peroxisome-staining-mammalian-cells-using-peroxisome-specific

Peroxisome Staining in Mammalian Cells Using Peroxisome-Specific Probes

Peroxisomes are ubiquitous organelles with essential roles in human health, and their dysfunction leads to a spectrum of genetic disorders. Visualization ...

University of Exeter

Type-II Diabetes Mellitus (T2DM) is one of the fastest growing public health issues of modern times, consuming 12% of worldwide health budgets and affecting an estimated 400 million people. A key pathological trait associated with this disease is the failure of normal glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells. Several lines of evidence suggest that vesicle trafficking events such as insulin secretion are regulated by the post-translational modification, SUMOylation, and indeed SUMOylation has been proposed to act as a 'brake' on insulin exocytosis. Here, we show that diabetic stimuli which inhibit GSIS are correlated with an increase in cellular protein SUMOylation, and that inhibition of deSUMOylation reduces GSIS. We demonstrate that manipulation of cellular protein SUMOylation levels, by overexpression of several different components of the SUMOylation pathway, have varied and complex effects on GSIS, indicating that SUMOylation regulates this process at multiple stages. We further demonstrate that inhibition of syntaxin1A SUMOylation, via a knockdown-rescue strategy, greatly enhances GSIS. Our data are therefore consistent with the model that SUMOylation acts as a brake on GSIS, and we have identified SUMOylation of syntaxin 1 A as a potential component of this brake. However, our data also demonstrate that the role of SUMOylation in GSIS is complex and may involve many substrates.

Protein SUMOylation regulates insulin secretion at multiple stages.

Type-II Diabetes Mellitus (T2DM) is one of the fastest growing public health issues today, consuming 12% of worldwide health budgets and affecting an estimated 400 million people. One of the key pathological traits of this disease is insulin resistance at 'glucose sink' tissues (mostly skeletal muscle), and this remains one of the features of this disease most intractable to therapeutic intervention. Several lines of evidence have implicated the post-translational modification, SUMOylation, in insulin signalling and insulin resistance in skeletal muscle. In this study, we examined this possibility by manipulation of cellular SUMOylation levels using multiple different tools, and assaying the effect on insulin-stimulated GLUT4 surface expression in differentiated L6 rat myocytes. Although insulin stimulation of L6 myocytes produced a robust decrease in total cellular SUMO1-ylation levels, manipulating cellular SUMOylation had no effect on insulin-responsive GLUT4 surface trafficking using any of the tools we employed. Whilst we cannot totally exclude the possibility that SUMOylation plays a role in the insulin signalling pathway in human health and disease, our data strongly argue that GLUT4 trafficking in response to insulin is not regulated by protein SUMOylation, and that SUMOylation does not therefore represent a viable therapeutic target for the treatment of insulin resistance.

Insulin-dependent GLUT4 trafficking is not regulated by protein SUMOylation in L6 myocytes.

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.

Mitochondrial fission factor (MFF) is a critical regulator of peroxisome maturation.

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.

Maintaining social contacts: The physiological relevance of organelle interactions.

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.

A Functional SMAD2/3 Binding Site in the β Promoter Identifies a Role for TGFβ in Peroxisome Proliferation in Humans.

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.

Determinants of Peroxisome Membrane Dynamics.

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.

PEX11β and FIS1 cooperate in peroxisome division independently of mitochondrial fission factor.

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.

Fission Impossible (?)-New Insights into Disorders of Peroxisome Dynamics.

Peroxisomes are dynamic subcellular organelles in mammals, playing essential roles in cellular lipid metabolism and redox homeostasis. They perform a wide spectrum of functions in human health and disease, with new roles, mechanisms, and regulatory pathways still being discovered. Recently elucidated biological roles of peroxisomes include as antiviral defense hubs, intracellular signaling platforms, immunomodulators, and protective organelles in sensory cells. Furthermore, peroxisomes are part of a complex inter-organelle interaction network, which involves metabolic cooperation and cross talk via membrane contacts. The detection of endogenous and/or overexpressed proteins within a cell by immunolabelling informs us about the organellar and even sub-organellar localization of both known and putative peroxisomal proteins. In turn, this can be exploited to characterize the effects of experimental manipulations on the morphology, distribution, and/or number of peroxisomes in a cell, which are key properties controlling peroxisome function. Here, we present a protocol used successfully in our laboratory for the immunolabelling of peroxisomal proteins in cultured mammalian cells. We present immunofluorescence and transfection techniques as well as reagents to determine the localization of endogenous and overexpressed peroxisomal proteins.

Immunolabeling for Detection of Endogenous and Overexpressed Peroxisomal Proteins in Mammalian Cells.

Peroxisomes are multifunctional, ubiquitous, and dynamic organelles. They are responsible for diverse metabolic and physiological functions and communicate with other organelles, including the ER, mitochondria, lipid droplets, and lysosomes, through membrane contact sites. However, despite their importance for healthy cell function, remarkably, little is known about how peroxisomes and peroxisomal proteins are regulated under physiological conditions in human cells. Here, we present a method to generate reporter cell lines to measure endogenous expression of peroxisomal proteins of interest. By CRISPR-mediated knock-in of an easily detectable protein-coding tag in-frame into the relevant genomic loci, endogenous levels of the protein of interest in a cell population can be quantified in a high-throughput manner under different conditions. This has important implications for the fundamental understanding of how peroxisomal proteins are regulated and may reveal the therapeutic potential of modulating peroxisomal protein expression to improve cell performance.

Generation of Reporter Cell Lines for Endogenous Expression Analysis of Peroxisomal Proteins.

Organelles within eukaryotic cells are not isolated static compartments, instead being morphologically diverse and highly dynamic in order to respond to cellular needs and carry out their diverse and cooperative functions. One phenomenon exemplifying this plasticity, and increasingly gaining attention, is the extension and retraction of thin tubules from organelle membranes. While these protrusions have been observed in morphological studies for decades, their formation, properties and functions are only beginning to be understood. In this review, we provide an overview of what is known and still to be discovered about organelle membrane protrusions in mammalian cells, focusing on the best-characterised examples of these membrane extensions arising from peroxisomes (ubiquitous organelles involved in lipid metabolism and reactive oxygen species homeostasis) and mitochondria. We summarise the current knowledge on the diversity of peroxisomal/mitochondrial membrane extensions, as well as the molecular mechanisms by which they extend and retract, necessitating dynamic membrane remodelling, pulling forces and lipid flow. We also propose broad cellular functions for these membrane extensions in inter-organelle communication, organelle biogenesis, metabolism and protection, and finally present a mathematical model that suggests that extending protrusions is the most efficient way for an organelle to explore its surroundings.

Organelle Membrane Extensions in Mammalian Cells.

Peroxisomes are highly dynamic, oxidative organelles with key metabolic functions in cellular lipid metabolism, such as the β-oxidation of fatty acids and the synthesis of myelin sheath lipids, as well as the regulation of cellular redox balance. Loss of peroxisomal functions causes severe metabolic disorders in humans. Furthermore, peroxisomes also fulfil protective roles in pathogen and viral defence and immunity, highlighting their wider significance in human health and disease. This has sparked increasing interest in peroxisome biology and their physiological functions. This review presents an update and a continuation of three previous review articles addressing the unsolved mysteries of this remarkable organelle. We continue to highlight recent discoveries, advancements, and trends in peroxisome research, and address novel findings on the metabolic functions of peroxisomes, their biogenesis, protein import, membrane dynamics and division, as well as on peroxisome-organelle membrane contact sites and organelle cooperation. Furthermore, recent insights into peroxisome organisation through super-resolution microscopy are discussed. Finally, we address new roles for peroxisomes in immune and defence mechanisms and in human disorders, and for peroxisomal functions in different cell/tissue types, in particular their contribution to organ-specific pathologies.

The peroxisome: an update on mysteries 3.0.

Seventy years of peroxisome research: current advances and future perspectives.

Peroxisomes are essential organelles, present in all nucleated cells, with key roles in lipid and redox homeostasis. They are important for maintaining healthy cell function, with defects in peroxisome biogenesis and/or metabolism leading to disease. Notably, patients with peroxisomal diseases exhibit predominantly neurological phenotypes, and peroxisomes are observed to be altered in a range of neurodegenerative conditions, highlighting the crucial roles they play in the brain. While most studies so far have focused on the contribution of peroxisomal metabolism, it is becoming apparent that many different aspects of peroxisome biology are necessary for healthy neural function. Peroxisomes are highly dynamic, responding to cellular needs with changes in number, shape and distribution. Furthermore, they do not act in isolation but instead interact and cooperate with a range of organelles to carry out their roles. This review summarizes our current knowledge on the importance of peroxisome dynamics and inter-organelle interactions in neuronal function and dysfunction. It considers their impact on neuronal physiology, and discusses the evidence that defects in these processes are associated with neurological pathophysiology and may thus represent a novel therapeutic target for treating diseases affecting the nervous system. Finally, the review outlines the current knowledge gaps relating to the mechanisms by which peroxisome dynamics and inter-organelle interactions influence neuronal (dys)function, proposing potential new research directions to address these and further our understanding of the multi-faceted roles peroxisomes play in brain health and disease.

Peroxisome dynamics and inter-organelle interactions in neuronal health and disease.

Maintenance of mitochondrial redox homeostasis is of fundamental importance to cellular health. Mitochondria harbor a host of intrinsic antioxidant defenses, but the contribution of extrinsic, nonmitochondrial antioxidant mechanisms is less well understood. We found a direct role for peroxisomes in maintaining mitochondrial redox homeostasis through contact-mediated reactive oxygen species (ROS) transfer. We found that ACBD5 and PTPIP51 form a contact between peroxisomes and mitochondria. The percentage of these contacts increased during mitochondrial oxidative stress and helped to maintain mitochondrial health through the transfer of mitochondrial ROS to the peroxisome lumen. Our findings reveal a multiorganelle layer of mitochondrial antioxidant defense-suggesting a direct mechanism by which peroxisomes contribute to mitochondrial health-and broaden the scope of known membrane contact site functions.

Article	Year
Protein SUMOylation regulates insulin secretion at multiple stages. Scientific reports\| PubMed ID: 30814610	2019
Insulin-dependent GLUT4 trafficking is not regulated by protein SUMOylation in L6 myocytes. Scientific reports\| PubMed ID: 31019221	2019
Mitochondrial fission factor (MFF) is a critical regulator of peroxisome maturation. Biochimica et biophysica acta. Molecular cell research\| PubMed ID: 32224193	2020
Maintaining social contacts: The physiological relevance of organelle interactions. Biochimica et biophysica acta. Molecular cell research\| PubMed ID: 32712071	2020
A Functional SMAD2/3 Binding Site in the β Promoter Identifies a Role for TGFβ in Peroxisome Proliferation in Humans. Frontiers in cell and developmental biology\| PubMed ID: 33195217	2020
Determinants of Peroxisome Membrane Dynamics. Frontiers in physiology\| PubMed ID: 35185625	2022
PEX11β and FIS1 cooperate in peroxisome division independently of mitochondrial fission factor. Journal of cell science\| PubMed ID: 35678336	2022
Fission Impossible (?)-New Insights into Disorders of Peroxisome Dynamics. Cells\| PubMed ID: 35741050	2022
Immunolabeling for Detection of Endogenous and Overexpressed Peroxisomal Proteins in Mammalian Cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 36952177	2023
Generation of Reporter Cell Lines for Endogenous Expression Analysis of Peroxisomal Proteins. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 36952191	2023
Organelle Membrane Extensions in Mammalian Cells. Biology\| PubMed ID: 37237478	2023
The peroxisome: an update on mysteries 3.0. Histochemistry and cell biology\| PubMed ID: 38244103	2024
Seventy years of peroxisome research: current advances and future perspectives. Histochemistry and cell biology\| PubMed ID: 39825042	2025
Peroxisome dynamics and inter-organelle interactions in neuronal health and disease. Frontiers in molecular neuroscience\| PubMed ID: 40621503	2025
ROS transfer at peroxisome-mitochondria contact regulates mitochondrial redox. Science (New York, N.Y.)\| PubMed ID: 40638754	2025

Ruth E. Carmichael

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