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.

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.

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.

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.

First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Tina Angelika Schrader and Ruth Carmichael are co-first authors on `PEX11 beta and FIS1 cooperate in peroxisome division independently of mitochondrial fission factor', published in JCS. Tina Angelika is a senior research technician in the lab of Prof. Michael Schrader at the College of Life and Environmental Sciences, Biosciences, University of Exeter, UK, where her area of expertise is culturing and manipulating mammalian cells, as well as implementing new scientific ideas and directions. Ruth is a Postdoctoral Research Fellow in the same lab, who is interested in the fundamental physiological mechanisms and regulation of organelle dynamics (e.g. their shape/morphology, number and interorganelle interactions) in mammalian cells, and the impact of this on health and disease.

Mitochondria are unavoidably subject to organellar stress resulting from exposure to a range of reactive molecular species. Consequently, cells operate a poorly understood quality control programme of mitophagy to facilitate elimination of dysfunctional mitochondria. Here, we used a model stressor, deferiprone (DFP), to investigate the molecular basis for stress-induced mitophagy. We show that mitochondrial fission 1 protein (Fis1) is required for DFP-induced mitophagy and that Fis1 is SUMOylated at K149, an amino acid residue critical for Fis1 mitochondrial localization. We find that DFP treatment leads to the stabilization of the SUMO protease SENP3, which is mediated by downregulation of the E3 ubiquitin (Ub) ligase CHIP. SENP3 is responsible for Fis1 deSUMOylation and depletion of SENP3 abolishes DFP-induced mitophagy. Furthermore, preventing Fis1 SUMOylation by conservative K149R mutation enhances Fis1 mitochondrial localization. Critically, expressing a Fis1 K149R mutant restores DFP-induced mitophagy in SENP3-depleted cells. Thus, we propose a model in which SENP3-mediated deSUMOylation facilitates Fis1 mitochondrial localization to underpin stress-induced mitophagy.

Primary cultured neurons represent a benchmark preparation for studying neuronal cell biology. However, due to their poor transfection efficiency, studies often require virus-mediated gene delivery to genetically alter primary neurons. Lentiviral systems, based on HIV, are a commonly used gene delivery system due to their efficient expression, ease of production, and low toxicity to target cells. A powerful technique in interrogating protein function is ablation of specific proteins from cells using RNAi. In particular, expression of shRNA against the target protein allows for stable, long-term reduction of the protein of interest. Here, we describe a lentiviral system we use to knock down target proteins in primary cultured neurons via expression of an shRNA. We outline the cloning steps required to produce an shRNA-expressing lentiviral construct and provide a detailed method that can be used to produce lentiviral particles to transduce cultured neurons. In addition, we describe how to concentrate the produced lentiviral particles, allowing their use for in vivo studies.

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.

Defining the molecular changes that underlie Alzheimer’s disease (AD) is an important question in neuroscience. Here, we examined changes in protein SUMOylation, and proteins involved in mitochondrial dynamics, in an in vitro model of AD induced by application of amyloid-β 1–42 (Aβ1–42) to cultured neurons. We observed Aβ1–42-induced decreases in global SUMOylation and in levels of the SUMO pathway enzymes SENP3, PIAS1/2, and SAE2. Aβ exposure also decreased levels of the mitochondrial fission proteins Drp1 and Mff and increased activation of caspase-3. To examine whether loss of SENP3 is cytoprotective we knocked down SENP3, which partially prevented the Aβ1–42-induced increase in caspase-3 activation. Together, these data support the hypothesis that altered SUMOylation may play a role in the mechanisms underlying AD.

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.

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.

The endosome-associated cargo adaptor sorting nexin-27 (SNX27) is linked to various neuropathologies through sorting of integral proteins to the synaptic surface, most notably AMPA receptors. To provide a broader view of SNX27-associated pathologies, we performed proteomics in rat primary neurons to identify SNX27-dependent cargoes, and identified proteins linked to excitotoxicity, epilepsy, intellectual disabilities, and working memory deficits. Focusing on the synaptic adhesion molecule LRFN2, we established that SNX27 binds to LRFN2 and regulates its endosomal sorting. Furthermore, LRFN2 associates with AMPA receptors and knockdown of LRFN2 results in decreased surface AMPA receptor expression, reduced synaptic activity, and attenuated hippocampal long-term potentiation. Overall, our study provides an additional mechanism by which SNX27 can control AMPA receptor-mediated synaptic transmission and plasticity indirectly through the sorting of LRFN2 and offers molecular insight into the perturbed function of SNX27 and LRFN2 in a range of neurological conditions.

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.

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.

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.

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.

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.

Differential trafficking of AMPA receptors (AMPARs) to and from the postsynaptic membrane is a key determinant of the strength of excitatory neurotransmission, and is thought to underlie learning and memory. The transcription factor MEF2 is a negative regulator of memory in vivo, in part by regulating trafficking of the AMPAR subunit GluA2, but the molecular mechanisms behind this have not been established. Here we show, via knockdown of endogenous MEF2A in primary neuronal culture, that MEF2A is specifically required for Group I metabotropic glutamate receptor (mGluR)-mediated GluA2 internalisation, but does not regulate AMPAR expression or trafficking under basal conditions. Furthermore, this process occurs independently of changes in expression of Arc/Arg3.1, a previously characterised MEF2 transcriptional target and mediator of mGluR-dependent long-term depression. These data demonstrate a novel MEF2A-dependent mechanism for the regulation of activity-dependent AMPAR trafficking.