploiting patient cells lacking mature peroxisomes, the McBride laboratory now assigns a role to ER and mitochondria in de novo mammalian peroxisome biogenesis by showing that the formation of immature pre-peroxisomes occurs through the fusion of Pex3-/Pex14-containing mitochondria-derived vesicles with Pex16-containing ER-derived vesicles.
Abstract.
Passmore JB, Pinho S, Gomez-Lazaro M, Schrader M (2017). The respiratory chain inhibitor rotenone affects peroxisomal dynamics via its microtubule-destabilising activity.
Histochemistry and Cell Biology,
148(3), 331-341.
Abstract:
The respiratory chain inhibitor rotenone affects peroxisomal dynamics via its microtubule-destabilising activity
Peroxisomes and mitochondria in mammalian cells are closely linked subcellular organelles, which maintain a redox-sensitive relationship. Their interplay and role in ROS signalling are supposed to impact on age-related and degenerative disorders. Whereas the generation of peroxisome-derived oxidative stress can affect mitochondrial morphology and function, little is known about the impact of mitochondria-derived oxidative stress on peroxisomes. Here, we investigated the effect of the mitochondrial complex I inhibitor rotenone on peroxisomal and mitochondrial membrane dynamics. We show that rotenone treatment of COS-7 cells alters peroxisome morphology and distribution. However, this effect is related to its microtubule-destabilising activity rather than to the generation of oxidative stress. Rotenone also induced alterations in mitochondrial morphology, which—in contrast to its effect on peroxisomes—were dependent on the generation of ROS but independent of its microtubule-active properties. The importance of our findings for the peroxisome-mitochondria redox relationship and the interpretation of in cellulo and in vivo studies with rotenone, which is widely used to study Parkinson’s disease, are discussed.
Abstract.
Schrader TA, Schrader M (2017). siRNA-mediated silencing of peroxisomal genes in mammalian cells.
,
1595, 69-79.
Abstract:
siRNA-mediated silencing of peroxisomal genes in mammalian cells
RNAi technologies are a valuable tool in the identification and investigation of proteins that are involved in peroxisome biogenesis and function. Small interfering RNA (siRNA) has developed into the most commonly used RNAi tool for the induction of transient, short-term silencing of protein coding genes. Although siRNA can induce gene knockdown in a variety of mammalian cell lines, their utility is limited by efficient uptake of synthetic oligonucleotides into the cells. Here, we describe different transfection methods that have been successfully used by us to silence peroxisomal genes in a variety of cell lines, including primary human skin fibroblasts, which are usually difficult to transfect.
Abstract.
2016
Lin C, Schuster M, Guimaraes SC, Ashwin P, Schrader M, Metz J, Hacker C, Gurr SJ, Steinberg G (2016). Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells.
Nature Communications,
7Abstract:
Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells
Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ∼95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes.
Abstract.
Aroso M, Agricola B, Hacker C, Schrader M (2016). Addendum to the paper: Proteoglycans support proper granule formation in pancreatic AR42J cells.
Histochem Cell Biol,
146(1).
Author URL.
Schrader M, Islinger M (2016). Editorial: Molecular mechanisms and physiological significance of organelle interactions and cooperation. Frontiers in Cell and Developmental Biology, 4(DEC).
Magalhães AC, Ferreira AR, Gomes S, Vieira M, Gouveia A, Valença I, Islinger M, Nascimento R, Schrader M, Kagan JC, et al (2016). Peroxisomes are platforms for cytomegalovirus' evasion from the cellular immune response.
Scientific Reports,
6Abstract:
Peroxisomes are platforms for cytomegalovirus' evasion from the cellular immune response
The human cytomegalovirus developed distinct evasion mechanisms from the cellular antiviral response involving vMIA, a virally-encoded protein that is not only able to prevent cellular apoptosis but also to inhibit signalling downstream from mitochondrial MAVS. vMIA has been shown to localize at mitochondria and to trigger their fragmentation, a phenomenon proven to be essential for the signalling inhibition. Here, we demonstrate that vMIA is also localized at peroxisomes, induces their fragmentation and inhibits the peroxisomal-dependent antiviral signalling pathway. Importantly, we demonstrate that peroxisomal fragmentation is not essential for vMIA to specifically inhibit signalling downstream the peroxisomal MAVS. We also show that vMIA interacts with the cytoplasmic chaperone Pex19, suggesting that the virus has developed a strategy to highjack the peroxisomal membrane proteins' transport machinery. Furthermore, we show that vMIA is able to specifically interact with the peroxisomal MAVS. Our results demonstrate that peroxisomes constitute a platform for evasion of the cellular antiviral response and that the human cytomegalovirus has developed a mechanism by which it is able to specifically evade the peroxisomal MAVS-dependent antiviral signalling.
Abstract.
Schrader M, Costello JL, Godinho LF, Azadi AS, Islinger M (2016). Proliferation and fission of peroxisomes - an update.
Biochimica et Biophysica Acta - Molecular Cell Research,
1863(5), 971-983.
Abstract:
Proliferation and fission of peroxisomes - an update
In mammals, peroxisomes perform crucial functions in cellular metabolism, signalling and viral defense which are essential to the health and viability of the organism. In order to achieve this functional versatility peroxisomes dynamically respond to molecular cues triggered by changes in the cellular environment. Such changes elicit a corresponding response in peroxisomes, which manifests itself as a change in peroxisome number, altered enzyme levels and adaptations to the peroxisomal structure. In mammals the generation of new peroxisomes is a complex process which has clear analogies to mitochondria, with both sharing the same division machinery and undergoing a similar division process. How the regulation of this division process is integrated into the cell's response to different stimuli, the signalling pathways and factors involved, remains somewhat unclear. Here, we discuss the mechanism of peroxisomal fission, the contributions of the various division factors and examine the potential impact of post-translational modifications, such as phosphorylation, on the proliferation process.We also summarize the signalling process and highlight the most recent data linking signalling pathways with peroxisome proliferation. This article is part of a Special Issue entitled: Peroxisomes edited by Ralf Erdmann.
Abstract.
2015
Silva AM, Varela-Moreira A, Gomes CP, Molinos M, Leite M, Almeida M, Ribeiro D, Schrader M, Figueiredo C, Barbosa M, et al (2015). Integrated Analysis of Biological Samples by Imaging Flow Cytometry. Microscopy and Microanalysis, 21, 95-96.
Camões F, Islinger M, Guimarães SC, Kilaru S, Schuster M, Godinho LF, Steinberg G, Schrader M (2015). New insights into the peroxisomal protein inventory: Acyl-CoA oxidases and -dehydrogenases are an ancient feature of peroxisomes.
Biochim Biophys Acta,
1853(1), 111-125.
Abstract:
New insights into the peroxisomal protein inventory: Acyl-CoA oxidases and -dehydrogenases are an ancient feature of peroxisomes.
Peroxisomes are ubiquitous organelles which participate in a variety of essential biochemical pathways. An intimate interrelationship between peroxisomes and mitochondria is emerging in mammals, where both organelles cooperate in fatty acid β-oxidation and cellular lipid homeostasis. As mitochondrial fatty acid β-oxidation is lacking in yeast and plants, suitable genetically accessible model systems to study this interrelationship are scarce. Here, we propose the filamentous fungus Ustilago maydis as a suitable model for those studies. We combined molecular cell biology, bioinformatics and phylogenetic analyses and provide the first comprehensive inventory of U. maydis peroxisomal proteins and pathways. Studies with a peroxisome-deficient Δpex3 mutant revealed the existence of parallel and complex, cooperative β-oxidation pathways in peroxisomes and mitochondria, mimicking the situation in mammals. Furthermore, we provide evidence that acyl-CoA dehydrogenases (ACADs) are bona fide peroxisomal proteins in fungi and mammals and together with acyl-CoA oxidases (ACOX) belong to the basic enzymatic repertoire of peroxisomes. A genome comparison with baker's yeast and human gained new insights into the basic peroxisomal protein inventory shared by humans and fungi and revealed novel peroxisomal proteins and functions in U. maydis. The importance of our findings for the evolution and function of the complex interrelationship between peroxisomes and mitochondria in fatty acid β-oxidation is discussed.
Abstract.
Author URL.
Schrader M, Costello J, Godinho LF, Islinger M (2015). Peroxisome-mitochondria interplay and disease.
Journal of Inherited Metabolic Disease,
38(4), 681-702.
Abstract:
Peroxisome-mitochondria interplay and disease
Peroxisomes and mitochondria are ubiquitous, highly dynamic organelles with an oxidative type of metabolism in eukaryotic cells. Over the years, substantial evidence has been provided that peroxisomes and mitochondria exhibit a close functional interplay which impacts on human health and development. The so-called “peroxisome-mitochondria connection” includes metabolic cooperation in the degradation of fatty acids, a redox-sensitive relationship, an overlap in key components of the membrane fission machineries and cooperation in anti-viral signalling and defence. Furthermore, combined peroxisome-mitochondria disorders with defects in organelle division have been revealed. In this review, we present the latest progress in the emerging field of peroxisomal and mitochondrial interplay in mammals with a particular emphasis on cooperative fatty acid β-oxidation, redox interplay, organelle dynamics, cooperation in anti-viral signalling and the resulting implications for disease.
Abstract.
Schrader M, Costello J, Godinho LF, Islinger M (2015). Peroxisome-mitochondria interplay and disease.
J Inherit Metab Dis,
38(4), 681-702.
Abstract:
Peroxisome-mitochondria interplay and disease.
Peroxisomes and mitochondria are ubiquitous, highly dynamic organelles with an oxidative type of metabolism in eukaryotic cells. Over the years, substantial evidence has been provided that peroxisomes and mitochondria exhibit a close functional interplay which impacts on human health and development. The so-called "peroxisome-mitochondria connection" includes metabolic cooperation in the degradation of fatty acids, a redox-sensitive relationship, an overlap in key components of the membrane fission machineries and cooperation in anti-viral signalling and defence. Furthermore, combined peroxisome-mitochondria disorders with defects in organelle division have been revealed. In this review, we present the latest progress in the emerging field of peroxisomal and mitochondrial interplay in mammals with a particular emphasis on cooperative fatty acid β-oxidation, redox interplay, organelle dynamics, cooperation in anti-viral signalling and the resulting implications for disease.
Abstract.
Author URL.
Guimaraes SC, Schuster M, Bielska E, Dagdas G, Kilaru S, Meadows BRA, Schrader M, Steinberg G (2015). Peroxisomes, lipid droplets, and endoplasmic reticulum "hitchhike" on motile early endosomes.
Journal of Cell Biology,
211(5), 945-954.
Abstract:
Peroxisomes, lipid droplets, and endoplasmic reticulum "hitchhike" on motile early endosomes
Intracellular transport is mediated by molecular motors that bind cargo to be transported along the cytoskeleton. Here, we report, for the first time, that peroxisomes (POs), lipid droplets (LDs), and the endoplasmic reticulum (ER) rely on early endosomes (EEs) for intracellular movement in a fungal model system. We show that POs undergo kinesin-3- and dynein-dependent transport along microtubules. Surprisingly, kinesin-3 does not colocalize with POs. Instead, the motor moves EEs that drag the POs through the cell. PO motility is abolished when EE motility is blocked in various mutants. Most LD and ER motility also depends on EE motility, whereas mitochondria move independently of EEs. Covisualization studies show that EE-mediated ER motility is not required for PO or LD movement, suggesting that the organelles interact with EEs independently. In the absence of EE motility, POs and LDs cluster at the growing tip, whereas ER is partially retracted to subapical regions. Collectively, our results show that moving EEs interact transiently with other organelles, thereby mediating their directed transport and distribution in the cell.
Abstract.
Aroso M, Agricola B, Hacker C, Schrader M (2015). Proteoglycans support proper granule formation in pancreatic acinar cells.
Histochemistry and Cell Biology,
144(4), 331-346.
Abstract:
Proteoglycans support proper granule formation in pancreatic acinar cells
© 2015, Springer-Verlag Berlin Heidelberg. Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. The molecular mechanisms of their biogenesis and the sorting of zymogens are still incompletely understood. Here, we investigated the role of proteoglycans in granule formation and secretion of zymogens in pancreatic AR42J cells, an acinar model system. Cupromeronic Blue cytochemistry and biochemical studies revealed an association of proteoglycans primarily with the granule membrane. Removal of proteoglycans by carbonate treatment led to a loss of membrane curvature indicating a supportive role in the maintenance of membrane shape and stability. Chemical inhibition of proteoglycan synthesis impaired the formation of normal electron-dense granules in AR42J cells and resulted in the formation of unusually small granule structures. These structures still contained the zymogen carboxypeptidase, a cargo molecule of secretory granules, but migrated to lighter fractions after density gradient centrifugation. Furthermore, the basal secretion of amylase was increased in AR42J cells after inhibitor treatment. In addition, irregular-shaped granules appeared in pancreatic lobules. We conclude that the assembly of a proteoglycan scaffold at the ZG membrane is supporting efficient packaging of zymogens and the proper formation of stimulus-competent storage granules in acinar cells of the pancreas.
Abstract.
Schrader M, Godinho LF, Costello JL, Islinger M (2015). The different facets of organelle interplay-An overview of organelle interactions.
Frontiers in Cell and Developmental Biology,
3(SEP).
Abstract:
The different facets of organelle interplay-An overview of organelle interactions
Membrane-bound organelles such as mitochondria, peroxisomes, or the endoplasmic reticulum (ER) create distinct environments to promote specific cellular tasks such as ATP production, lipid breakdown, or protein export. During recent years, it has become evident that organelles are integrated into cellular networks regulating metabolism, intracellular signaling, cellular maintenance, cell fate decision, and pathogen defence. In order to facilitate such signaling events, specialized membrane regions between apposing organelles bear distinct sets of proteins to enable tethering and exchange of metabolites and signaling molecules. Such membrane associations between the mitochondria and a specialized site of the ER, the mitochondria associated-membrane (MAM), as well as between the ER and the plasma membrane (PAM) have been partially characterized at the molecular level. However, historical and recent observations imply that other organelles like peroxisomes, lysosomes, and lipid droplets might also be involved in the formation of such apposing membrane contact sites. Alternatively, reports on so-called mitochondria derived-vesicles (MDV) suggest alternative mechanisms of organelle interaction. Moreover, maintenance of cellular homeostasis requires the precise removal of aged organelles by autophagy-a process which involves the detection of ubiquitinated organelle proteins by the autophagosome membrane, representing another site of membrane associated-signaling. This review will summarize the available data on the existence and composition of organelle contact sites and the molecular specializations each site uses in order to provide a timely overview on the potential functions of organelle interaction.
Abstract.
Williams C, Opalinski L, Landgraf C, Costello J, Schrader M, Krikken AM, Knoops K, Kram AM, Volkmer R, Van Der Klei IJ, et al (2015). The membrane remodeling protein Pex11p activates the GTPase Dnm1p during peroxisomal fission.
Proceedings of the National Academy of Sciences of the United States of America,
112(20), 6377-6382.
Abstract:
The membrane remodeling protein Pex11p activates the GTPase Dnm1p during peroxisomal fission
The initial phase of peroxisomal fission requires the peroxisomal membrane protein Peroxin 11 (Pex11p), which remodels the membrane, resulting in organelle elongation. Here, we identify an additional function for Pex11p, demonstrating that Pex11p also plays a crucial role in the final step of peroxisomal fission: dynamin-like protein (DLP)-mediated membrane scission. First, we demonstrate that yeast Pex11p is necessary for the function of the GTPase Dynamin-related 1 (Dnm1p) in vivo. In addition, our data indicate that Pex11p physically interacts with Dnm1p and that inhibiting this interaction compromises peroxisomal fission. Finally, we demonstrate that Pex11p functions as a GTPase activating protein (GAP) for Dnm1p in vitro. Similar observations were made for mammalian Pex11β and the corresponding DLP Drp1, indicating that DLP activation by Pex11p is conserved. Our work identifies a previously unknown requirement for a GAP in DLP function.
Abstract.
2014
Magalhaes AC, Gomes S, Ferreira AR, Oes FC, Nascimento R, Schrader M, Ribeiro D (2014). A role for peroxisomes on the cellular antiviral response to Cytomegalovirus infection.
Author URL.
Gouveia AMS, Gomes S, Guimaraes S, Lazaro MG, Sampaio P, Schrader M, Ribeiro D (2014). Functional specificity of the tail-anchored proteins Mff and Fis1 on peroxisomal and mitochondrial dynamics.
Author URL.
Schrader M, Castro I, Fahimi HD, Islinger M (2014). Peroxisome morphology in pathologies. In (Ed)
Molecular Machines Involved in Peroxisome Biogenesis and Maintenance, 125-151.
Abstract:
Peroxisome morphology in pathologies
Abstract.
Camoes F, Islinger M, Costello J, Gomes S, Bonekamp N, Almeida M, Castro I, Ribeiro D, Schrader M (2014). Targeting of tail-anchored proteins to peroxisomes and mitochondria in mammalian cells.
Author URL.
2013
Huber N, Guimaraes S, Schrader M, Suter U, Niemann A (2013). Charcot-Marie-Tooth disease-associated mutants of GDAP1 dissociate its roles in peroxisomal and mitochondrial fission.
EMBO Reports,
14(6), 545-552.
Abstract:
Charcot-Marie-Tooth disease-associated mutants of GDAP1 dissociate its roles in peroxisomal and mitochondrial fission
Mitochondria and peroxisomes can be fragmented by the process of fission. The fission machineries of both organelles share a set of proteins. GDAP1 is a tail-anchored protein of mitochondria and induces mitochondrial fragmentation. Mutations in GDAP1 lead to Charcot-Marie-Tooth disease (CMT), an inherited peripheral neuropathy, and affect mitochondrial dynamics. Here, we show that GDAP1 is also targeted to peroxisomes mediated by the import receptor Pex19. Knockdown of GDAP1 leads to peroxisomal elongation that can be rescued by re-expressing GDAP1 and by missense mutated forms found in CMT patients. GDAP1-induced peroxisomal fission is dependent on the integrity of its hydrophobic domain 1, and on Drp1 and Mff, as is mitochondrial fission. Thus, GDAP1 regulates mitochondrial and peroxisomal fission by a similar mechanism. However, our results reveal also a more critical role of the amino-terminal GDAP1 domains, carrying most CMT-causing mutations, in the regulation of mitochondrial compared to peroxisomal fission. © 2013 European Molecular Biology Organization.
Abstract.
Bonekamp NA, Islinger M, Lázaro MG, Schrader M (2013). Cytochemical detection of peroxisomes and mitochondria.
Methods Mol Biol,
931, 467-482.
Abstract:
Cytochemical detection of peroxisomes and mitochondria.
Peroxisomes and mitochondria are essential subcellular organelles in mammals. Interestingly, recent studies have elucidated that these highly dynamic and plastic organelles exhibit a much closer interrelationship than previously assumed. Peroxisomes and mitochondria are metabolically linked organelles, which are cooperating and cross-talking. They share key components of their division machinery and cooperate in antiviral signaling and defense. As peroxisomal alterations in metabolism, biogenesis, dynamics, and proliferation have the potential to influence mitochondrial morphology and functions (and vice versa), there is currently great interest in the detection of both organelles under different experimental conditions. Here, we present protocols used successfully in our laboratory for the dual detection of peroxisomes and mitochondria in cultured mammalian cells. We address double immunofluorescence and fluorescence-based techniques as well as reagents to investigate organelle dynamics, morphological alterations, and organelle-specific targeting of proteins. In addition, we describe the application of diaminobenzidine cytochemistry on cultured cells to specifically label peroxisomes in ultrastructural studies.
Abstract.
Author URL.
Castro I, Gouveia A, Ribeiro D, Schrader M (2013). Miro1 regulates microtubule-dependent motility of peroxisomes in mammalian cells.
MOLECULAR BIOLOGY OF THE CELL,
24 Author URL.
Schrader M, Grille S, Fahimi HD, Islinger M (2013). Peroxisome interactions and cross-talk with other subcellular compartments in animal cells.
Subcell Biochem,
69, 1-22.
Abstract:
Peroxisome interactions and cross-talk with other subcellular compartments in animal cells.
Peroxisomes are remarkably plastic and dynamic organelles, which fulfil important functions in hydrogen peroxide and lipid metabolism rendering them essential for human health and development. Despite great advances in the identification and characterization of essential components and molecular mechanisms associated with the biogenesis and function of peroxisomes, our understanding of how peroxisomes are incorporated into metabolic pathways and cellular communication networks is just beginning to emerge. Here we address the interaction of peroxisomes with other subcellular compartments including the relationship with the endoplasmic reticulum, the peroxisome-mitochondria connection and the association with lipid droplets. We highlight metabolic cooperations and potential cross-talk and summarize recent findings on peroxisome-peroxisome interactions and the interaction of peroxisomes with microtubules in mammalian cells.
Abstract.
Author URL.
Bonekamp NA, Grille S, Cardoso MJ, Almeida M, Aroso M, Gomes S, Magalhaes AC, Ribeiro D, Islinger M, Schrader M, et al (2013). Self-interaction of human Pex11pβ during peroxisomal growth and division.
PLoS One,
8(1).
Abstract:
Self-interaction of human Pex11pβ during peroxisomal growth and division.
Pex11 proteins are involved in membrane elongation and division processes associated with the multiplication of peroxisomes. Human Pex11pβ has recently been linked to a new disorder affecting peroxisome morphology and dynamics. Here, we have analyzed the exact membrane topology of Pex11pβ. Studies with an epitope-specific antibody and protease protection assays show that Pex11pβ is an integral membrane protein with two transmembrane domains flanking an internal region exposed to the peroxisomal matrix and N- and C-termini facing the cytosol. A glycine-rich internal region within Pex11pβ is dispensable for peroxisome membrane elongation and division. However, we demonstrate that an amphipathic helix (Helix 2) within the first N-terminal 40 amino acids is crucial for membrane elongation and self-interaction of Pex11pβ. Interestingly, we find that Pex11pβ self-interaction strongly depends on the detergent used for solubilization. We also show that N-terminal cysteines are not essential for membrane elongation, and that putative N-terminal phosphorylation sites are dispensable for Pex11pβ function. We propose that self-interaction of Pex11pβ regulates its membrane deforming activity in conjunction with membrane lipids.
Abstract.
Author URL.
Islinger M, Gomes S, Bonekamp N, Magalhaes AC, Camoes F, Castro I, Almeida M, Schrader M (2013). Targeting of tail-anchored proteins to peroxisomes requires Pex19 and a highly positively charged tail.
MOLECULAR BIOLOGY OF THE CELL,
24 Author URL.
2012
Bonekamp NA, Islinger M, Lázaro MG, Schrader M (2012). Cytochemical detection of peroxisomes and mitochondria.
Methods in Molecular Biology,
931, 467-482.
Abstract:
Cytochemical detection of peroxisomes and mitochondria
Peroxisomes and mitochondria are essential subcellular organelles in mammals. Interestingly, recent studies have elucidated that these highly dynamic and plastic organelles exhibit a much closer interrelationship than previously assumed. Peroxisomes and mitochondria are metabolically linked organelles, which are cooperating and cross-talking. They share key components of their division machinery and cooperate in antiviral signaling and defense. As peroxisomal alterations in metabolism, biogenesis, dynamics, and proliferation have the potential to in fluence mitochondrial morphology and functions (and vice versa), there is currently great interest in the detection of both organelles under different experimental conditions. Here, we present protocols used successfully in our laboratory for the dual detection of peroxisomes and mitochondria in cultured mammalian cells. We address double immuno fluorescence and fluorescencebased techniques as well as reagents to investigate organelle dynamics, morphological alterations, and organelle-speci fic targeting of proteins. In addition, we describe the application of diaminobenzidine cytochemistry on cultured cells to speci fically label peroxisomes in ultrastructural studies ©Springer Science+Business Media, LLC 2012.
Abstract.
Schrader M, Bonekamp NA, Islinger M (2012). Fission and proliferation of peroxisomes.
Biochimica et Biophysica Acta - Molecular Basis of Disease,
1822(9), 1343-1357.
Abstract:
Fission and proliferation of peroxisomes
Peroxisomes are remarkably dynamic, multifunctional organelles, which react to physiological changes in their cellular environment and adopt their morphology, number, enzyme content and metabolic functions accordingly. At the organelle level, the key molecular machinery controlling peroxisomal membrane elongation and remodeling as well as membrane fission is becoming increasingly established and defined. Key players in peroxisome division are conserved in animals, plants and fungi, and key fission components are shared with mitochondria. However, the physiological stimuli and corresponding signal transduction pathways regulating and modulating peroxisome maintenance and proliferation are, despite a few exceptions, largely unexplored. There is emerging evidence that peroxisomal dynamics and proper regulation of peroxisome number and morphology are crucial for the physiology of the cell, as well as for the pathology of the organism. Here, we discuss several key aspects of peroxisomal fission and proliferation and highlight their association with certain diseases. We address signaling and transcriptional events resulting in peroxisome proliferation, and focus on novel findings concerning the key division components and their interplay. Finally, we present an updated model of peroxisomal growth and division. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease. © 2012 Elsevier B.V.
Abstract.
Rinn C, Aroso M, Prüssing J, Islinger M, Schrader M (2012). Modulating zymogen granule formation in pancreatic AR42J cells.
Exp Cell Res,
318(15), 1855-1866.
Abstract:
Modulating zymogen granule formation in pancreatic AR42J cells.
Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. To investigate ZG biogenesis, cargo sorting and packaging, suitable cellular model systems are required. Here, we demonstrate that granule formation in pancreatic AR42J cells, an acinar model system, can be modulated by altering the growth conditions in cell culture. We find that cultivation of AR42J cells in Panserin™ 401, a serum-free medium, enhances the induction of granule formation in the presence or absence of dexamethasone when compared to standard conditions including serum. Biochemical and morphological studies revealed an increase in ZG markers on the mRNA and protein level, as well as in granule size compared to standard conditions. Our data indicate that this effect is related to pronounced differentiation of AR42J cells. To address if enhanced expression of ZG proteins promotes granule formation, we expressed several zymogens and ZG membrane proteins in unstimulated AR42J cells and in constitutively secreting COS-7 cells. Neither single expression nor co-expression was sufficient to initiate granule formation in AR42J cells or the formation of granule-like structures in COS-7 cells as described for neuroendocrine cargo proteins. The importance of our findings for granule formation in exocrine cells is discussed.
Abstract.
Author URL.
Ribeiro D, Castro I, Dariush Fahimi H, Schrader M (2012). Peroxisome morphology in pathology.
Histology and Histopathology,
27(6), 661-676.
Abstract:
Peroxisome morphology in pathology
Peroxisomes are remarkably dynamic and versatile organelles that are essential for human health and development. They respond to physiological changes in the cellular environment by adapting their morphology, number, enzyme content and metabolic functions accordingly. With the discovery of the first key peroxisomal morphology proteins, the investigation of peroxisomal shape, distribution and dynamics has become an exciting new field in cell biology and biomedical sciences because of its relation to organelle functionality and its impact on developmental and physiological processes. In this review, we summarize recent findings on peroxisome biology, dynamics and the modulation of peroxisome morphology, especially in mammals. Furthermore, we discuss the roles of peroxisome dynamics and morphology in cell pathology and present recent examples for alterations in peroxisome morphology under disease conditions. Besides defects in the peroxisomal morphology machinery, we also address peroxisome biogenesis disorders, alterations of peroxisome number during carcinogenesis and liver cirrhosis, and morphological alterations of peroxisomes during viral infection.
Abstract.
Schrader M, Almeida M, Grille S (2012). Postfixation detergent treatment liberates the membrane modelling protein Pex11β from peroxisomal membranes.
Histochem Cell Biol,
138(3), 541-547.
Abstract:
Postfixation detergent treatment liberates the membrane modelling protein Pex11β from peroxisomal membranes.
Pex11 proteins are involved in membrane remodelling processes of peroxisomes, and are key components of peroxisomal division and proliferation. In mammals, three Pex11 isoforms, Pex11α, Pex11β, and Pex11γ exist. Here we demonstrate that Pex11β, but not Pex11α or Pex11γ, is almost exclusively extracted from peroxisomal membranes of paraformaldehyde-fixed cells by permeabilisation with the non-ionic detergent Triton X-100. This results in diminished detection of Myc-Pex11β in immunofluorescence preparations and appearance of the protein in the Triton X-100 extract. To our knowledge, Pex11β is the first peroxisomal membrane protein showing such a peculiar behaviour. Loss of Pex11β can be avoided by permeabilisation with digitonin, the addition of glutaraldehyde to the fixative, or the expression of a Pex11 fusion protein with a larger protein tag (e.g. YFP). Our observations further point to different functions and biochemical properties of the Pex11 isoforms within the peroxisomal membrane and during peroxisome proliferation.
Abstract.
Author URL.
Islinger M, Grille S, Fahimi HD, Schrader M (2012). The peroxisome: an update on mysteries.
Histochem Cell Biol,
137(5), 547-574.
Abstract:
The peroxisome: an update on mysteries.
Peroxisomes contribute to several crucial metabolic processes such as β-oxidation of fatty acids, biosynthesis of ether phospholipids and metabolism of reactive oxygen species, which render them indispensable to human health and development. Peroxisomes are highly dynamic organelles that rapidly assemble, multiply and degrade in response to metabolic needs. In recent years, the interest in peroxisomes and their physiological functions has significantly increased. This review intends to highlight recent discoveries and trends in peroxisome research, and represents an update as well as a continuation of a former review article. Novel exciting findings on the biological functions, biogenesis, formation and degradation of peroxisomes, on peroxisomal dynamics and division, as well as on the interaction and cross-talk of peroxisomes with other subcellular compartments are addressed. Furthermore, recent findings on the role of peroxisomes in the brain are discussed.
Abstract.
Author URL.
Bonekamp NA, Sampaio P, de Abreu FV, Lüers GH, Schrader M (2012). Transient Complex Interactions of Mammalian Peroxisomes Without Exchange of Matrix or Membrane Marker Proteins.
Traffic,
13(7), 960-978.
Abstract:
Transient Complex Interactions of Mammalian Peroxisomes Without Exchange of Matrix or Membrane Marker Proteins
Peroxisomes and mitochondria show a much closer interrelationship than previously anticipated. They co-operate in the metabolism of fatty acids and reactive oxygen species, but also share components of their fission machinery. If peroxisomes - like mitochondria - also fuse in mammalian cells is a matter of debate and was not yet systematically investigated. To examine potential peroxisomal fusion and interactions in mammalian cells, we established an in vivo fusion assay based on hybridoma formation by cell fusion. Fluorescence microscopy in time course experiments revealed a merge of different peroxisomal markers in fused cells. However, live cell imaging revealed that peroxisomes were engaged in transient and long-term contacts, without exchanging matrix or membrane markers. Computational analysis showed that transient peroxisomal interactions are complex and can potentially contribute to the homogenization of the peroxisomal compartment. However, peroxisomal interactions do not increase after fatty acid or H 2O 2 treatment. Additionally, we provide the first evidence that mitochondrial fusion proteins do not localize to peroxisomes. We conclude that mammalian peroxisomes do not fuse with each other in a mechanism similar to mitochondrial fusion. However, they show an extensive degree of interaction, the implication of which is discussed. © 2012 John Wiley & Sons A/S.
Abstract.
Bonekamp NA, Sampaio P, de Abreu FV, Lüers GH, Schrader M (2012). Transient complex interactions of mammalian peroxisomes without exchange of matrix or membrane marker proteins.
Traffic,
13(7), 960-978.
Abstract:
Transient complex interactions of mammalian peroxisomes without exchange of matrix or membrane marker proteins.
Peroxisomes and mitochondria show a much closer interrelationship than previously anticipated. They co-operate in the metabolism of fatty acids and reactive oxygen species, but also share components of their fission machinery. If peroxisomes - like mitochondria - also fuse in mammalian cells is a matter of debate and was not yet systematically investigated. To examine potential peroxisomal fusion and interactions in mammalian cells, we established an in vivo fusion assay based on hybridoma formation by cell fusion. Fluorescence microscopy in time course experiments revealed a merge of different peroxisomal markers in fused cells. However, live cell imaging revealed that peroxisomes were engaged in transient and long-term contacts, without exchanging matrix or membrane markers. Computational analysis showed that transient peroxisomal interactions are complex and can potentially contribute to the homogenization of the peroxisomal compartment. However, peroxisomal interactions do not increase after fatty acid or H(2) O(2) treatment. Additionally, we provide the first evidence that mitochondrial fusion proteins do not localize to peroxisomes. We conclude that mammalian peroxisomes do not fuse with each other in a mechanism similar to mitochondrial fusion. However, they show an extensive degree of interaction, the implication of which is discussed.
Abstract.
Author URL.
Bonekamp NA, Schrader M (2012). Transient complex peroxisomal interactions: a new facet of peroxisome dynamics in mammalian cells.
Communicative and Integrative Biology,
5(6), 534-537.
Abstract:
Transient complex peroxisomal interactions: a new facet of peroxisome dynamics in mammalian cells
Mitochondria and peroxisomes are ubiquitous subcellular organelles that fulfill essential metabolic functions, rendering them indispensable for human development and health. Both are highly dynamic organelles that can undergo remarkable changes in morphology and number to accomplish cellular needs. While mitochondrial dynamics are also regulated by frequent fusion events, the fusion of mature peroxisomes in mammalian cells remained a matter of debate. In our recent study, we clarified systematically that there is no complete fusion of mature peroxisomes analogous to mitochondria. Moreover, in contrast to key division components such as DLP1, Fis1 or Mff, mitochondrial fusion proteins were not localized to peroxisomes. However, we discovered and characterized novel transient, complex interactions between individual peroxisomes which may contribute to the homogenization of the often heterogeneous peroxisomal compartment, e.g. by distribution of metabolites, signals or other "molecular information" via interperoxisomal contact sites. © 2012 Landes Bioscience.
Abstract.
2011
Islinger M, Schrader M (2011). Peroxisomes.
Curr Biol,
21(19), R800-R801.
Author URL.
Delille HK, Dodt G, Schrader M (2011). Pex11pβ-mediated maturation of peroxisomes. Communicative & Integrative Biology, 4(1), 51-54.
Delille HK, Dodt G, Schrader M (2011). Pex11pβ-mediated maturation of peroxisomes.
Commun Integr Biol,
4(1), 51-54.
Abstract:
Pex11pβ-mediated maturation of peroxisomes.
Peroxisomes are highly dynamic, multifunctional organelles that display remarkable changes in morphology, number and enzyme content. Peroxisomes multiply by growth and division of pre-existing organelles, but they can also form de novo from the ER. Growth and division of peroxisomes in mammalian cells involves elongation, membrane constriction and final fission and requires the peroxisome biogenesis Pex11 proteins as well as the recruitment of Dynamin-like protein DLP1/Drp1. We recently exploited the division-inhibiting properties of a unique Pex11pβ-YFP fusion protein to further dissect the process of peroxisomal growth and division. By applying life cell imaging and the HaloTag technology, our study revealed that Pex11pβ-mediated growth (elongation) and division of peroxisomes follows a multistep maturation pathway, which is initiated by the formation of an early peroxisomal membrane compartment from a pre-existing peroxisome and its stepwise conversion into a mature, metabolically active peroxisome compartment. Our observations support the view that peroxisomes formed by growth and division of pre-existing ones contain new membrane and matrix components. Peroxisome division is an asymmetric process, which is more complex than simple (symmetric) division of a preexisting organelle and equal distribution of the protein content. Our findings are in favor of Pex11pβ acting as a peroxisomal membrane shaping protein.
Abstract.
Author URL.
2010
Borta H, Aroso M, Rinn C, Gomez-Lazaro M, Vitorino R, Zeuschner D, Grabenbauer M, Amado F, Schrader M (2010). Analysis of low abundance membrane-associated proteins from rat pancreatic zymogen granules.
J Proteome Res,
9(10), 4927-4939.
Abstract:
Analysis of low abundance membrane-associated proteins from rat pancreatic zymogen granules.
Zymogen granules (ZG) are specialized storage organelles in the exocrine pancreas that allow the sorting, packaging, and regulated apical secretion of digestive enzymes. As there is a critical need for further understanding of the key processes in regulated secretion to develop new therapeutic options in medicine, we applied a suborganellar proteomics approach to identify peripheral membrane-associated ZG proteins. We focused on the analysis of a "basic" group (pH range 6.2-11) with about 46 spots among which 44 were identified by tandem mass spectrometry. These spots corresponded to 16 unique proteins, including rat mast cell chymase (RMCP-1) and peptidyl-prolyl cis-trans isomerase B (PpiB; cyclophilin B), an ER-resident protein. To confirm that these proteins were specific to zymogen granules and not contaminants of the preparation, we conducted a series of validation experiments. Immunoblotting of ZG subfractions revealed that chymase and PpiB behaved like bona fide peripheral membrane proteins. Their expression in rat pancreas was regulated by feeding behavior. Ultrastructural and immunofluorescence studies confirmed their ZG localization. Furthermore, a chymase-YFP fusion protein was properly targeted to ZG in pancreatic AR42J cells. Interestingly, for both proteins, proteoglycan-binding properties have been reported. The importance of our findings for sorting and packaging during ZG formation is discussed.
Abstract.
Author URL.
Islinger M, Cardoso MJR, Schrader M (2010). Be different--the diversity of peroxisomes in the animal kingdom.
Biochim Biophys Acta,
1803(8), 881-897.
Abstract:
Be different--the diversity of peroxisomes in the animal kingdom.
Peroxisomes represent so-called "multipurpose organelles" as they contribute to various anabolic as well as catabolic pathways. Thus, with respect to the physiological specialization of an individual organ or animal species, peroxisomes exhibit a functional diversity, which is documented by significant variations in their proteome. These differences are usually regarded as an adaptational response to the nutritional and environmental life conditions of a specific organism. Thus, human peroxisomes can be regarded as an in part physiologically unique organellar entity fulfilling metabolic functions that differ from our animal model systems. In line with this, a profound understanding on how peroxisomes acquired functional heterogeneity in terms of an evolutionary and mechanistic background is required. This review summarizes our current knowledge on the heterogeneity of peroxisomal physiology, providing insights into the genetic and cell biological mechanisms, which lead to the differential localization or expression of peroxisomal proteins and further gives an overview on peroxisomal biochemical pathways, which are specialized in different animal species and organs. Moreover, it addresses the impact of proteome studies on our understanding of differential peroxisome function describing the utility of mass spectrometry and computer-assisted algorithms to identify peroxisomal target sequences for the detection of new organ- or species-specific peroxisomal proteins.
Abstract.
Author URL.
Bonekamp NA, Vormund K, Jacob R, Schrader M (2010). Dynamin-like protein 1 at the Golgi complex: a novel component of the sorting/targeting machinery en route to the plasma membrane.
Exp Cell Res,
316(20), 3454-3467.
Abstract:
Dynamin-like protein 1 at the Golgi complex: a novel component of the sorting/targeting machinery en route to the plasma membrane.
The final step in the liberation of secretory vesicles from the trans-Golgi network (TGN) involves the mechanical action of the large GTPase dynamin as well as conserved dynamin-independent fission mechanisms, e.g. mediated by Brefeldin A-dependent ADP-ribosylated substrate (BARS). Another member of the dynamin family is the mammalian dynamin-like protein 1 (DLP1/Drp1) that is known to constrict and tubulate membranes, and to divide mitochondria and peroxisomes. Here, we examined a potential role for DLP1 at the Golgi complex. DLP1 localized to the Golgi complex in some but not all cell lines tested, thus explaining controversial reports on its cellular distribution. After silencing of DLP1, an accumulation of the apical reporter protein YFP-GL-GPI, but not the basolateral reporter VSVG-SP-GFP at the Golgi complex was observed. A reduction in the transport of YFP-GL-GPI to the plasma membrane was confirmed by surface immunoprecipitation and TGN-exit assays. In contrast, YFP-GL-GPI trafficking was not disturbed in cells silenced for BARS, which is involved in basolateral sorting and trafficking of VSVG-SP-GFP in COS-7 cells. Our data indicate a new role for DLP1 at the Golgi complex and thus a role for DLP1 as a novel component of the apical sorting machinery at the TGN is discussed.
Abstract.
Author URL.
Delille HK, Agricola B, Guimaraes SC, Borta H, Lüers GH, Fransen M, Schrader M (2010). Pex11pbeta-mediated growth and division of mammalian peroxisomes follows a maturation pathway.
J Cell Sci,
123(Pt 16), 2750-2762.
Abstract:
Pex11pbeta-mediated growth and division of mammalian peroxisomes follows a maturation pathway.
Peroxisomes are ubiquitous subcellular organelles, which multiply by growth and division but can also form de novo via the endoplasmic reticulum. Growth and division of peroxisomes in mammalian cells involves elongation, membrane constriction and final fission. Dynamin-like protein (DLP1/Drp1) and its membrane adaptor Fis1 function in the later stages of peroxisome division, whereas the membrane peroxin Pex11pbeta appears to act early in the process. We have discovered that a Pex11pbeta-YFP(m) fusion protein can be used as a specific tool to further dissect peroxisomal growth and division. Pex11pbeta-YFP(m) inhibited peroxisomal segmentation and division, but resulted in the formation of pre-peroxisomal membrane structures composed of globular domains and tubular extensions. Peroxisomal matrix and membrane proteins were targeted to distinct regions of the peroxisomal structures. Pex11pbeta-mediated membrane formation was initiated at pre-existing peroxisomes, indicating that growth and division follows a multistep maturation pathway and that formation of mammalian peroxisomes is more complex than simple division of a pre-existing organelle. The implications of these findings on the mechanisms of peroxisome formation and membrane deformation are discussed.
Abstract.
Author URL.
Gómez-Lázaro M, Rinn C, Aroso M, Amado F, Schrader M (2010). Proteomic analysis of zymogen granules.
Expert Rev Proteomics,
7(5), 735-747.
Abstract:
Proteomic analysis of zymogen granules.
Zymogen granules (ZGs) are specialized storage organelles in the exocrine pancreas that allow the sorting, packaging and regulated apical secretion of digestive enzymes. ZG constituents play important roles in pancreatic injury and disease. The molecular mechanisms underlying these processes are still poorly defined. Thus, there is currently great interest in the identification and characterization of ZG components. Recent proteomic studies have greatly enhanced our knowledge regarding potential new 'players' in ZG biogenesis and regulated secretion. In this article, we present the latest advancements in and insights into the analysis of the ZG proteome by the combination of organelle isolation, protein separation, mass spectrometry and validation of protein identification. Recent developments in the analysis of ZG proteins from pancreatic juice and related proteins from saliva are also discussed.
Abstract.
Author URL.
Fardilha M, Schrader M, da Cruz E Silva OAB, da Cruz E Silva EF (2010). Understanding fatty acid metabolism through an active learning approach.
Biochem Mol Biol Educ,
38(2), 65-69.
Abstract:
Understanding fatty acid metabolism through an active learning approach.
A multi-method active learning approach (MALA) was implemented in the Medical Biochemistry teaching unit of the Biomedical Sciences degree at the University of Aveiro, using problem-based learning as the main learning approach. In this type of learning strategy, students are involved beyond the mere exercise of being taught by listening. Less emphasis is placed on transmitting information and the focus is shifted toward developing higher order thinking (analysis, synthesis, and evaluation). However, MALA should always involve clearly identified objectives and well-defined targets. Understanding fatty acid metabolism was one of the proposed goals of the Medical Biochemistry unit. To this end, students were challenged with a variety of learning strategies to develop skills associated with group conflict resolution, critical thinking, information access, and retrieval, as well as oral and written communication skills. Overall, students and learning facilitators were highly motivated by the diversity of learning activities, particularly due to the emphasis on correlating theoretical knowledge with human health and disease. As a quality control exercise, the students were asked to answer a questionnaire on their evaluation of the whole teaching/learning experience. Our initial analysis of the learning outcomes permits us to conclude that the approach undertaken yields results that surpass the traditional teaching methods.
Abstract.
Author URL.
2009
Schrader M (2009). Anniversaries, peroxisomes and reactive oxygen species. Histochemistry and Cell Biology, 131(4), 435-436.
Delille HK, Alves R, Schrader M (2009). Biogenesis of peroxisomes and mitochondria: linked by division.
Histochem Cell Biol,
131(4), 441-446.
Abstract:
Biogenesis of peroxisomes and mitochondria: linked by division.
Peroxisomes and mitochondria are metabolically linked organelles, which are crucial to human health and development. The search for components involved in their dynamics and maintenance led to the interesting finding that mitochondria and peroxisomes share components of their division machinery. Recently, it became clear that this is a common strategy used by mammals, fungi and plants. Furthermore, a closer interrelationship between peroxisomes and mitochondria has been proposed, which might have an impact on functionality and disease conditions. Here, we briefly highlight the major findings, views and open questions concerning peroxisomal formation, division, and interrelationship with mitochondria.
Abstract.
Author URL.
Delille HK, Schrader M (2009). Hypertubulation of peroxisomes by multiple stimuli.
EUROPEAN JOURNAL OF CELL BIOLOGY,
88, 68-68.
Author URL.
Camões F, Bonekamp NA, Delille HK, Schrader M (2009). Organelle dynamics and dysfunction: a closer link between peroxisomes and mitochondria.
J Inherit Metab Dis,
32(2), 163-180.
Abstract:
Organelle dynamics and dysfunction: a closer link between peroxisomes and mitochondria.
Mitochondria and peroxisomes are ubiquitous subcellular organelles, which fulfil an indispensable role in the cellular metabolism of higher eukaryotes. Moreover, they are highly dynamic and display large plasticity. There is growing evidence now that both organelles exhibit a closer interrelationship than previously appreciated. This connection includes metabolic cooperations and cross-talk, a novel putative mitochondria-to-peroxisome vesicular trafficking pathway, as well as an overlap in key components of their fission machinery. Thus, peroxisomal alterations in metabolism, biogenesis, dynamics and proliferation can potentially influence mitochondrial functions, and vice versa. In this review, we present the latest progress in the emerging field of peroxisomal and mitochondrial interrelationship with a particular emphasis on organelle dynamics and its implication in diseases.
Abstract.
Author URL.
Bonekamp NA, Völkl A, Fahimi HD, Schrader M (2009). Reactive oxygen species and peroxisomes: struggling for balance.
Biofactors,
35(4), 346-355.
Abstract:
Reactive oxygen species and peroxisomes: struggling for balance.
Reactive oxygen species (ROS) can surely be considered as multifunctional biofactors within the cell. They are known to participate in regular cell functions, for example, as signal mediators, but overproduction under oxidative stress conditions leads to deleterious cellular effects, cell death and diverse pathological conditions. Peroxisomal function has long been linked to oxygen metabolism due to the high concentration of H(2)O(2)-generating oxidases in peroxisomes and their set of antioxidant enzymes, especially catalase. Still, mitochondria have been very much placed in the centre of ROS metabolism and oxidative stress. This review discusses novel findings concerning the relationship between ROS and peroxisomes, as they revealed to be a key player in the dynamic spin of ROS metabolism and oxidative injury. An overview of ROS generating enzymes as well as their antioxidant counterparts will be given, exemplifying the precise fine-tuning between the opposing systems. Various conditions in which the balance between generation and scavenging of ROS in peroxisomes is perturbed, for example, exogenous manipulation, ageing and peroxisomal disorders, are addressed. Furthermore, peroxisome-derived oxidative stress and its effect on mitochondria (and vice versa) are discussed, highlighting the close interrelationship of both organelles.
Abstract.
Author URL.
2008
Gomez-Lazaro M, Bonekamp NA, Galindo MF, Jordán J, Schrader M (2008). 6-Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrial fragmentation in SH-SY5Y cells.
Free Radical Biology and Medicine,
44(11), 1960-1969.
Abstract:
6-Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrial fragmentation in SH-SY5Y cells
Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used neurotoxin to study Parkinson's disease. Herein we studied the potential effects of 6-OHDA on mitochondrial morphology in SH-SY5Y neuroblastoma cells. By immunofluorescence and time-lapse fluorescence microscopy we demonstrated that 6-OHDA induced profound mitochondrial fragmentation in SH-SY5Y cells, an event that was similar to mitochondrial fission induced by overexpression of Fis1p, a membrane adaptor for the dynamin-related protein 1 (DLP1/Drp1). 6-OHDA failed to induce any changes in peroxisome morphology. Biochemical experiments revealed that 6-OHDA-induced mitochondrial fragmentation is an early event preceding the collapse of the mitochondrial membrane potential and cytochrome c release in SH-SY5Y cells. Silencing of DLP1/Drp1, which is involved in mitochondrial and peroxisomal fission, prevented 6-OHDA-induced fragmentation of mitochondria. Furthermore, in cells silenced for Drp1, 6-OHDA-induced cell death was reduced, indicating that a block in mitochondrial fission protects SH-SY5Y cells against 6-OHDA toxicity. Experiments in mouse embryonic fibroblasts deficient in Bax or p53 revealed that both proteins are not essential for 6-OHDA-induced mitochondrial fragmentation. Our data demonstrate for the first time an involvement of mitochondrial fragmentation and Drp1 function in 6-OHDA-induced apoptosis.
Abstract.
Vormund K, Bonekamp NA, Jacob R, Schrader M (2008). A novel function of dynamin-like protein 1 at the Golgi complex.
Author URL.
Faust F, Gomez-Lazaro M, Borta H, Agricola B, Schrader M (2008). Rab8 is involved in zymogen granule formation in pancreatic acinar AR42J cells.
Traffic,
9(6), 964-979.
Abstract:
Rab8 is involved in zymogen granule formation in pancreatic acinar AR42J cells.
Zymogen granules (ZGs) are specialized storage organelles in the exocrine pancreas, which allow digestive enzyme storage and regulated apical secretion. To understand the function of these important organelles, we are conducting studies to identify and characterize ZG membrane proteins. Small guanosine triphosphatases (GTPases) of the Rab family are key protein components involved in vesicular/granular trafficking and membrane fusion in eukaryotic cells. In this study, we show by morphological studies that Rab8 (Rab8A) localizes to ZGs in acinar cells of the pancreas. We find that Rab8 is present on isolated ZGs from rat pancreas and in the ZG membrane fraction obtained after granule subfractionation. To address a putative role of Rab8 in granule biogenesis, we conducted RNA interference experiments to 'knock down' the expression of Rab8 in pancreatic AR42J cells. Silencing of Rab8 (but not of Rab3) resulted in a decrease in the number of ZGs and in an accumulation of granule marker proteins within the Golgi complex. By contrast, the trafficking of lysosomal and plasma membrane proteins was not affected. These data provide first evidence for a role of Rab8 early on in ZG formation at the Golgi complex and thus, apical trafficking of digestive enzymes in acinar cells of the pancreas.
Abstract.
Author URL.
Delille HK, Schrader M (2008). Targeting of hFis1 to peroxisomes is mediated by Pex19p.
J Biol Chem,
283(45), 31107-31115.
Abstract:
Targeting of hFis1 to peroxisomes is mediated by Pex19p.
The processes of peroxisome formation and proliferation are still a matter of debate. We have previously shown that peroxisomes share some components of their division machinery with mitochondria. hFis1, a tail-anchored membrane protein, regulates the membrane fission of both organelles by DLP1/Drp1 recruitment, but nothing is known about the mechanisms of the dual targeting of hFis1. Here we demonstrate for the first time that peroxisomal targeting of hFis1 depends on Pex19p, a peroxisomal membrane protein import factor. hFis1/Pex19p binding was demonstrated by expression and co-immunoprecipitation studies. Using mutated versions of hFis1 an essential binding region for Pex19p was located within the last 26 C-terminal amino acids of hFis1, which are required for proper targeting to both mitochondria and peroxisomes. The basic amino acids in the very C terminus are not essential for Pex19p binding and peroxisomal targeting, but are instead required for mitochondrial targeting. Silencing of Pex19p by small interference RNA reduced the targeting of hFis1 to peroxisomes, but not to mitochondria. In contrast, overexpression of Pex19p alone was not sufficient to shift the targeting of hFis1 to peroxisomes. Our findings indicate that targeting of hFis1 to peroxisomes and mitochondria are independent events and support a direct, Pex19p-dependent targeting of peroxisomal tail-anchored proteins.
Abstract.
Author URL.
Schrader M, Fahimi HD (2008). The peroxisome: still a mysterious organelle.
Histochem Cell Biol,
129(4), 421-440.
Abstract:
The peroxisome: still a mysterious organelle.
More than half a century of research on peroxisomes has revealed unique features of this ubiquitous subcellular organelle, which have often been in disagreement with existing dogmas in cell biology. About 50 peroxisomal enzymes have so far been identified, which contribute to several crucial metabolic processes such as beta-oxidation of fatty acids, biosynthesis of ether phospholipids and metabolism of reactive oxygen species, and render peroxisomes indispensable for human health and development. It became obvious that peroxisomes are highly dynamic organelles that rapidly assemble, multiply and degrade in response to metabolic needs. However, many aspects of peroxisome biology are still mysterious. This review addresses recent exciting discoveries on the biogenesis, formation and degradation of peroxisomes, on peroxisomal dynamics and division, as well as on the interaction and cross talk of peroxisomes with other subcellular compartments. Furthermore, recent advances on the role of peroxisomes in medicine and in the identification of novel peroxisomal proteins are discussed.
Abstract.
Author URL.
2007
Schrader M, Yoon Y (2007). Mitochondria and peroxisomes: are the 'big brother' and the 'little sister' closer than assumed?.
Bioessays,
29(11), 1105-1114.
Abstract:
Mitochondria and peroxisomes: are the 'big brother' and the 'little sister' closer than assumed?
Mitochondria and peroxisomes are essential subcellular organelles in mammals. Despite obvious differences, both organelles display certain morphological and functional similarities. Recent studies have elucidated that these highly dynamic and plastic organelles share components of their division machinery. Mitochondria and peroxisomes are metabolically linked organelles, which are cooperating and cross-talking. This review addresses the dynamics and division of mitochondria and peroxisomes as well as their functional similarities to provide insight as to why these organelles share the fission machinery in evolutionary aspects.
Abstract.
Author URL.
2006
Schrader M, Fahimi HD (2006). Growth and division of peroxisomes.
Int Rev Cytol,
255, 237-290.
Abstract:
Growth and division of peroxisomes.
Peroxisomes are ubiquitous subcellular organelles, which are highly dynamic and display large plasticity in response to cellular and environmental conditions. Novel proteins and pathways that mediate and control peroxisome formation, growth, and division continue to be discovered, and the cellular machineries that act together to regulate peroxisome number and size are under active investigation. Here, advances in the field of peroxisomal dynamics and proliferation in mammals and yeast are reviewed. The authors address the signals, conditions, and proteins that affect, regulate, and control the number and size of this essential organelle, especially the components involved in the division of peroxisomes. Special emphasis is on the function of dynamin-related proteins (DRPs), on Fis1, a putative adaptor for DRPs, on the role of the Pex11 family of peroxisomal membrane proteins, and the cytoskeleton.
Abstract.
Author URL.
Delille HK, Bonekamp NA, Schrader M (2006). Peroxisomes and disease - an overview.
Int J Biomed Sci,
2(4), 308-314.
Abstract:
Peroxisomes and disease - an overview.
Peroxisomes are indispensable for human health and development. They represent ubiquitous subcellular organelles which compartmentalize enzymes responsible for several crucial metabolic processes such as β-oxidation of specific fatty acids, biosynthesis of ether phospholipids and metabolism of reactive oxygen species. Peroxisomes are highly flexible organelles that rapidly assemble, multiply and degrade in response to metabolic needs. Basic research on the biogenesis of peroxisomes and their metabolic functions have improved our knowledge about their crucial role in several inherited disorders and in other pathophysiological conditions. The goal of this review is to give a comprehensive overview of the role of peroxisomes in disease. Besides the genetic peroxisomal disorders in humans, the role of peroxisomes in carcinogenesis and in situations related to oxidative stress such as inflammation, ischemia-reperfusion, and diabetes will be addressed.
Abstract.
Author URL.
Schrader M, Fahimi HD (2006). Peroxisomes and oxidative stress.
Biochim Biophys Acta,
1763(12), 1755-1766.
Abstract:
Peroxisomes and oxidative stress.
The discovery of the colocalization of catalase with H2O2-generating oxidases in peroxisomes was the first indication of their involvement in the metabolism of oxygen metabolites. In past decades it has been revealed that peroxisomes participate not only in the generation of reactive oxygen species (ROS) with grave consequences for cell fate such as malignant degeneration but also in cell rescue from the damaging effects of such radicals. In this review the role of peroxisomes in a variety of physiological and pathological processes involving ROS mainly in animal cells is presented. At the outset the enzymes generating and scavenging H2O2 and other oxygen metabolites are reviewed. The exposure of cultured cells to UV light and different oxidizing agents induces peroxisome proliferation with formation of tubular peroxisomes and apparent upregulation of PEX genes. Significant reduction of peroxisomal volume density and several of their enzymes is observed in inflammatory processes such as infections, ischemia-reperfusion injury and hepatic allograft rejection. The latter response is related to the suppressive effects of TNFalpha on peroxisomal function and on PPARalpha. Their massive proliferation induced by a variety of xenobiotics and the subsequent tumor formation in rodents is evidently due to an imbalance in the formation and scavenging of ROS, and is mediated by PPARalpha. In PEX5-/- mice with the absence of functional peroxisomes severe abnormalities of mitochondria in different organs are observed which resemble closely those in respiratory chain disorders associated with oxidative stress. Interestingly, no evidence of oxidative damage to proteins or lipids, nor of increased peroxide production has been found in that mouse model. In this respect the role of PPARalpha, which is highly activated in those mice, in prevention of oxidative stress deserves further investigation.
Abstract.
Author URL.
Schrader M (2006). Shared components of mitochondrial and peroxisomal division.
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH,
1763(5-6), 531-541.
Author URL.
Schrader M (2006). Shared components of mitochondrial and peroxisomal division.
Biochim Biophys Acta,
1763(5-6), 531-541.
Abstract:
Shared components of mitochondrial and peroxisomal division.
Mitochondria and peroxisomes are ubiquitous subcellular organelles, which are highly dynamic and display large plasticity. Recent studies have led to the surprising finding that both organelles share components of their division machinery, namely the dynamin-related protein DLP1/Drp1 and hFis1, which recruits DLP1/Drp1 to the organelle membranes. This review addresses the current state of knowledge concerning the dynamics and fission of peroxisomes, especially in relation to mitochondrial morphology and division in mammalian cells.
Abstract.
Author URL.
Bach J-P, Borta H, Ackermann W, Faust F, Borchers O, Schrader M (2006). The secretory granule protein syncollin localizes to HL-60 cells and neutrophils.
J Histochem Cytochem,
54(8), 877-888.
Abstract:
The secretory granule protein syncollin localizes to HL-60 cells and neutrophils.
The secretory granule protein syncollin was first identified in the exocrine pancreas where a population of the protein is associated with the luminal surface of the zymogen granule membrane. In this study we provide first morphological and biochemical evidence that, in addition to its pancreatic localization, syncollin is also present in neutrophilic granulocytes of rat and human origin. By immunohistological studies, syncollin was detected in neutrophilic granulocytes of the spleen. Furthermore, syncollin is expressed by the promyelocytic HL-60 cells, where it is stored in azurophilic granules and in a vesicular compartment. These findings were confirmed by fractionation experiments and immunoelectron microscopy. Treatment with a phorbol ester triggered the release of syncollin indicating that in HL-60 cells it is a secretory protein that can be mobilized upon stimulation. A putative role for syncollin in host defense is discussed.
Abstract.
Author URL.
Faust F, Borta H, Schrader M (2006). The small GTPase Rab8 is involved in zymogen granule formation in acinar cells of the exocrine pancreas.
Author URL.
2005
Koch A, Yoon Y, Bonekamp NA, McNiven MA, Schrader M (2005). A role for Fis1 in both mitochondrial and peroxisomal fission in mammalian cells.
Mol Biol Cell,
16(11), 5077-5086.
Abstract:
A role for Fis1 in both mitochondrial and peroxisomal fission in mammalian cells.
The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.
Abstract.
Author URL.
Schrader M, Gould SJ (2005). Assay and functional analysis of dynamin-like protein 1 in peroxisome division.
Methods Enzymol,
404, 586-597.
Abstract:
Assay and functional analysis of dynamin-like protein 1 in peroxisome division.
Recent studies have demonstrated that peroxisome division requires at least one dynamin-like protein, Vps1p, in the yeast Saccharomyces cerevisiae and DLP1 (DRP1) in mammalian cells. Although the requirement for these proteins in peroxisome division is supported by many lines of evidence, their roles in peroxisome division have yet to be identified. Given the independence of peroxisomes from other organelle systems, the peroxisome system appears to have unique attributes for studying the function of dynamin-like proteins in organelle division. Here, we present methods that have been used for studying the role of DLP1 in peroxisome biogenesis and division.
Abstract.
Author URL.
Boll A, Schrader M (2005). Elongation of peroxisomes as an indicator for efficient dynamin-like protein 1 knock down in mammalian cells.
J Histochem Cytochem,
53(8), 1037-1040.
Abstract:
Elongation of peroxisomes as an indicator for efficient dynamin-like protein 1 knock down in mammalian cells.
RNA interference has become a valuable tool to identify and investigate proteins involved in the formation of peroxisomes. We demonstrate that the elongation of peroxisomes serves as an excellent indicator for efficient knock down of dynamin-like protein 1 (DLP1) in mammalian cells. We took advantage of the silencing-dependent morphological changes of peroxisomes to compare different transfection methods and show that a single transfection of DLP1 siRNA by electroporation is sufficient to effectively silence DLP1. We present a fast, easy, and convenient protocol for efficient gene silencing in a large number of cells, which can be used for quantitative and biochemical studies.
Abstract.
Author URL.
2004
Schrader M, Fahimi HD (2004). Mammalian peroxisomes and reactive oxygen species.
Histochem Cell Biol,
122(4), 383-393.
Abstract:
Mammalian peroxisomes and reactive oxygen species.
The central role of peroxisomes in the generation and scavenging of hydrogen peroxide has been well known ever since their discovery almost four decades ago. Recent studies have revealed their involvement in metabolism of oxygen free radicals and nitric oxide that have important functions in intra- and intercellular signaling. The analysis of the role of mammalian peroxisomes in a variety of physiological and pathological processes involving reactive oxygen species (ROS) is the subject of this review. The general characteristics of peroxisomes and their enzymes involved in the metabolism of ROS are briefly reviewed. An expansion of the peroxisomal compartment with proliferation of tubular peroxisomes is observed in cells exposed to UV irradiation and various oxidants and is apparently accompanied by upregulation of PEX genes. Significant reduction of peroxisomes and their enzymes is observed in inflammatory processes including infections, ischemia-reperfusion injury, and allograft rejection and seems to be related to the suppressive effect of tumor necrosis factor-alpha on peroxisome function and peroxisome proliferator activated receptor-alpha. Xenobiotic-induced proliferation of peroxisomes in rodents is accompanied by the formation of hepatic tumors, and evidently the imbalance in generation and decomposition of ROS plays an important role in this process. In PEX5-/- knockout mice lacking functional peroxisomes severe alterations of mitochondria in various organs are observed which seem to be due to a generalized increase in oxidative stress confirming the important role of peroxisomes in homeostasis of ROS and the implications of its disturbances for cell pathology.
Abstract.
Author URL.
Schrader M (2004). Membrane targeting in secretion.
Subcell Biochem,
37, 391-421.
Abstract:
Membrane targeting in secretion.
Regulated secretion and exocytosis require the selective packaging of regulated secretory proteins in secretory storage organelles and the controlled docking and fusion of these organelles with the plasma membrane. Secretory granule biogenesis involves sorting of secretory proteins and membrane components both at the level of the trans-Golgi network and the immature secretory granule. Sorting is thought to be mediated by selective protein aggregation and the interaction of these proteins with specific membrane domains. There is now considerable interest in the understanding of the complex lipid-protein and protein-protein interactions at the trans-Golgi network and the granule membrane. A role for lipid microdomains and associated sorting receptors in membrane targeting and granule formation is vividly discussed for (neuro)endocrine cells. In exocrine cells, however, little has been known of granule membrane composition and membrane protein function. With the cloning and characterization of granule membrane proteins and their interactions at the inner leaflet of zymogen granules of pancreatic acinar cells, it is now possible to elucidate their function in membrane targeting and sorting of zymogens at the molecular level.
Abstract.
Author URL.
Koch A, Schneider G, Lüers GH, Schrader M (2004). Peroxisome elongation and constriction but not fission can occur independently of dynamin-like protein 1.
J Cell Sci,
117(Pt 17), 3995-4006.
Abstract:
Peroxisome elongation and constriction but not fission can occur independently of dynamin-like protein 1.
The mammalian dynamin-like protein DLP1 belongs to the dynamin family of large GTPases, which have been implicated in tubulation and fission events of cellular membranes. We have previously shown that the expression of a dominant-negative DLP1 mutant deficient in GTP hydrolysis (K38A) inhibited peroxisomal division in mammalian cells. In this study, we conducted RNA interference experiments to 'knock down' the expression of DLP1 in COS-7 cells stably expressing a GFP construct bearing the C-terminal peroxisomal targeting signal 1. The peroxisomes in DLP1-silenced cells were highly elongated with a segmented morphology. Ultrastructural and quantitative studies confirmed that the tubular peroxisomes induced by DLP1-silencing retained the ability to constrict their membranes but were not able to divide into spherical organelles. Co-transfection of DLP1 siRNA with Pex11pbeta, a peroxisomal membrane protein involved in peroxisome proliferation, induced further elongation and network formation of the peroxisomal compartment. Time-lapse microscopy of living cells silenced for DLP1 revealed that the elongated peroxisomes moved in a microtubule-dependent manner and emanated tubular projections. DLP1-silencing in COS-7 cells also resulted in a pronounced elongation of mitochondria, and in more dispersed, elongated Golgi structures, whereas morphological changes of the rER, lysosomes and the cytoskeleton were not detected. These observations clearly demonstrate that DLP1 acts on multiple membranous organelles. They further indicate that peroxisomal elongation, constriction and fission require distinct sets of proteins, and that the dynamin-like protein DLP1 functions primarily in the latter process.
Abstract.
Author URL.
Kovacs WJ, Schrader M, Walter I, Stangl H (2004). The hypolipidemic compound cetaben induces changes in Golgi morphology and vesicle movement.
Histochemistry and Cell Biology,
122(2), 95-109.
Abstract:
The hypolipidemic compound cetaben induces changes in Golgi morphology and vesicle movement
The human hepatoma cell line HepG2 was used to study the effect of cetaben, a non-fibrate hypolipidemic drug, on cell morphology and vesicle distribution. Cetaben treatment correlated with a fragmentation and/or condensation of Golgi cisternae and the appearance of large electron-lucent vesicles. The Golgi apparatus, demonstrated, for example, by fluorescence-lectin histochemistry, was fragmented after cetaben treatment. The lectin-positive remnants were dispersed throughout the cytoplasm, but with a preference for being transported to tips of cells. However, microtubules and the intermediate filaments as well as the actin microfilaments were unchanged after cetaben treatment indicating that changes in Golgi morphology are not caused by alterations in the cytoskeleton. Cetaben decreases the cholesterol content due to inhibition of cholesterol biosynthesis. Changes in the intracellular cholesterol content are known to influence the intracellular vesicle distribution and are most likely responsible for cetaben-induced Golgi alterations, as depletion of cellular cholesterol by starvation or lovastatin and/or cyclodextrin treatment resulted in a similar redistribution of Golgi-derived wheat germ agglutinin vesicles. These lectin-stained vesicles colocalized with lysosomal marker proteins such as Limp-1 and Lamp-2, but not with the early endosomal markers Rab5 and EEA1. Upon removal of cetaben the lectin- and Limp-1/ Lamp-2-costained vesicles dissociated and were transported back to the perinuclear region. Thus, cetaben-induced changes such as fragmentation of the Golgi apparatus and the dispersion of lysosomes away from their juxtanuclear location were reversible. © Springer-Verlag 2004.
Abstract.
2003
Koch A, Thiemann M, Grabenbauer M, Yoon Y, McNiven MA, Schrader M (2003). Dynamin-like protein 1 is involved in peroxisomal fission.
JOURNAL OF BIOLOGICAL CHEMISTRY,
278(10), 8597-8605.
Author URL.
Schrader M, Thiemann M, Fahimi HD (2003). Peroxisomal motility and interaction with microtubules.
Microsc Res Tech,
61(2), 171-178.
Abstract:
Peroxisomal motility and interaction with microtubules.
Recent in vivo observations have revealed that peroxisomes are more dynamic and interactive than previously assumed. The growing recognition of the tubular and reticular morphology of peroxisomes in living cells, their association with microtubules, and the dynamic movements of peroxisomes in vivo and in vitro have inspired the query into the investigation of the cellular machinery that mediates such a complex behaviour. The characterisation of the underlying molecular components of this machinery is providing insight into the mechanisms regulating peroxisomal morphology and intracellular distribution.
Abstract.
Author URL.
2002
KALUS I, HODEL A, Annett K, KLEENE R, EDWARDSON JM, SCHRADER M (2002). Interaction of syncollin with GP-2, the major membrane protein of pancreatic zymogen granules, and association with lipid microdomains. Biochemical Journal, 362(2), 433-442.
Kalus I, Hodel A, Koch A, Kleene R, Edwardson JM, Schrader M (2002). Interaction of syncollin with GP-2, the major membrane protein of pancreatic zymogen granules, and association with lipid microdomains.
Biochem J,
362(Pt 2), 433-442.
Abstract:
Interaction of syncollin with GP-2, the major membrane protein of pancreatic zymogen granules, and association with lipid microdomains.
Syncollin, a novel pancreatic zymogen granule protein, is present on the luminal side of the granule membrane. To address the function of syncollin, we searched for putative binding partners. Cross-linking experiments with purified syncollin, and granule content and membrane proteins revealed a direct interaction between syncollin and GP-2, a major glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein. An interaction was also observed when cross-linking was performed with recombinant GP-2. In addition, syncollin could be cross-linked to itself, supporting the suggestion that it exists as a homo-oligomer. Cleavage of the GPI anchor of GP-2 by treatment of granule membranes with phosphatidylinositol-specific phospholipase C had no effect on the membrane attachment of syncollin, indicating that it is not mediated exclusively via an interaction with GP-2. Syncollin was found to be associated with detergent-insoluble cholesterol/glycolipid-enriched complexes. These complexes floated to the lighter fractions of sucrose-density gradients and also contained GP-2, the lectin ZG16p, sulphated matrix proteoglycans and the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) syntaxin 3 and synaptobrevin 2. Our results indicate that membrane-associated syncollin is a component of lipid rafts, where it interacts both with GP-2 and membrane lipids. We suggest that the syncollin-GP-2 complex might play a role in signal transduction across the granule membrane.
Abstract.
Author URL.
Schmidt K, Agricola B, Kleene R, Schrader M (2002). Submembranous proteoglycans are required for granule formation and regulated secretion in the acinar cells of the exocrine pancreas.
Author URL.
2001
HODEL A, AN SJ, HANSEN NJ, LAWRENCE J, WÄSLE B, SCHRADER M, EDWARDSON JM (2001). Cholesterol-dependent interaction of syncollin with the membrane of the pancreatic zymogen granule. Biochemical Journal, 356(3), 843-850.
Hodel A, an SJ, Hansen NJ, Lawrence J, Wäsle B, Schrader M, Edwardson JM (2001). Cholesterol-dependent interaction of syncollin with the membrane of the pancreatic zymogen granule.
Biochem J,
356(Pt 3), 843-850.
Abstract:
Cholesterol-dependent interaction of syncollin with the membrane of the pancreatic zymogen granule.
Syncollin is a protein of the pancreatic zymogen granule that was isolated through its ability to bind to syntaxin. Despite this in vitro interaction, it is now clear that syncollin is present on the luminal side of the zymogen granule membrane. Here we show that there are two pools of syncollin within the zymogen granule: one free in the lumen and the other tightly associated with the granule membrane. When unheated or cross-linked samples of membrane-derived syncollin are analysed by SDS/PAGE, higher-order forms are seen in addition to the monomer, which has an apparent molecular mass of 16 kDa. Extraction of cholesterol from the granule membrane by treatment with methyl-beta-cyclodextrin causes the detachment of syncollin, and this effect is enhanced at a high salt concentration. Purified syncollin is able to bind to brain liposomes at pH 5.0, but not at pH 11.0, a condition that also causes its extraction from granule membranes. Syncollin binds only poorly to dioleoyl phosphatidylcholine liposomes, but binding is dramatically enhanced by the inclusion of cholesterol. Finally, cholesterol can be co-immunoprecipitated with syncollin. We conclude that syncollin is able to interact directly with membrane lipids, and to insert into the granule membrane in a cholesterol-dependent manner. Membrane-associated syncollin apparently exists as a homo-oligomer, possibly consisting of six subunits, and its association with the membrane may be stabilized by electrostatic interactions with either other proteins or phospholipids.
Abstract.
Author URL.
Schmidt K, Schrader M, Kern HF, Kleene R (2001). Regulated apical secretion of zymogens in rat pancreas. Involvement of the glycosylphosphatidylinositol-anchored glycoprotein GP-2, the lectin ZG16p, and cholesterol-glycosphingolipid-enriched microdomains.
J Biol Chem,
276(17), 14315-14323.
Abstract:
Regulated apical secretion of zymogens in rat pancreas. Involvement of the glycosylphosphatidylinositol-anchored glycoprotein GP-2, the lectin ZG16p, and cholesterol-glycosphingolipid-enriched microdomains.
We examined the role of glycosphingolipid- and cholesterol-enriched microdomains, or rafts, in the sorting of digestive enzymes into zymogen granules destined for apical secretion and in granule formation. Isolated membranes of zymogen granules from pancreatic acinar cells showed an enrichment in cholesterol and sphingomyelin and formed detergent-insoluble glycolipid-enriched complexes. These complexes floated to the lighter fractions of sucrose density gradients and contained the glycosylphosphatidylinositol (GPI)-anchored glycoprotein GP-2, the lectin ZG16p, and sulfated matrix proteoglycans. Morphological and pulse-chase studies with isolated pancreatic lobules revealed that after inhibition of GPI-anchor biosynthesis by mannosamine or the fungal metabolite YW 3548, granule formation was impaired leading to an accumulation of newly synthesized proteins in the Golgi apparatus and the rough endoplasmic reticulum. Furthermore, the membrane attachment of matrix proteoglycans was diminished. After cholesterol depletion or inhibition of glycosphingolipid synthesis by fumonisin B1, the formation of zymogen granules as well as the formation of detergent-insoluble complexes was reduced. In addition, cholesterol depletion led to constitutive secretion of newly synthesized proteins, e.g. amylase, indicating that zymogens were missorted. Together, these data provide first evidence that in polarized acinar cells of the exocrine pancreas GPI-anchored proteins, e.g. GP-2, and cholesterol-sphingolipid-enriched microdomains are required for granule formation as well as for regulated secretion of zymogens and may function as sorting platforms for secretory proteins destined for apical delivery.
Abstract.
Author URL.
Schrader M (2001). Tubulo-reticular clusters of peroxisomes in living COS-7 cells: dynamic behavior and association with lipid droplets.
J Histochem Cytochem,
49(11), 1421-1429.
Abstract:
Tubulo-reticular clusters of peroxisomes in living COS-7 cells: dynamic behavior and association with lipid droplets.
We characterized more complex peroxisomal structures, i.e. tubulo-reticular peroxisomal clusters, in greater detail under in vivo conditions in COS-7 cells that were transfected with a GFP-PTS1 fusion protein. Live cell imaging revealed the dynamic nature of peroxisomal clusters and allowed a detailed analysis of the motile properties of a heterogeneous peroxisome population. Furthermore, peroxisomal clusters were found to be associated with lipid droplets. The frequency of peroxisomal clusters correlated with an increase in cell density and in the size of lipid droplets. These data provide further evidence for the dynamic nature of the peroxisomal compartment and indicate that peroxisomal clusters have a function in lipid metabolism.
Abstract.
Author URL.
2000
Schrader M, King SJ, Stroh TA, Schroer TA (2000). Evidence for peroxisomal reticuli in COS-7 cells: Association with lipid droplets.
MOLECULAR BIOLOGY OF THE CELL,
11, 425A-425A.
Author URL.
Hillebrand M, Schmidt K, Kern HF, Kleene R, Schrader M (2000). Inhibitors of glycosylation, GPI-anchor- and cholesterol biosynthesis interfere with granule formation and regulated secretion in the acinar cells of the exocrine rat pancreas.
MOLECULAR BIOLOGY OF THE CELL,
11, 499A-499A.
Author URL.
Thiemann M, Schrader M, Völkl A, Baumgart E, Fahimi HD (2000). Interaction of peroxisomes with microtubules. In vitro studies using a novel peroxisome-microtubule binding assay.
Eur J Biochem,
267(20), 6264-6275.
Abstract:
Interaction of peroxisomes with microtubules. In vitro studies using a novel peroxisome-microtubule binding assay.
The association of membrane-bounded cell organelles to microtubules is crucial for determination of their shape, intracellular localization and translocation. We have shown previously the high affinity binding of peroxisomes to microtubules which appears to be of static nature as in vivo studies indicate that only a few peroxisomes move along the microtubular tracks. In order to characterize the interactions of peroxisomes with microtubules, we have developed a semiquantitative in vitro binding assay, which is based on the association of highly purified rat liver peroxisomes to microtubules coated onto microtiterplates. The binding was visualized by differential interference contrast and immunofluorescence using a confocal laser scanning microscope. The binding was concentration dependent and saturable, being affected by time, temperature, and pH. Addition of ATP or the motor proteins kinesin and dynein increased the binding capacity, while ATP-depletion or microtubule associated proteins (MAPs) decreased it. KCl treatment of peroxisomes reduced the binding, which was restored by dialyzed KCl-stripping eluate as well as by rat liver cytosol. The reconstituting effect of cytosol was abolished by its pretreatment with proteases or N-ethylmaleimide. Moreover, the treatment of peroxisomes with proteases or N-ethylmaleimide reduced their binding, which was not reversed by cytosol. These results suggest the involvement of a peroxisomal membrane protein and cytosolic factor(s) in the binding of peroxisomes to microtubules. This notion is supported by the observation that distinct subfractions of dialyzed KCl-stripping eluate obtained by gel chromatography augmented the binding. Those subfractions, as well as purified peroxisome fractions, exhibited strong immunoreactivity with an antibody to cytoplasmic linker protein (CLIP)-115, revealing a 70-kDa polypeptide. Moreover, immunodepletion of KCl-stripping eluate and its subfractions with an antibody to the conserved microtubule binding domain of CLIPs, abolished their promoting effect on the binding, thus suggesting the involvement of a CLIP-related protein in the binding of peroxisomes to microtubules.
Abstract.
Author URL.
Schrader M, King SJ, Stroh TA, Schroer TA (2000). Real time imaging reveals a peroxisomal reticulum in living cells.
J Cell Sci,
113 ( Pt 20), 3663-3671.
Abstract:
Real time imaging reveals a peroxisomal reticulum in living cells.
We have directly imaged the dynamic behavior of a variety of morphologically different peroxisomal structures in HepG2 and COS-7 cells transfected with a construct encoding GFP bearing the C-terminal peroxisomal targeting signal 1. Real time imaging revealed that moving peroxisomes interacted with each other and were engaged in transient contacts, and at higher magnification, tubular peroxisomes appeared to form a peroxisomal reticulum. Local remodeling of these structures could be observed involving the formation and detachment of tubular processes that interconnected adjacent organelles. Inhibition of cytoplasmic dynein based motility by overexpression of the dynactin subunit, dynamitin (p50), inhibited the movement of peroxisomes in vivo and interfered with the reestablishment of a uniform distribution of peroxisomes after recovery from nocodazole treatment. Isolated peroxisomes moved in vitro along microtubules in the presence of a microtubule motor fraction. Our data reveal that peroxisomal behavior in vivo is significantly more dynamic and interactive than previously thought and suggest a role for the dynein/dynactin motor in peroxisome motility.
Abstract.
Author URL.
Kleene R, Classen B, Zdzieblo J, Schrader M (2000). SH3 binding sites of ZG29p mediate an interaction with amylase and are involved in condensation-sorting in the exocrine rat pancreas.
Biochemistry,
39(32), 9893-9900.
Abstract:
SH3 binding sites of ZG29p mediate an interaction with amylase and are involved in condensation-sorting in the exocrine rat pancreas.
ZG29p, a novel pancreas-specific zymogen granule protein, has been proposed to act as a 'helper protein' in granule formation. To address its function in more detail, we searched for putative binding partners of ZG29p. In zymogen complexes isolated by nondenaturing isoelectric focusing, ZG29p was associated with a protein complex consisting of amylase and cationic trysinogen. Amylase also coeluted with ZG29p after immunoaffinity chromatography using an antibody to recombinant ZG29p. Cross-linking experiments with granule content proteins revealed a direct interaction between recombinant ZG29p and amylase. An interaction was also observed when purified amylase was used, whereas no interaction with recombinant or purified cationic trypsinogen was seen. ZG29p could also be cross-linked to three membrane proteins with molecular masses of 40, 18, and 16 kDa. The binding of ZG29p to amylase and to the membrane proteins was inhibited in the presence of synthetic peptides matching the consensus sequence of proline-rich SH3 binding sites present in ZG29p. The synthetic peptides could be cross-linked to amylase and to three yet unidentified acidic content proteins with molecular masses of about 30 kDa. The peptides also interacted with purified or recombinant amylase, but not with recombinant or purified cationic trypsinogen. In a condensation-sorting assay, the binding (sorting) of zymogen complexes to the granule membrane was reduced in the presence of the peptides. Our results indicate that the interaction of ZG29p with amylase is mediated by SH3 binding domains and that these domains are involved in the sorting of amylase to the granule membrane.
Abstract.
Author URL.
1999
Schrader M, Wodopia R, Fahimi HD (1999). Induction of tubular peroxisomes by UV irradiation and reactive oxygen species in HepG2 cells.
J Histochem Cytochem,
47(9), 1141-1148.
Abstract:
Induction of tubular peroxisomes by UV irradiation and reactive oxygen species in HepG2 cells.
Peroxisomes in the human hepatoblastoma cell line HepG2 exhibit a high degree of plasticity. Whereas in confluent cultures they appear as small (0.1-0.3 micrometer) spherical particles, they undergo dramatic changes, forming elongated tubules measuring up to 5 micrometer on separation of cells and cultivation at low density. We recently showed that several growth factors, including nerve growth factor (NGF), induce the formation of tubular peroxisomes and that this induction is sensitive to K 252b, a specific tyrosine kinase inhibitor, suggesting the involvement of this signal transduction pathway. Because tyrosine kinase is also involved in signal transduction via the reactive oxygen species (ROS), we have analyzed in this study the effects of UV irradiation, H(2)O(2), and oxygen on tubulation of peroxisomes. UVC irradiation induced a significant increase in formation of tubular peroxisomes (40-50% of cells) and this effect was dose-dependently inhibited by pretreatment with N-acetyl cysteine, confirming the involvement of ROS in the UV effect. Furthermore, H(2)O(2) also directly induced the tubulation of peroxisomes, although to a lesser extent. Finally, cultivation under hypoxic conditions (1.5% O(2)) drastically reduced the inducing effect of fetal calf serum on tubulation of peroxisomes, suggesting the involvement of oxygen-mediated signaling. Taken together, our observations indicate that ROS induce the tubulation of peroxisomes in HepG2 cells. Because peroxisomes harbor most of the enzymes for catabolism of ROS, the tubulation and expansion of the peroxisome compartment could have a cell rescue function against the destructive effects of ROS.
Abstract.
Author URL.
Valetti C, Wetzel DM, Schrader M, Hasbani MJ, Gill SR, Kreis TE, Schroer TA (1999). Role of dynactin in endocytic traffic: Effects of dynamitin overexpression and colocalization with CLIP-170.
Molecular Biology of the Cell,
10(12), 4107-4120.
Abstract:
Role of dynactin in endocytic traffic: Effects of dynamitin overexpression and colocalization with CLIP-170
The flow of material from peripheral, early endosomes to late endosomes requires microtubules and is thought to be facilitated by the minus end- directed motor cytoplasmic dynein and its activator dynactin. The microtubule-binding protein CLIP-170 may also play a role by providing an early link to endosomes. Here, we show that perturbation of dynactin function in vivo affects endosome dynamics and trafficking. Endosome movement, which is normally bidirectional, is completely inhibited. Receptor-mediated uptake and recycling occur normally, but cells are less susceptible to infection by enveloped viruses that require delivery to late endosomes, and they show reduced accumulation of lysosomally targeted probes. Dynactin colocalizes at microtubule plus ends with CLIP-170 in a way that depends on CLIP-170's putative cargo-binding domain. Overexpression studies using p150(Glued), the microtubule-binding subunit of dynactin, and mutant and wild-type forms of CLIP-170 indicate that CLIP-170 recruits dynactin to microtubule ends. These data suggest a new model for the formation of motile complexes of endosomes and microtubules early in the endocytic pathway.
Abstract.
1998
Ihrke G, Martin GV, Shanks MR, Schrader M, Schroer TA, Hubbard AL (1998). Apical plasma membrane proteins and endolyn-78 travel through a subapical compartment in polarized WIF-B hepatocytes.
J Cell Biol,
141(1), 115-133.
Abstract:
Apical plasma membrane proteins and endolyn-78 travel through a subapical compartment in polarized WIF-B hepatocytes.
We studied basolateral-to-apical transcytosis of three classes of apical plasma membrane (PM) proteins in polarized hepatic WIF-B cells and then compared it to the endocytic trafficking of basolaterally recycling membrane proteins. We used antibodies to label the basolateral cohort of proteins at the surface of living cells and then followed their trafficking at 37 degreesC by indirect immunofluorescence. The apical PM proteins aminopeptidase N, 5'nucleotidase, and the polymeric IgA receptor were efficiently transcytosed. Delivery to the apical PM was confirmed by microinjection of secondary antibodies into the bile canalicular-like space and by EM studies. Before acquiring their apical steady-state distribution, the trafficked antibodies accumulated in a subapical compartment, which had a unique tubulovesicular appearance by EM. In contrast, antibodies to the receptors for asialoglycoproteins and mannose-6-phosphate or to the lysosomal membrane protein, lgp120, distributed to endosomes or lysosomes, respectively, without accumulating in the subapical area. However, the route taken by the endosomal/lysosomal protein endolyn-78 partially resembled the transcytotic pathway, since anti-endolyn-78 antibodies were found in a subapical compartment before delivery to lysosomes. Our results suggest that in WIF-B cells, transcytotic molecules pass through a subapical compartment that functions as a second sorting site for a subset of basolaterally endocytosed membrane proteins reaching this compartment.
Abstract.
Author URL.
Fahimi HD, Grabenbauer M, Schrader M, Stier H, Baumgart E (1998). Biogenesis and maturation of peroxisomes in mammalian cells.
Author URL.
Schrader M, Reuber BE, Morrell JC, Jimenez-Sanchez G, Obie C, Stroh TA, Valle D, Schroer TA, Gould SJ (1998). Expression of PEX11β Mediates Peroxisome Proliferation in the Absence of Extracellular Stimuli. Journal of Biological Chemistry, 273(45), 29607-29614.
Schrader M, Krieglstein K, Fahimi HD (1998). Tubular peroxisomes in HepG2 cells: selective induction by growth factors and arachidonic acid.
Eur J Cell Biol,
75(2), 87-96.
Abstract:
Tubular peroxisomes in HepG2 cells: selective induction by growth factors and arachidonic acid.
We showed recently the plasticity of the peroxisomal compartment in the human hepatoblastoma cell line HepG2 as evidenced by the presence of elongated tubular peroxisomes measuring up to 5 microm next to much smaller spherical or rod-shaped ones (0.1-0.3 microm). Since the occurrence of tubular peroxisomes in a given cell in culture is synchronized, with neighboring cells containing either small spherical or elongated tubular peroxisomes, cell counting of immunofluorescence preparations stained for catalase was used for the quantitative assessment of the dynamics of the peroxisomal compartment and the factors regulating this process. Initial studies revealed that the formation of tubular peroxisomes is primarily influenced by the cell density as well as by lipid- and protein-factors in fetal calf serum, being independent of an intact microtubular network. Biochemical studies showed that the occurrence of tubular peroxisomes correlated with the expression of the mRNA for 70 kDa peroxisomal membrane protein (PMP70), but not with that of matrix proteins. By cultivation of cells in serum- and protein-free media specific factors were identified which influenced the formation of tubular peroxisomes. Among several growth factors tested, nerve growth factor (NGF) was the most potent one inducing tubular peroxisomes and its effect was blocked by K252b, a specific inhibitor of neurotrophin receptor pathway, suggesting the involvement of signal transduction in this process. Furthermore, from several polyunsaturated fatty acids (PUFA) which all induced tubular peroxisomes, the arachidonic acid (AA) was the most potent one. Our observations suggest that tubular peroxisomes are transient structures in the process of rapid expansion of the peroxisomal compartment which are induced either by specific growth factors or by polyunsaturated fatty acids both of which are involved in intracellular signaling.
Abstract.
Author URL.
1997
Kinscherf R, Kamencic H, Deigner HP, Pill J, Schmiedt W, Schrader M, Metz J (1997). Effect of alterations of blood cholesterol levels on macrophages in the myocardium of New Zealand White rabbits.
J Leukoc Biol,
62(6), 719-725.
Abstract:
Effect of alterations of blood cholesterol levels on macrophages in the myocardium of New Zealand White rabbits.
We investigated the effect of alterations of blood cholesterol levels on macrophages (mphi) in the myocardium of New Zealand White (NZW) rabbits. Three groups of NZW rabbits were used: controls, rabbits fed a 0.5% cholesterol-enriched diet (CH-D) for 96 days, and rabbits fed a 0.5% CH-D for 96 days followed by normal chow for 4 months. Immunohistochemical analysis by mAbs directed against mphi (RAM-11) and Mn superoxide dismutase (MnSOD) were quantified by computer-assisted morphometry. Using cultured human and rabbit mphi, a cross-reaction of the human MnSOD mAbs was found as well as the predominant localization of MnSOD-immunoreactivity (IR) in mitochondria. In group 1, only a very few RAM-11-immunoreactive (ir) mphi occurred in the interstitial space of the myocardium. In group II blood cholesterol levels significantly increased in parallel with the numbers of mphi, which often contained lipid droplets (foam cells). Although blood cholesterol concentrations regressed about 10-fold in group III, mphi in the myocardium were found to be reduced only about 20%. Most mphi were also MnSOD-ir. In atherosclerotic coronary arteries RAM-11-IR was located in mphi and also extracellularly, whereas MnSOD-IR was found only in mphi. Drastically induced MnSOD in the mitochondria of mphi is suggested as an indicator of increased oxidative stress caused by in vitro conditions or by phagocytosis of low-density lipoprotein in vivo. Elevation of the cholesterol level leads to a long-term increase and its regression results in a delayed reduction of such mphi, which seem to play a key role in the atherogenesis of the coronary arteries as well.
Abstract.
Author URL.
Kinscherf R, Deigner HP, Usinger C, Pill J, Wagner M, Kamencic H, Hou D, Chen M, Schmiedt W, Schrader M, et al (1997). Induction of mitochondrial manganese superoxide dismutase in macrophages by oxidized LDL: its relevance in atherosclerosis of humans and heritable hyperlipidemic rabbits.
FASEB J,
11(14), 1317-1328.
Abstract:
Induction of mitochondrial manganese superoxide dismutase in macrophages by oxidized LDL: its relevance in atherosclerosis of humans and heritable hyperlipidemic rabbits.
The objective of the study was to analyze the intracellular antioxidative response of macrophages (Mphi) exposed to increased levels of low density lipoprotein (LDL). We studied manganese superoxide dismutase (MnSOD) and, in part, GSH in cultured human and rabbit Mphi, and in atheromatous arterial tissue of humans and heritable hyperlipidemic (HHL) rabbits. Incubation of human Mphi with oxidized-LDL (ox-LDL) resulted in an induction of MnSOD mRNA production as shown by RT-PCR. MnSOD immunoreactivity (IR) was found to be located in the mitochondria of Mphi. In HHL rabbits, MnSOD activity and GSH concentration were significantly increased in atherosclerotic intima compared to the media of the aorta, but significantly decreased (P
Abstract.
Author URL.
1996
Schrader M, Burkhardt JK, Baumgart E, Lüers G, Spring H, Völkl A, Fahimi HD (1996). Interaction of microtubules with peroxisomes. Tubular and spherical peroxisomes in HepG2 cells and their alterations induced by microtubule-active drugs.
Eur J Cell Biol,
69(1), 24-35.
Abstract:
Interaction of microtubules with peroxisomes. Tubular and spherical peroxisomes in HepG2 cells and their alterations induced by microtubule-active drugs.
We have studied the interaction of microtubules with peroxisomes and the influence of changes in the microtubular network on the peroxisomal compartment. From the several cell lines analyzed for this purpose, HepG2 cells proved to be the best candidate exhibiting both a well-developed cytoskeleton and a peroxisomal compartment with great plasticity. Three distinct types of peroxisomes: small spherical (0.1-0.3 micron), rod-shaped (0.5 micron) and elongated tubular (up to 5 microns) ones were identified in this cell line. A shift of the elongated tubular forms to spherical particles was noted by increasing the density of cells in culture, whereas no correlation between the distinct peroxisomal forms and the cellular proliferation could be observed. At time points when the elongated tubular peroxisomes were disappearing, many spherical peroxisomes arranged like 'chains of beads on a string' were observed, suggesting that the fission of elongated tubular forms may give rise to newly developing spherical peroxisomes. A clear association of spherical peroxisomes with microtubules was visualized by double immunofluorescence in combination with confocal laser scanning microscopy (CLSM). Treatment with a variety of microtubule-depolymerizing drugs (colcemid, nocodazole, vinblastine) induced a significant increase in the frequency of tubular peroxisomes and led to the formation of peroxisomal clusters. These effects were reversible since already 1 to 2 h after removal of the drugs from the culture medium, a uniform distribution of spherical peroxisomes was reestablished. Taxol, a microtubule-stabilizing drug, on the other hand exerted no significant effects on the peroxisomal compartment. The direct interaction of microtubules with peroxisomes in vitro was demonstrated using highly purified rat liver peroxisomes and taxol-stabilized microtubules from bovine or pig brain. The binding of peroxisomes to microtubules was visualized by video-enhanced contrast microscopy (VECM) and was abolished by pretreatment of peroxisomes with 100 mM KCl ('stripping'), proteinase K or trypsin. Incubation with cytosol restored the binding capacity of KCl-treated peroxisomes, but did not complement the protease treatment. The data presented provide for the first time evidence for a direct interaction of microtubules with the peroxisomal compartment indicating that this cytoskeletal system plays an important role in the morphogenesis and intracellular distribution of peroxisomes.
Abstract.
Author URL.
Schrader M, Burkhardt JK, Baumgart E, Lüers G, Völkl A, Fahimi HD (1996). The importance of microtubules in determination of shape and intracellular distribution of peroxisomes.
Ann N Y Acad Sci,
804, 669-671.
Author URL.
Schrader M, Fahimi HD (1996). Tubular peroxisomes in HepG2 cells. The role of nutritional factors in their formation.
MOLECULAR BIOLOGY OF THE CELL,
7, 2879-2879.
Author URL.
1995
Schrader M, Baumgart E, Fahimi HD (1995). Effects of fixation on the preservation of peroxisomal structures for immunofluorescence studies using HepG2 cells as a model system.
Histochem J,
27(8), 615-619.
Abstract:
Effects of fixation on the preservation of peroxisomal structures for immunofluorescence studies using HepG2 cells as a model system.
The immunofluorescence technique has become an important tool for the investigation of peroxisomes in cell culture. We have used this method for the study of peroxisomes in the human hepatoblastoma cell line HepG2. A marked heterogeneity of peroxisomal forms was detected. Besides spherical (about 100 nm) and rod-shaped structures (about 300 nm) many elongated, undulating tubular forms (up to 5 microns) were found. Further observations indicate that the appearance of the peroxisomal forms in immunofluorescence is dependent on the fixation procedure used. Whereas the fixation with methanol-acetone (-20 degrees C) or ethanol results in a punctate pattern with spherical particles, the use of formaldehyde/Triton X-100 fixation shows well-preserved tubules and rods. These observations may be of special importance for studies on the biogenesis of peroxisomes.
Abstract.
Author URL.
1994
Schrader M, Temm-Grove CJ, Lessard JL, Jockusch BM (1994). Chicken antibodies to rabbit muscle actin with a restricted repertoire of F-actin recognition.
Eur J Cell Biol,
63(2), 326-335.
Abstract:
Chicken antibodies to rabbit muscle actin with a restricted repertoire of F-actin recognition.
This report describes a polyclonal antibody against actin with unexpected and unusual properties. The antibody was raised in chicken immunized with a complex of DNase I and rabbit skeletal muscle actin, and purified from egg yolk by affinity chromatography. In Western blots, it reacted with alpha, beta and gamma isoforms of actin. In immunofluorescence and dot blot assays, however, it recognized selectively actin filaments in myofibrils, microvilli of brush border-type epithelium and the "comet tails" of the intracellular parasite Listeria monocytogenes, while it did not react with stress fibers and peripheral belts of fibroblasts and epithelial cells, respectively. This reactivity pattern is reminiscent of that previously described for a monoclonal mouse antibody raised against smooth muscle actin (Sawtell et al. Cell Motil. Cytoskel. 11, 318, 1988). The data presented in this study are consistent with the hypothesis that the chicken antibody recognizes an actin epitope/actin epitopes either accessible in only a subpopulation of microfilaments, or expressed only in a particular conformation of F-actin.
Abstract.
Author URL.
Schrader M, Baumgart E, Völkl A, Fahimi HD (1994). Heterogeneity of peroxisomes in human hepatoblastoma cell line HepG2. Evidence of distinct subpopulations.
Eur J Cell Biol,
64(2), 281-294.
Abstract:
Heterogeneity of peroxisomes in human hepatoblastoma cell line HepG2. Evidence of distinct subpopulations.
Peroxisomes in human hepatoblastoma cell line HepG2 have been studied using immunocytochemical, ultrastructural and biochemical techniques. By immunofluorescence, small spherical peroxisomes were seen next to rod-shaped and elongated forms. By electron microscopy and catalase cytochemistry, small particles with a diameter of 90 to 100 nm were found next to larger ones measuring up to 300 nm and some exhibiting tail-like extensions. Both the intensity of DAB-staining and the immunolabeling density for catalase were heterogenous in different peroxisomes. Contrary to a recent biochemical study, the enzyme alanine-glyoxylate-aminotransferase was found by double immunofluorescence and immunoelectron microscopy to be localized exclusively in peroxisomes of HepG2 cells. By Metrizamide density gradient centrifugation two populations of peroxisomes were isolated: a regular fraction banding at 1.20 g/cm3 with a mean diameter of 222 nm and a lighter peroxisome fraction banding at 1.14 g/cm3. The ratio of lipid beta-oxidation enzymes to catalase was significantly higher in peroxisomes with lower density than in the regular ones. These observations show clearly the existence of heterogenous populations of peroxisomes in HepG2 cells which may provide a useful model system for the investigation of biogenesis and metabolism of peroxisomes of human origin.
Abstract.
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