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.

PEX11ß 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 PEX11ß (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 PEX11ß 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 PEX11ß and restores peroxisome morphology in PEX11ß-deficient patient cells. Moreover, targeting of PEX11ß to mitochondria induces mitochondrial division, indicating the potential for PEX11ß 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.

Short Depuration of Oysters Intended for Human Consumption Is Effective at Reducing Exposure to Nanoplastics.

Nanoplastics (NPs; <1 µm) have greater availability to marine organisms than microplastics (1-5000 µm). Understanding NP uptake and depuration in marine organisms intended for human consumption is imperative for food safety, but until now it has been limited due to analytical constraints. Oysters (Crassostrea gigas) were exposed to polystyrene NPs doped with palladium (Pd), allowing the measurements of their uptake into tissues by inductively coupled plasma mass spectrometry (ICP-MS) combined with electron microscopy. Oysters were exposed for 6 days (d) to "Smooth" or "Raspberry" NPs, followed by 30 d of depuration with the aim of assessing the NP concentration in C. gigas following exposure, inferring the accumulation and elimination rates, and understanding the clearance of Pd NPs during the depuration period. After 6 d, the most significant accumulation was found in the digestive gland (106.6 and 135.3 µg g-1 dw, for Smooth and Raspberry NPs, respectively) and showed the most evident depuration (elimination rate constant KSmooth = 2 d-1 and KRaspberry = 0.2 d-1). Almost complete depuration of the Raspberry NPs occurred after 30 d. While a post-harvesting depuration period of 24-48 h for oysters could potentially reduce the NP content by 75%, more research to validate these findings, including depuration studies of oysters from the field, is required to inform practices to reduce human exposure through consumption.

Development of the electric organ in embryos and larvae of the knifefish, Brachyhypopomus gauderio.

South American Gymnotiform knifefish possess electric organs that generate electric fields for electro-location and electro-communication. Electric organs in fish can be derived from either myogenic cells (myogenic electric organ/mEO) or neurogenic cells (neurogenic electric organ/nEO). To date, the embryonic development of EOs has remained obscure. Here we characterize the development of the mEO in the Gymnotiform bluntnose knifefish, Brachyhypopomus gauderio. We find that EO primordial cells arise during embryonic stages in the ventral edge of the tail myotome, translocate into the ventral fin and develop into syncytial electrocytes at early larval stages. We also describe a pair of thick nerve cords that flank the dorsal aorta, the location and characteristic morphology of which are reminiscent of the nEO in Apteronotid species, suggesting a common evolutionary origin of these tissues. Taken together, our findings reveal the embryonic origins of the mEO and provide a basis for elucidating the mechanisms of evolutionary diversification of electric charge generation by myogenic and neurogenic EOs.

Cardiovascular Effects and Molecular Mechanisms of Bisphenol A and Its Metabolite MBP in Zebrafish.

The plastic monomer bisphenol A (BPA) is one of the highest production volume chemicals in the world and is frequently detected in wildlife and humans, particularly children. BPA has been associated with numerous adverse health outcomes relating to its estrogenic and other hormonal properties, but direct causal links are unclear in humans and animal models. Here we simulated measured (1×) and predicted worst-case (10× ) maximum fetal exposures for BPA, or equivalent concentrations of its metabolite MBP, using fluorescent reporter embryo-larval zebrafish, capable of quantifying Estrogen Response Element (ERE) activation throughout the body. Heart valves were primary sites for ERE activation by BPA and MBP, and transcriptomic analysis of microdissected heart tissues showed that both chemicals targeted several molecular pathways constituting biomarkers for calcific aortic valve disease (CAVD), including extra-cellular matrix (ECM) alteration. ECM collagen deficiency and impact on heart valve structural integrity were confirmed by histopathology for high-level MBP exposure, and structural defects (abnormal curvature) of the atrio-ventricular valves corresponded with impaired cardiovascular function (reduced ventricular beat rate and blood flow). Our results are the first to demonstrate plausible mechanistic links between ERE activation in the heart valves by BPA's reactive metabolite MBP and the development of valvular-cardiovascular disease states.

ATP prevents Woronin bodies from sealing septal pores in unwounded cells of the fungus Zymoseptoria tritici.

Septa of filamentous ascomycetes are perforated by septal pores that allow communication between individual hyphal compartments. Upon injury, septal pores are plugged rapidly by Woronin bodies (WBs), thereby preventing extensive cytoplasmic bleeding. The mechanism by which WBs translocate into the pore is not known, but it has been suggested that wound-induced cytoplasmic bleeding "flushes" WBs into the septal opening. Alternatively, contraction of septum-associated tethering proteins may pull WBs into the septal pore. Here, we investigate WB dynamics in the wheat pathogen Zymoseptoria tritici. Ultrastructural studies showed that 3.4 ± 0.2 WBs reside on each side of a septum and that single WBs of 128.5 ± 3.6 nm in diameter seal the septal pore (41 ± 1.5 nm). Live cell imaging of green fluorescent ZtHex1, a major protein in WBs, and the integral plasma membrane protein ZtSso1 confirms WB translocation into the septal pore. This was associated with the occasional formation of a plasma membrane "balloon," extruding into the dead cell, suggesting that the plasma membrane rapidly seals the wounded septal pore wound. Minor amounts of fluorescent ZtHex1-enhanced green fluorescent protein (eGFP) appeared associated with the "ballooning" plasma membrane, indicating that cytoplasmic ZtHex1-eGFP is recruited to the extending plasma membrane. Surprisingly, in ~15% of all cases, WBs moved from the ruptured cell into the septal pore. This translocation against the cytoplasmic flow suggests that an active mechanism drives WB plugging. Indeed, treatment of unwounded and intact cells with the respiration inhibitor carbonyl cyanide m-chlorophenyl hydrazone induced WB translocation into the pores. Moreover, carbonyl cyanide m-chlorophenyl hydrazone treatment recruited cytoplasmic ZtHex1-eGFP to the lateral plasma membrane of the cells. Thus, keeping the WBs out of the septal pores, in Z. tritici, is an ATP-dependent process.

Phospholipase C-η2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth.

Neurite growth is central to the formation and differentiation of functional neurons, and recently, an essential role for phospholipase C-η2 (PLCη2) in neuritogenesis was revealed. Here we investigate the function of PLCη2 in neuritogenesis using Neuro2A cells, which upon stimulation with retinoic acid differentiate and form neurites. We first investigated the role of the PLCη2 calcium-binding EF-hand domain, a domain that is known to be required for PLCη2 activation. To do this, we quantified neurite outgrowth in Neuro2A cells, stably overexpressing wild-type PLCη2 and D256A (EF-hand) and H460Q (active site) PLCη2 mutants. Retinoic acid-induced neuritogenesis was highly dependent on PLCη2 activity, with the H460Q mutant exhibiting a strong dominant-negative effect. Expression of the D256A mutant had little effect on neurite growth relative to the control, suggesting that calcium-directed activation of PLCη2 is not essential to this process. We next investigated which cellular compartments contain endogenous PLCη2 by comparing immunoelectron microscopy signals over control and knockdown cell lines. When signals were analyzed to reveal specific labeling for PLCη2, it was found to be localized predominantly over the nucleus and cytosol. Furthermore in these compartments (and also in growing neurites), a proximity ligand assay revealed that PLCη2 specifically interacts with LIMK-1 in Neuro2A cells. Taken together, these data emphasize the importance of the PLCη2 EF-hand domain and articulation of PLCη2 with LIMK-1 in regulating neuritogenesis.

Plasma membrane-located purine nucleotide transport proteins are key components for host exploitation by microsporidian intracellular parasites.

Microsporidia are obligate intracellular parasites of most animal groups including humans, but despite their significant economic and medical importance there are major gaps in our understanding of how they exploit infected host cells. We have investigated the evolution, cellular locations and substrate specificities of a family of nucleotide transport (NTT) proteins from Trachipleistophora hominis, a microsporidian isolated from an HIV/AIDS patient. Transport proteins are critical to microsporidian success because they compensate for the dramatic loss of metabolic pathways that is a hallmark of the group. Our data demonstrate that the use of plasma membrane-located nucleotide transport proteins (NTT) is a key strategy adopted by microsporidians to exploit host cells. Acquisition of an ancestral transporter gene at the base of the microsporidian radiation was followed by lineage-specific events of gene duplication, which in the case of T. hominis has generated four paralogous NTT transporters. All four T. hominis NTT proteins are located predominantly to the plasma membrane of replicating intracellular cells where they can mediate transport at the host-parasite interface. In contrast to published data for Encephalitozoon cuniculi, we found no evidence for the location for any of the T. hominis NTT transporters to its minimal mitochondria (mitosomes), consistent with lineage-specific differences in transporter and mitosome evolution. All of the T. hominis NTTs transported radiolabelled purine nucleotides (ATP, ADP, GTP and GDP) when expressed in Escherichia coli, but did not transport radiolabelled pyrimidine nucleotides. Genome analysis suggests that imported purine nucleotides could be used by T. hominis to make all of the critical purine-based building-blocks for DNA and RNA biosynthesis during parasite intracellular replication, as well as providing essential energy for parasite cellular metabolism and protein synthesis.

Proteoglycans support proper granule formation in pancreatic acinar cells.

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.

Strategies for maximizing ATP supply in the microsporidian Encephalitozoon cuniculi: direct binding of mitochondria to the parasitophorous vacuole and clustering of the mitochondrial porin VDAC.

Microsporidia are obligate intracellular parasites with extremely reduced genomes and a dependence on host-derived ATP. The microsporidium Encephalitozoon cuniculi proliferates within a membranous vacuole and we investigated how the ATP supply is optimized at the vacuole-host interface. Using spatial EM quantification (stereology), we found a single layer of mitochondria coating substantial proportions of the parasitophorous vacuole. Mitochondrial binding occurred preferentially over the vegetative 'meront' stages of the parasite, which bulged into the cytoplasm, thereby increasing the membrane surface available for mitochondrial interaction. In a broken cell system mitochondrial binding was maintained and was typified by electron dense structures (< 10 nm long) bridging between outer mitochondrial and vacuole membranes. In broken cells mitochondrial binding was sensitive to a range of protease treatments. The function of directly bound mitochondria, as measured by the membrane potential sensitive dye JC-1, was indistinguishable from other mitochondria in the cell although there was a generalized depression of the membrane potential in infected cells. Finally, quantitative immuno-EM revealed that the ATP-delivering mitochondrial porin, VDAC, was concentrated atthe mitochondria-vacuole interaction site. Thus E. cuniculi appears to maximize ATP supply by direct binding of mitochondria to the parasitophorous vacuole bringing this organelle within 0.020 microns of the growing vegetative form of the parasite. ATP-delivery is further enhanced by clustering of ATP transporting porins in those regions of the outer mitochondrial membrane lying closest to the parasite.

Probing the structure of the mechanosensitive channel of small conductance in lipid bilayers with pulsed electron-electron double resonance.

Mechanosensitive channel proteins are important safety valves against osmotic shock in bacteria, and are involved in sensing touch and sound waves in higher organisms. The mechanosensitive channel of small conductance (MscS) has been extensively studied. Pulsed electron-electron double resonance (PELDOR or DEER) of detergent-solubilized protein confirms that as seen in the crystal structure, the outer ring of transmembrane helices do not pack against the pore-forming helices, creating an apparent void. The relevance of this void to the functional form of MscS in the bilayer is the subject of debate. Here, we report PELDOR measurements of MscS reconstituted into two lipid bilayer systems: nanodiscs and bicelles. The distance measurements from multiple mutants derived from the PELDOR data are consistent with the detergent-solution arrangement of the protein. We conclude, therefore, that the relative positioning of the transmembrane helices is preserved in mimics of the cell bilayer, and that the apparent voids are not an artifact of detergent solution but a property of the protein that will have to be accounted for in any molecular mechanism of gating.

The genome of the obligate intracellular parasite Trachipleistophora hominis: new insights into microsporidian genome dynamics and reductive evolution.

The dynamics of reductive genome evolution for eukaryotes living inside other eukaryotic cells are poorly understood compared to well-studied model systems involving obligate intracellular bacteria. Here we present 8.5 Mb of sequence from the genome of the microsporidian Trachipleistophora hominis, isolated from an HIV/AIDS patient, which is an outgroup to the smaller compacted-genome species that primarily inform ideas of evolutionary mode for these enormously successful obligate intracellular parasites. Our data provide detailed information on the gene content, genome architecture and intergenic regions of a larger microsporidian genome, while comparative analyses allowed us to infer genomic features and metabolism of the common ancestor of the species investigated. Gene length reduction and massive loss of metabolic capacity in the common ancestor was accompanied by the evolution of novel microsporidian-specific protein families, whose conservation among microsporidians, against a background of reductive evolution, suggests they may have important functions in their parasitic lifestyle. The ancestor had already lost many metabolic pathways but retained glycolysis and the pentose phosphate pathway to provide cytosolic ATP and reduced coenzymes, and it had a minimal mitochondrion (mitosome) making Fe-S clusters but not ATP. It possessed bacterial-like nucleotide transport proteins as a key innovation for stealing host-generated ATP, the machinery for RNAi, key elements of the early secretory pathway, canonical eukaryotic as well as microsporidian-specific regulatory elements, a diversity of repetitive and transposable elements, and relatively low average gene density. Microsporidian genome evolution thus appears to have proceeded in at least two major steps: an ancestral remodelling of the proteome upon transition to intracellular parasitism that involved reduction but also selective expansion, followed by a secondary compaction of genome architecture in some, but not all, lineages.

Generating open-source 3D phytoplankton models by integrating photogrammetry with scanning electron microscopy.

The community structure and ecological function of marine ecosystems are critically dependent on phytoplankton. However, our understanding of phytoplankton is limited due to the lack of detailed information on their morphology. To address this gap, we developed a framework that combines scanning electron microscopy (SEM) with photogrammetry to create realistic 3D (three-dimensional) models of phytoplankton. The workflow of this framework is demonstrated using two marine algal species, one dinoflagellate Prorocentrum micans and one diatom Halamphora sp. The resulting 3D models are made openly available and allow users to interact with phytoplankton and their complex structures virtually (digitally) and tangibly (3D printing). They also allow for surface area and biovolume calculations of phytoplankton, as well as the exploration of their light scattering properties, which are both important for ecosystem modeling. Additionally, by presenting these models to the public, it bridges the gap between scientific inquiry and education, promoting broader awareness on the importance of phytoplankton.

Biogenesis, inheritance, and 3D ultrastructure of the microsporidian mitosome.

During the reductive evolution of obligate intracellular parasites called microsporidia, a tiny remnant mitochondrion (mitosome) lost its typical cristae, organellar genome, and most canonical functions. Here, we combine electron tomography, stereology, immunofluorescence microscopy, and bioinformatics to characterise mechanisms of growth, division, and inheritance of this minimal mitochondrion in two microsporidia species (grown within a mammalian RK13 culture-cell host). Mitosomes of Encephalitozoon cuniculi (2-12/cell) and Trachipleistophora hominis (14-18/nucleus) displayed incremental/non-phasic growth and division and were closely associated with an organelle identified as equivalent to the fungal microtubule-organising centre (microsporidian spindle pole body; mSPB). The mitosome-mSPB association was resistant to treatment with microtubule-depolymerising drugs nocodazole and albendazole. Dynamin inhibitors (dynasore and Mdivi-1) arrested mitosome division but not growth, whereas bioinformatics revealed putative dynamins Drp-1 and Vps-1, of which, Vps-1 rescued mitochondrial constriction in dynamin-deficient yeast (Schizosaccharomyces pombe). Thus, microsporidian mitosomes undergo incremental growth and dynamin-mediated division and are maintained through ordered inheritance, likely mediated via binding to the microsporidian centrosome (mSPB).

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.

Antagonistic Mobile Genetic Elements Can Counteract Each Other's Effects on Microbial Community Composition.

Bacteriophages ("phages") are hypothesized to be key drivers of bacterial population dynamics, driving microbial community composition, but empirical support for this is mixed. One reason why phages may have a less-than-expected impact on community composition is that many different phages and other mobile genetic elements (MGEs) interact with each bacterium. For instance, the same phage may have higher or lower costs to different bacterial strains or species. Assuming that resistance or susceptibility to MGE infection is not consistent across all MGEs, a simple prediction is that the net effect of MGEs on each bacterial taxon may converge with an increasing number of interactions with different MGEs. We formalized this prediction using in silico population dynamics simulations and then carried out experiments using three bacterial species, one generalist conjugative plasmid, and three species-specific phages. While the presence of only phages or only the plasmid altered community structure, these differential effects on community structure canceled out when both were together. The effects of MGEs were largely indirect and could not be explained by simple pairwise bipartite interactions (i.e., between each MGE and each bacterial species). Our results suggest that the effects of MGEs may be overestimated by studies that focus on a single MGE and not on interactions among multiple MGEs. IMPORTANCE While bacteriophages ("phages") are often cited as some of the key drivers of microbial diversity, evidence for this is greatly mixed. We demonstrate, in silico and experimentally, that the impact of phages, an example of a mobile genetic element (MGE), on community structure can diminish with increasing MGE diversity. This is because MGEs can have diverse effects on host fitness, and therefore as diversity increases, their individual effects cancel out, returning communities back to an MGE-free state. In addition, interactions in mixed-species and MGE communities could not be predicted from simple pairwise interactions, highlighting the difficulty in generalizing a MGE's effect from pairwise studies.

Scoping intergenerational effects of nanoplastic on the lipid reserves of Antarctic krill embryos.

Antarctic krill (Euphausia superba) plays a central role in the Antarctic marine food web and biogeochemical cycles and has been identified as a species that is potentially vulnerable to plastic pollution. While plastic pollution has been acknowledged as a potential threat to Southern Ocean marine ecosystems, the effect of nanoplastics (<1000 nm) is poorly understood. Deleterious impacts of nanoplastic are predicted to be higher than that of larger plastics, due to their small size which enables their permeation of cell membranes and potentially provokes toxicity. Here, we investigated the intergenerational impact of exposing Antarctic krill to nanoplastics. We focused on whether embryonic energy resources were affected when gravid female krill were exposed to nanoplastic by determining lipid and fatty acid compositions of embryos produced in incubation. Embryos were collected from females who had spawned under three different exposure treatments (control, nanoplastic, nanoplastic + algae). Embryos collected from each maternal treatment were incubated for a further 6 days under three nanoplastic exposure treatments (control, low concentration nanoplastic, and high concentration nanoplastic). Nanoplastic additions to seawater did not impact lipid metabolism (total lipid or fatty acid composition) across the maternal or direct embryo treatments, and no interactive effects were observed. The provision of a food source during maternal exposure to nanoplastic had a positive effect on key fatty acids identified as important during embryogenesis, including higher total polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) when compared to the control and nanoplastic treatments. Whilst the short exposure time was ample for lipids from maternally digested algae to be incorporated into embryos, we discuss why the nanoplastic-fatty acid relationship may be more complex. Our study is the first to scope intergeneration effects of nanoplastic on Antarctic krill lipid and fatty acid reserves. From this, we suggest directions for future research including long term exposures, multi-stressor scenarios and exploring other critical energy reserves such as proteins.

The Use of Off-Label Medications in Newborn Infants Despite an Approved Alternative Being Available-Results of a National Survey.

Neonates continue to be treated with off-label or unlicensed drugs while in hospital. However, some medications that have previously been used in adults underwent clinical testing and licensure for use with a different indication in the neonatal and pediatric population. Almost always, the marketing of these newly approved substances in a niche indication is accompanied by a steep increase in the price of the compound. We investigated the use of the approved formulation or the cheaper off-label alternative of Ibuprofen (Pedea®), Propanolol (Hemangiol®) and Caffeine Citrate (Peyona®) in neonatal clinical practice by conducting a National Survey of 214 Perinatal Centers in Germany. We also assessed price differences between on- and off-label alternatives and the extend of the clinical development program of the on-label medication in the neonatal population. On-label medication was more frequently used than the off-label alternative in all indications (PDA: on-label to off-label ratio 1:0.26, Apnea: 1:0.56, Hemangioma 1:0.76). All sponsors did conduct placebo-controlled Phase III trials with efficacy and safety endpoints in the target population and the number of participants in the target population varied between 82 and 497. Costs for the three drugs in their approved and marketed formulations increased in median 405-fold compared with the corresponding off-label alternative. Overall, about one out of three neonatologists prescribed an off-label or non-approved drug to patients despite an alternative medication that is approved for the indication in the target population being available.

Differential routing and disposition of the long-chain saturated fatty acid palmitate in rodent vs human beta-cells.

BACKGROUND: Rodent and human ß-cells are differentially susceptible to the "lipotoxic" effects of long-chain saturated fatty acids (LC-SFA) but the factors accounting for this are unclear. Here, we have studied the intracellular disposition of the LC-SFA palmitate in human vs rodent ß-cells and present data that reveal new insights into the factors regulating ß-cell lipotoxicity. METHODS: The subcellular distribution of the LC-SFA palmitate was studied in rodent (INS-1E and INS-1 823/13 cells) and human (EndoC-ßH1) ß-cells using confocal fluorescence and electron microscopy (EM). Protein expression was assessed by Western blotting and cell viability, by vital dye staining. RESULTS: Exposure of INS-1 cells to palmitate for 24 h led to loss of viability, whereas EndoC-ßH1 cells remained viable even after 72 h of treatment with a high concentration (1 mM) of palmitate. Use of the fluorescent palmitate analogue BODIPY FL C16 revealed an early localisation of the LC-SFA to the Golgi apparatus in INS-1 cells and this correlated with distention of intracellular membranes, visualised under the EM. Despite this, the PERK-dependent ER stress pathway was not activated under these conditions. By contrast, BODIPY FL C16 did not accumulate in the Golgi apparatus in EndoC-ßH1 cells but, rather, co-localised with the lipid droplet-associated protein, PLIN2, suggesting preferential routing into lipid droplets. When INS-1 cells were treated with a combination of palmitate plus oleate, the toxic effects of palmitate were attenuated and BODIPY FL C16 localised primarily with PLIN2 but not with a Golgi marker. CONCLUSION: In rodent ß-cells, palmitate accumulates in the Golgi apparatus at early time points whereas, in EndoC- ßH1 cells, it is routed preferentially into lipid droplets. This may account for the differential sensitivity of rodent vs human ß-cells to "lipotoxicity" since manoeuvres leading to the incorporation of palmitate into lipid droplets is associated with the maintenance of cell viability in both cell types.

Regulating peroxisome-ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3ß.

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 GSK3ß (glycogen synthase kinase-3 ß) 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.