GelMap: intrinsic calibration and deformation mapping for expansion microscopy.

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy by physically expanding biological specimen in three dimensions. Nonetheless, using ExM for quantitative or diagnostic applications requires robust quality control methods to precisely determine expansion factors and to map deformations due to anisotropic expansion. Here we present GelMap, a flexible workflow to introduce a fluorescent grid into pre-expanded hydrogels that scales with expansion and reports deformations. We demonstrate that GelMap can be used to precisely determine the local expansion factor and to correct for deformations without the use of cellular reference structures or pre-expansion ground-truth images. Moreover, we show that GelMap aids sample navigation for correlative uses of expansion microscopy. Finally, we show that GelMap is compatible with expansion of tissue and can be readily implemented as a quality control step into existing ExM workflows.

Differential roles for ACBD4 and ACBD5 in peroxisome-ER interactions and lipid metabolism.

Peroxisomes and the endoplasmic reticulum (ER) are intimately linked subcellular organelles, physically connected at membrane contact sites. While collaborating in lipid metabolism, for example, of very long-chain fatty acids (VLCFAs) and plasmalogens, the ER also plays a role in peroxisome biogenesis. Recent work identified tethering complexes on the ER and peroxisome membranes that connect the organelles. These include membrane contacts formed via interactions between the ER protein VAPB (vesicle-associated membrane protein-associated protein B) and the peroxisomal proteins ACBD4 and ACBD5 (acyl-coenzyme A-binding domain protein). Loss of ACBD5 has been shown to cause a significant reduction in peroxisome-ER contacts and accumulation of VLCFAs. However, the role of ACBD4 and the relative contribution these two proteins make to contact site formation and recruitment of VLCFAs to peroxisomes remain unclear. Here, we address these questions using a combination of molecular cell biology, biochemical, and lipidomics analyses following loss of ACBD4 or ACBD5 in HEK293 cells. We show that the tethering function of ACBD5 is not absolutely required for efficient peroxisomal ß-oxidation of VLCFAs. We demonstrate that loss of ACBD4 does not reduce peroxisome-ER connections or result in the accumulation of VLCFAs. Instead, the loss of ACBD4 resulted in an increase in the rate of ß-oxidation of VLCFAs. Finally, we observe an interaction between ACBD5 and ACBD4, independent of VAPB binding. Overall, our findings suggest that ACBD5 may act as a primary tether and VLCFA recruitment factor, whereas ACBD4 may have regulatory functions in peroxisomal lipid metabolism at the peroxisome-ER interface.

From observing to controlling: Inducible control of organelle dynamics and interactions.

The dynamics and interactions of cellular organelles underlie many aspects of cellular functioning. Until recently, assessment of organelle dynamics has been primarily observational or required whole-cell perturbations to assess the implications of altered organelle motility and positioning. However, thanks to recently developed and optimized intervention strategies, we now have the ability to control organelles in their unperturbed state, altering organelle positioning, membrane trafficking pathways, as well as organelle interactions. This can be performed both globally and locally, giving fine control over the range, reversibility, and extent of organelle dynamics. Here, we describe how these tools are currently used for controlling organelles and give insight into the exciting future of this emerging field.

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.

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 PEX11ß 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 .

Fluorescent tools to analyse peroxisome-ER interactions in mammalian cells.

Peroxisomes and the endoplasmic reticulum (ER) cooperate extensively in lipid-related metabolic pathways, and the ER also provides phospholipids to enable the peroxisomal membrane to expand prior to division. Recently, we identified peroxisomal proteins ACBD5 and ACBD4, and the ER protein VAPB as tethering components which physically interact to foster peroxisome-ER associations at membrane contact sites. Overexpression or loss of these tether proteins alters the extent of peroxisome-ER interactions, impacting on lipid exchange between these two compartments. To facilitate further studies into peroxisome-ER associations at the level of membrane contact sites, their role, composition and regulation, we have developed two fluorescence-based systems to monitor peroxisome-ER interactions. We modified a proximity ligation assay and a split-fluorescence reporter system using split superfolder green fluorescent protein. Using the proximity ligation assay we were able to measure changes in peroxisome-ER interactions whilst the split-fluorescence reporter was more limited and only allowed us to label ER-peroxisome contacts. We show that both techniques can be useful additions to the toolkit of methods to study peroxisome-ER associations and explore the relative merits of each.

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. Overexpression 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.

Multi-localized Proteins: The Peroxisome-Mitochondria Connection.

Peroxisomes and mitochondria are dynamic, multifunctional organelles that play pivotal cooperative roles in the metabolism of cellular lipids and reactive oxygen species. Their functional interplay, the "peroxisome-mitochondria connection", also includes cooperation in anti-viral signalling and defence, as well as coordinated biogenesis by sharing key division proteins. In this review, we focus on multi-localised proteins which are shared by peroxisomes and mitochondria in mammals. We first outline the targeting and sharing of matrix proteins which are involved in metabolic cooperation. Next, we discuss shared components of peroxisomal and mitochondrial dynamics and division, and we present novel insights into the dual targeting of tail-anchored membrane proteins. Finally, we provide an overview of what is currently known about the role of shared membrane proteins in disease. What emerges is that sharing of proteins between these two organelles plays a key role in their cooperative functions which, based on new findings, may be more extensive than originally envisaged. Gaining a better insight into organelle interplay and the targeting of shared proteins is pivotal to understanding how organelle cooperation contributes to human health and disease.

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.

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.