Endoplasmic Reticulum Plasma Membrane Research Articles

Altered GM1 catabolism affects NMDAR-mediated Ca2+ signaling at ER-PM junctions and increases synaptic spine formation in a GM1-gangliosidosis model

Endoplasmic reticulum-plasma membrane (ER-PM) junctions mediate Ca2+ flux across neuronal membranes. The properties of these membrane contact sites are defined by their lipid content, but little attention has been given to glycosphingolipids (GSLs). Here, we show that GM1-ganglioside, an abundant GSL in neuronal membranes, is integral to ER-PM junctions; it interacts with synaptic proteins/receptors and regulates Ca2+ signaling. In a model of the neurodegenerative lysosomal storage disease, GM1-gangliosidosis, pathogenic accumulation of GM1 at ER-PM junctions due to β-galactosidase deficiency drastically altersneuronal Ca2+ homeostasis. Mechanistically, we show that GM1 interacts with the phosphorylated N-methyl D-aspartate receptor (NMDAR) Ca2+ channel, thereby increasing Ca2+ flux, activating extracellular signal-regulated kinase (ERK) signaling, and increasing the number of synaptic spines without increasing synaptic connectivity. Thus, GM1 clustering at ER-PM junctions alters synaptic plasticity and worsens the generalized neuronal cell death characteristic of GM1-gangliosidosis.

The VAMP-associated protein VAP27-1 plays a crucial role in plant resistance to ER stress by modulating ER–PM contact architecture in Arabidopsis

The endoplasmic reticulum (ER) and the plasma membrane (PM) form ER–PM contact sites (EPCSs) that allow the ER and PM to exchange materials and information. Stress-induced disruption of protein folding triggers ER stress, and the cell initiates the unfolded protein response (UPR) to resist the stress. However, whether EPCSs play a role in ER stress in plants remains unclear. VESICLE-ASSOCIATED MEMBRANE PROTEIN (VAMP)-ASSOCIATED PROTEIN 27-1 (VAP27-1) functions in EPCS tethering and is encoded by a family of 10 genes (VAP27-1–10) in Arabidopsis thaliana. Here, we used CRISPR-Cas9-mediated genome editing to obtain a homozygous vap27-1 vap27-3 vap27-4 (vap27-1/3/4) triple mutant lacking three of the key VAP27 family members in Arabidopsis. The vap27-1/3/4 mutant exhibits defects in ER–PM connectivity and EPCS architecture, as well as excessive UPR signaling. We further showed that relocation of VAP27-1 to the PM mediates specific VAP27-1-related EPCS remodeling and expansion under ER stress. Moreover, the spatiotemporal dynamics of VAP27-1 at the PM increase ER–PM connectivity and enhance Arabidopsis resistance to ER stress. In addition, we revealed an important role for intracellular calcium homeostasis in the regulation of UPR signaling. Taken together, these results broaden our understanding of the molecular and cellular mechanisms of ER stress and UPR signaling in plants, providing additional clues for improving plant broad-spectrum resistance to different stresses.

Routing of Kv7.1 to endoplasmic reticulum plasma membrane junctions.

The voltage-gated Kv7.1 channel, in association with the regulatory subunit KCNE1, contributes to the IKs current in the heart. However, both proteins travel to the plasma membrane using different routes. While KCNE1 follows a classical Golgi-mediated anterograde pathway, Kv7.1 is located in endoplasmic reticulum-plasma membrane junctions (ER-PMjs), where it associates with KCNE1 before being delivered to the plasma membrane. To characterize the channel routing to these spots we used a wide repertoire of methodologies, such as protein expression analysis (i.e. protein association and biotin labeling), confocal (i.e. immunocytochemistry, FRET, and FRAP), and dSTORM microscopy, transmission electron microscopy, proteomics, and electrophysiology. We demonstrated that Kv7.1 targeted ER-PMjs regardless of the origin or architecture of these structures. Kv2.1, a neuronal channel that also contributes to a cardiac action potential, and JPHs, involved in cardiac dyads, increased the number of ER-PMjs in nonexcitable cells, driving and increasing the level of Kv7.1 at the cell surface. Both ER-PMj inducers influenced channel function and dynamics, suggesting that different protein structures are formed. Although exhibiting no physical interaction, Kv7.1 resided in more condensed clusters (ring-shaped) with Kv2.1 than with JPH4. Moreover, we found that VAMPs and AMIGO, which are Kv2.1 ancillary proteins also associated with Kv7.1. Specially, VAP B, showed higher interaction with the channel when ER-PMjs were stimulated by Kv2.1. Our results indicated that Kv7.1 may bind to different structures of ER-PMjs that are induced by different mechanisms. This variable architecture can differentially affect the fate of cardiac Kv7.1 channels.

The Endoplasmic Reticulum-Plasma Membrane Tethering Protein Ice2 Controls Lipid Droplet Size via the Regulation of Phosphatidylcholine in Candida albicans.

Lipid droplets (LDs) are intracellular organelles that play important roles in cellular lipid metabolism; they change their sizes and numbers in response to both intracellular and extracellular signals. Changes in LD size reflect lipid synthesis and degradation and affect many cellular activities, including energy supply and membrane synthesis. Here, we focused on the function of the endoplasmic reticulum-plasma membrane tethering protein Ice2 in LD dynamics in the fungal pathogen Candida albicans (C. albicans). Nile red staining and size quantification showed that the LD size increased in the ice2Δ/Δ mutant, indicating the critical role of Ice2 in the regulation of LD dynamics. A lipid content analysis further demonstrated that the mutant had lower phosphatidylcholine levels. As revealed with GFP labeling and fluorescence microscopy, the methyltransferase Cho2, which is involved in phosphatidylcholine synthesis, had poorer localization in the plasma membrane in the mutant than in the wild-type strain. Interestingly, the addition of the phosphatidylcholine precursor choline led to the recovery of normal-sized LDs in the mutant. These results indicated that Ice2 regulates LD size by controlling intracellular phosphatidylcholine levels and that endoplasmic reticulum-plasma membrane tethering proteins play a role in lipid metabolism regulation in C. albicans. This study provides significant findings for further investigation of the lipid metabolism in fungi.

T cell Ca2+ microdomains through the lens of computational modeling.

Cellular Ca2+ signaling is highly organized in time and space. Locally restricted and short-lived regions of Ca2+ increase, called Ca2+ microdomains, constitute building blocks that are differentially arranged to create cellular Ca2+ signatures controlling physiological responses. Here, we focus on Ca2+ microdomains occurring in restricted cytosolic spaces between the plasma membrane and the endoplasmic reticulum, called endoplasmic reticulum-plasma membrane junctions. In T cells, these microdomains have been finely characterized. Enough quantitative data are thus available to develop detailed computational models of junctional Ca2+ dynamics. Simulations are able to predict the characteristics of Ca2+ increases at the level of single channels and in junctions of different spatial configurations, in response to various signaling molecules. Thanks to the synergy between experimental observations and computational modeling, a unified description of the molecular mechanisms that create Ca2+ microdomains in the first seconds of T cell stimulation is emerging.

Localization of the tubby domain, a PI(4,5)P2 biosensor, to E-Syt3-rich endoplasmic reticulum-plasma membrane junctions.

The phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] acts as a signaling lipid at the plasma membrane (PM) with pleiotropic regulatory actions on multiple cellular processes. Signaling specificity might result from spatiotemporal compartmentalization of the lipid and from combinatorial binding of PI(4,5)P2 effector proteins to additional membrane components. Here, we analyzed the spatial distribution of tubbyCT, a paradigmatic PI(4,5)P2-binding domain, in live mammalian cells by total internal reflection fluorescence (TIRF) microscopy and molecular dynamics simulations. We found that unlike other well-characterized PI(4,5)P2 recognition domains, tubbyCT segregates into distinct domains within the PM. TubbyCT enrichment occurred at contact sites between PM and endoplasmic reticulum (ER) (i.e. at ER-PM junctions) as shown by colocalization with ER-PM markers. Localization to these sites was mediated in a combinatorial manner by binding to PI(4,5)P2 and by interaction with a cytosolic domain of extended synaptotagmin 3 (E-Syt3), but not other E-Syt isoforms. Selective localization to these structures suggests that tubbyCT is a novel selective reporter for a ER-PM junctional pool of PI(4,5)P2. Finally, we found that association with ER-PM junctions is a conserved feature of tubby-like proteins (TULPs), suggesting an as-yet-unknown function of TULPs.

Phosphatidylinositol 4,5-bisphosphate and calcium at ER-PM junctions - Complex interplay of simple messengers.

Endoplasmic reticulum-plasma membrane contact sites (ER-PM MCS) are a specialised domain involved in the control of Ca2+ dynamics and various Ca2+-dependent cellular processes. Intracellular Ca2+ signals are broadly supported by Ca2+ release from intracellular Ca2+ channels such as inositol 1,4,5-trisphosphate receptors (IP3Rs) and subsequent store-operated Ca2+ entry (SOCE) across the PM to replenish store content. IP3Rs sit in close proximity to the PM where they can easily access newly synthesised IP3, interact with binding partners such as actin, and localise adjacent to ER-PM MCS populated by the SOCE machinery, STIM1-2 and Orai1-3, to possibly form a locally regulated unit of Ca2+ influx. PtdIns(4,5)P2 is a multiplex regulator of Ca2+ signalling at the ER-PM MCS interacting with multiple proteins at these junctions such as actin and STIM1, whilst also being consumed as a substrate for phospholipase C to produce IP3 in response to extracellular stimuli. In this review, we consider the mechanisms regulating the synthesis and turnover of PtdIns(4,5)P2 via the phosphoinositide cycle and its significance for sustained signalling at the ER-PM MCS. Furthermore, we highlight recent insights into the role of PtdIns(4,5)P2 in the spatiotemporal organization of signalling at ER-PM junctions and raise outstanding questions on how this multi-faceted regulation occurs.

Porcine reproductive and respiratory syndrome virus infection triggers autophagy via ER stress-induced calcium signaling to facilitate virus replication.

Calcium (Ca2+), a ubiquitous second messenger, plays a crucial role in many cellular functions. Viruses often hijack Ca2+ signaling to facilitate viral processes such as entry, replication, assembly, and egress. Here, we report that infection by the swine arterivirus, porcine reproductive and respiratory syndrome virus (PRRSV), induces dysregulated Ca2+ homeostasis, subsequently activating calmodulin-dependent protein kinase-II (CaMKII) mediated autophagy, and thus fueling viral replication. Mechanically, PRRSV infection induces endoplasmic reticulum (ER) stress and forms a closed ER-plasma membrane (PM) contacts, resulting the opening of store operated calcium entry (SOCE) channel and causing the ER to take up extracellular Ca2+, which is then released into the cytoplasm by inositol trisphosphate receptor (IP3R) channel. Importantly, pharmacological inhibition of ER stress or CaMKII mediated autophagy blocks PRRSV replication. Notably, we show that PRRSV protein Nsp2 plays a dominant role in the PRRSV induced ER stress and autophagy, interacting with stromal interaction molecule 1 (STIM1) and the 78 kDa glucose-regulated protein 78 (GRP78). The interplay between PRRSV and cellular calcium signaling provides a novel potential approach to develop antivirals and therapeutics for the disease outbreaks.

Endoplasmic Reticulum-Plasma Membrane Junctions as Sites of Depolarization-Induced Ca2+ Signaling in Excitable Cells.

Membrane contact sites between endoplasmic reticulum (ER) and plasma membrane (PM), or ER-PM junctions, are found in all eukaryotic cells. In excitable cells they play unique roles in organizing diverse forms of Ca2+ signaling as triggered by membrane depolarization. ER-PM junctions underlie crucial physiological processes such as excitation-contraction coupling, smooth muscle contraction and relaxation, and various forms of activity-dependent signaling and plasticity in neurons. In many cases the structure and molecular composition of ER-PM junctions in excitable cells comprise important regulatory feedback loops linking depolarization-induced Ca2+ signaling at these sites to the regulation of membrane potential. Here, we describe recent findings on physiological roles and molecular composition of native ER-PM junctions in excitable cells. We focus on recent studies that provide new insights into canonical forms of depolarization-induced Ca2+ signaling occurring at junctional triads and dyads of striated muscle, as well as the diversity of ER-PM junctions in these cells and in smooth muscle and neurons.

ER-PM Junctions on GABAergic Interneurons Are Organized by Neuregulin 2/VAP Interactions and Regulated by NMDA Receptors.

Neuregulins (NRGs) signal via ErbB receptors to regulate neural development, excitability, synaptic and network activity, and behaviors relevant to psychiatric disorders. Bidirectional signaling between NRG2/ErbB4 and NMDA receptors is thought to homeostatically regulate GABAergic interneurons in response to increased excitatory neurotransmission or elevated extracellular glutamate levels. Unprocessed proNRG2 forms discrete clusters on cell bodies and proximal dendrites that colocalize with the potassium channel Kv2.1 at specialized endoplasmic reticulum-plasma membrane (ER-PM) junctions, and NMDA receptor activation triggers rapid dissociation from ER-PM junctions and ectodomain shedding by ADAM10. Here, we elucidate the mechanistic basis of proNRG2 clustering at ER-PM junctions and its regulation by NMDA receptors. Importantly, we demonstrate that proNRG2 promotes the formation of ER-PM junctions by directly binding the ER-resident membrane tether VAP, like Kv2.1. The proNRG2 intracellular domain harbors two non-canonical, low-affinity sites that cooperatively mediate VAP binding. One of these is a cryptic and phosphorylation-dependent VAP binding motif that is dephosphorylated following NMDA receptor activation, thus revealing how excitatory neurotransmission promotes the dissociation of proNRG2 from ER-PM junctions. Therefore, proNRG2 and Kv2.1 can independently function as VAP-dependent organizers of neuronal ER-PM junctions. Based on these and prior studies, we propose that proNRG2 and Kv2.1 serve as co-regulated downstream effectors of NMDA receptors to homeostatically regulate GABAergic interneurons.

The Tricalbin-Family Endoplasmic Reticulum-Plasma Membrane Tethering Proteins Attenuate ROS-Involved Caspofungin Sensitivity in Candida albicans.

The endoplasmic reticulum-plasma membrane (ER-PM) contacts are one kind of important membrane contact structures in eukaryotic cells, which mediate material and message exchange between the ER and the PM. However, the specific types and functions of ER-PM tethering proteins are poorly understood in the human fungal pathogen Candida albicans. In this study, we observed that the two tricalbin-family proteins, i.e., Tcb1 and Tcb3, were colocalized with the ER-PM contacts in C. albicans. Deletion of the tricalbin-encoding genes TCB1 and TCB3 remarkably reduced ER-PM contacts, suggesting that tricalbins are ER-PM tethering proteins of C. albicans. Stress sensitivity assays showed that the TCB-deleted strains, including tcb1Δ/Δ, tcb3Δ/Δ, and tcb1Δ/Δ tcb3Δ/Δ, exhibited hypersensitivity to cell wall stress induced by caspofungin. Further investigation revealed that caspofungin induced drastic reactive oxygen species (ROS) accumulation in the mutants, which was attributed to enhanced oxidation of Ero1 in the ER lumen. Removal of intracellular ROS by the ROS scavenger vitamin C rescued the growth of the mutants under caspofungin treatment, indicating that Ero1 oxidation-related ROS accumulation was involved in caspofungin hypersensitivity of the mutants. Moreover, deletion of the TCB genes decreased secretion of extracellular aspartyl proteinases, reduced transport of the cell wall protein Hwp1 from the cytoplasm to the cell wall, and attenuated virulence of the fungal pathogen. This study sheds a light on the role of ER-PM tethering proteins in maintenance of cell wall integrity and virulence in fungal pathogens. IMPORTANCE The endoplasmic reticulum-plasma membrane contacts are important membrane contact structures in eukaryotic cells, functioning in material and message exchange between the ER and the PM. We observed that the two tricalbin-family endoplasmic reticulum-plasma membrane contact proteins are required for tolerance to caspofungin-induced cell wall stress in the pathogenic fungus Candida albicans. The tricalbin mutants exhibited hypersensitivity to cell wall stress induced by caspofungin. Further investigation revealed that Ero1 oxidation-related reactive species oxygen accumulation was involved in caspofungin hypersensitivity of the tricalbin mutants. Moreover, loss of tricalbins reduced secretion of extracellular aspartyl proteinases, decreased transport of the cell wall proteins from the cytoplasm to the cell wall, and attenuated virulence of the fungal pathogen. This study uncovers the role of ER-PM tethering proteins in sustaining protein secretion, maintenance of cell wall integrity and virulence in fungal pathogens.

CSIG-27. MODULATION OF GPR133 (ADGRD1) SIGNALING BY ITS INTRACELLULAR INTERACTION PARTNER EXTENDED SYNAPTOTAGMIN 1 (ESYT1)

Abstract GPR133 (ADGRD1), a member of the adhesion GPCR (aGPCR) family, has been recently implicated in the pathogenesis of glioblastoma. Like other aGPCRs, GPR133 is characterized by a large N-terminus, which possesses a conserved GPCR autoproteolysis-inducing (GAIN) domain catalyzing cleavage at a GPCR proteolysis site (GPS), resulting in a C-terminal fragment (CTF) and an N-terminal fragment (NTF). We showed that dissociation of the cleaved NTF and CTF at the plasma membrane correlates with increased GPR133 signaling, which is mediated by coupling to Gs and increase in cytosolic cAMP. Since GPR133 is absent in normal brain cells and de novo expressed in GBM, it represents a potential target for GBM treatment. However, little is known about the receptor’s protein interactome and its effects on receptor function and signaling. To identify intracellular interactors of GPR133, we used a proximity biotinylation/mass spectrometry approach in HEK293T cells expressing the wild-type or a signaling-incompetent mutant GPR133. We identified Extended Synaptotagmin 1 (ESYT1), a protein responsible for Ca2+-dependent formation of endoplasmic reticulum-plasma membrane bridges, as the strongest interaction partner for both versions of the receptor. Co-immunoprecipitation confirmed robust binding of ESYT1 to GPR133. Knockdown/knockout of ESYT1 increased GPR133 signaling, while overexpression of ESYT1 had the opposite effect. This effect was not mediated by changes in GPR133 expression or plasma membrane trafficking. We are currently performing additional studies to elucidate the mechanism whereby ESYT1 modulates GPR133 signaling. Collectively, these data suggest that GPR133 signaling is modulated through a novel cytosolic, signaling-independent interaction with ESYT1.

Nuanced Interactions between AKAP79 and STIM1 with Orai1 Ca2+ Channels at Endoplasmic Reticulum-Plasma Membrane Junctions Sustain NFAT Activation.

A-kinase anchoring protein 79 (AKAP79) is a human scaffolding protein that organizes Ca2+/calmodulin-dependent protein phosphatase calcineurin, calmodulin, cAMP-dependent protein kinase, protein kinase C, and the transcription factor nuclear factor of activated T cells (NFAT1) into a signalosome at the plasma membrane. Upon Ca2+ store depletion, AKAP79 interacts with the N-terminus of STIM1-gated Orai1 Ca2+ channels, enabling Ca2+ nanodomains to stimulate calcineurin. Calcineurin then dephosphorylates and activates NFAT1, which then translocates to the nucleus. A fundamental question is how signalosomes maintain long-term signaling when key effectors are released and therefore removed beyond the reach of the activating signal. Here, we show that the AKAP79-Orai1 interaction is considerably more transient than that of STIM1-Orai1. Free AKAP79, with calcineurin and NFAT1 in tow, is able to replace rapidly AKAP79 devoid of NFAT1 on Orai1, in the presence of continuous Ca2+ entry. We also show that Ca2+ nanodomains near Orai1 channels activate almost the entire cytosolic pool of NFAT1. Recycling of inactive NFAT1 from the cytoplasm to AKAP79 in the plasma membrane, coupled with the relatively weak interaction between AKAP79 and Orai1, maintain excitation-transcription coupling. By measuring rates for AKAP79-NFAT interaction, we formulate a mathematical model that simulates NFAT dynamics at the plasma membrane.

Chronic reduction of store operated Ca2+ entry is viable therapeutically but is associated with cardiovascular complications.

Loss of function mutations in store-operated Ca2+ entry (SOCE) are associated with severe paediatric disorders in humans, including combined immunodeficiency, anaemia, thrombocytopenia, anhidrosis and muscle hypotonia. Given its central role in immune cell activation, SOCE has been a therapeutic target for autoimmune and inflammatory diseases. Treatment for such chronic diseases would require prolonged SOCE inhibition. It is, however, unclear whether chronic SOCE inhibition is viable therapeutically. Here we address this issue using a novel genetic mouse model (SOCE hypomorph) with deficient SOCE, nuclear factor of activated T cells activation, and T cell cytokine production. SOCE hypomorph mice develop and reproduce normally and do not display muscle weakness or overt anhidrosis. They do, however, develop cardiovascular complications, including hypertension and tachycardia, which we show are due to increased sympathetic autonomic nervous system activity and not cardiac or vascular smooth muscle autonomous defects. These results assert that chronic SOCE inhibition is viable therapeutically if the cardiovascular complications can be managed effectively clinically. They further establish the SOCE hypomorph line as a genetic model to define the therapeutic window of SOCE inhibition and dissect toxicities associated with chronic SOCE inhibition in a tissue-specific fashion. KEY POINTS: A floxed stromal interaction molecule 1 (STIM1)hypomorph mouse model was generated with significant reduction in Ca2+ influx through store-operated Ca2+ entry (SOCE), resulting in defective nuclear translocation of nuclear factor of activated T cells, cytokine production and inflammatory response. The hypomorph mice are viable and fertile, with no overt defects. Decreased SOCE in the hypomorph mice is due to poor translocation of the mutant STIM1 to endoplasmic reticulum-plasma membrane contact sites resulting in fewer STIM1 puncta. Hypomorph mice have similar susceptibility to controls to develop diabetes but exhibit tachycardia and hypertension. The hypertension is not due to increased vascular smooth muscle contractility or vascular remodelling. The tachycardia is not due to heart-specific defects but rather seems to be due to increased circulating catecholamines in the hypomorph. Therefore, long term SOCE inhibition is viable if the cardiovascular defects can be managed clinically.

Unbiased proteomic and forward genetic screens reveal that mechanosensitive ion channel MSL10 functions at ER-plasma membrane contact sites in Arabidopsis thaliana.

Mechanosensitive (MS) ion channels are an evolutionarily conserved way for cells to sense mechanical forces and transduce them into ionic signals. The channel properties of Arabidopsis thaliana MscS-Like (MSL)10 have been well studied, but how MSL10 signals remains largely unknown. To uncover signaling partners of MSL10, we employed a proteomic screen and a forward genetic screen; both unexpectedly implicated endoplasmic reticulum-plasma membrane contact sites (EPCSs) in MSL10 function. The proteomic screen revealed that MSL10 associates with multiple proteins associated with EPCSs. Of these, only VAMP-associated proteins (VAP)27-1 and VAP27-3 interacted directly with MSL10. The forward genetic screen, for suppressors of a gain-of-function MSL10 allele (msl10-3G, MSL10S640L), identified mutations in the synaptotagmin (SYT)5 and SYT7 genes. We also found that EPCSs were expanded in leaves of msl10-3G plants compared to the wild type. Taken together, these results indicate that MSL10 associates and functions with EPCS proteins, providing a new cell-level framework for understanding MSL10 signaling. In addition, placing a mechanosensory protein at EPCSs provides new insight into the function and regulation of this type of subcellular compartment.

The resealing factor S100A11 interacts with annexins and extended synaptotagmin-1 in the course of plasma membrane wound repair

After damage, cells repair their plasma membrane in an active process that is driven by Ca2+ entering through the wound. This triggers a range of Ca2+-regulated events such as the translocation of different Ca2+-binding proteins to the wound site which likely function in the repair process. The translocated proteins include Ca2+/phospholipid binding proteins of the annexin (ANX) family and S100A11, an EF hand-type Ca2+-binding protein which can interact with ANX. The molecular mechanism by which S100A11 mediates PM wound repair remains poorly understood although it likely involves interactions with ANX. Here, using S100A11 knockout endothelial cells and expression of S100A11 mutants, we show that endothelial S100A11 is essential for efficient plasma membrane wound repair and engages in Ca2+-dependent interactions with ANXA1 and ANXA2 through its C-terminal extension (residues 93–105). ANXA2 but not ANXA1 translocation to the wound is substantially inhibited in the absence of S100A11; however, the repair defect in S100A11 knockout cells is rescued by ectopic expression of an ANX interaction-defective S100A11 mutant, suggesting an ANX-independent role of S100A11 in membrane wound repair. In search for other interaction partners that could mediate this action of S100A11 we identify extended synaptotagmin 1 (E-Syt1), a protein tether that regulates endoplasmic reticulum-plasma membrane contact sites. E-Syt1 binds to S100A11 in the presence of Ca2+ and depletion of E-Syt1 interferes with wound site recruitment of S100A11 and proper membrane resealing. Thus, the role of S100A11 in membrane wound repair does not exclusively dependent on ANX interactions and a Ca2+-regulated S100A11-E-Syt1 complex acts as a yet unrecognized component of the membrane resealing machinery.

A partnership between the lipid scramblase XK and the lipid transfer protein VPS13A at the plasma membrane

Chorea-acanthocytosis (ChAc) and McLeod syndrome are diseases with shared clinical manifestations caused by mutations in VPS13A and XK, respectively. Key features of these conditions are the degeneration of caudate neurons and the presence of abnormally shaped erythrocytes. XK belongs to a family of plasma membrane (PM) lipid scramblases whose action results in exposure of PtdSer at the cell surface. VPS13A is an endoplasmic reticulum (ER)-anchored lipid transfer protein with a putative role in the transport of lipids at contacts of the ER with other membranes. Recently VPS13A and XK were reported to interact by still unknown mechanisms. So far, however, there is no evidence for a colocalization of the two proteins at contacts of the ER with the PM, where XK resides, as VPS13A was shown to be localized at contacts between the ER and either mitochondria or lipid droplets. Here we show that VPS13A can also localize at ER-PM contacts via the binding of its PH domain to a cytosolic loop of XK, that such interaction is regulated by an intramolecular interaction within XK, and that both VPS13A and XK are highly expressed in the caudate neurons. Binding of the PH domain of VPS13A to XK is competitive with its binding to intracellular membranes that mediate other tethering functions of VPS13A. Our findings support a model according to which VPS13A-dependent lipid transfer between the ER and the PM is coupled to lipid scrambling within the PM. They raise the possibility that defective cell surface exposure of PtdSer may be responsible for neurodegeneration.

The ultrastructural organization of endoplasmic reticulum-plasma membrane contacts is conserved in epithelial cells.

Contacts between the endoplasmic reticulum and the plasma membrane (ER-PM contacts) have important roles in membrane lipid and calcium dynamics, yet their organization in polarized epithelial cells has not been thoroughly described. Here we examine ER-PM contacts in hepatocytes in mouse liver using electron microscopy, providing the first comprehensive ultrastructural study of ER-PM contacts in a mammalian epithelial tissue. Our quantitative analyses reveal strikingly distinct ER-PM contact architectures spatially linked to apical, lateral, and basal PM domains. Notably, we find that an extensive network of ER-PM contacts exists at lateral PM domains that form intercellular junctions between hepatocytes. Moreover, the spatial organization of ER-PM contacts is conserved in epithelial spheroids, suggesting that ER-PM contacts may serve conserved roles in epithelial cell architecture. Consistent with this notion, we show that ORP5 activity at ER-PM contacts modulates the apical-basolateral aspect ratio in HepG2 cells. Thus ER-PM contacts have a conserved distribution and crucial roles in PM domain architecture across epithelial cell types.

Validated Growth Rate-Dependent Regulation of Lipid Metabolism in Yarrowia lipolytica.

Given the strong potential of Yarrowia lipolytica to produce lipids for use as renewable fuels and oleochemicals, it is important to gain in-depth understanding of the molecular mechanism underlying its lipid accumulation. As cellular growth rate affects biomass lipid content, we performed a comparative proteomic analysis of Y. lipolytica grown in nitrogen-limited chemostat cultures at different dilution rates. After confirming the correlation between growth rate and lipid accumulation, we were able to identify various cellular functions and biological mechanisms involved in oleaginousness. Inspection of significantly up- and downregulated proteins revealed nonintuitive processes associated with lipid accumulation in this yeast. This included proteins related to endoplasmic reticulum (ER) stress, ER–plasma membrane tether proteins, and arginase. Genetic engineering of selected targets validated that some genes indeed affected lipid accumulation. They were able to increase lipid content and were complementary to other genetic engineering strategies to optimize lipid yield.

Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia

SUMMARYActivated microglia release extracellular vesicles (EVs) as modulators of brain homeostasis and innate immunity. However, the molecules critical for regulating EV production from microglia are poorly understood. Here we establish a murine microglial cell model to monitor EV secretion by measuring the fluorescence signal of tdTomato, which is linked to tetraspanin CD63. Stimulation of tdTomato+ cells with ATP induces rapid secretion of EVs and a reduction in cellular tdTomato intensity, reflecting EV secretion. We generate a GFP+ tdTomato+ cell library expressing TurboGFP and barcoded short hairpin RNAs for genome-wide screening using next-generation sequencing. We identify Mcfd2, Sepp1, and Sdc1 as critical regulators of ATP-induced EV secretion from murine microglia. Small interfering RNA (siRNA-based) silencing of each of these genes suppresses lipopolysaccharide- and ATP-induced inflammasome activation, as determined by interleukin-1β release from primary cultured murine microglia. These molecules are critical for microglial EV secretion and are potential therapeutic targets for neuroinflammatory disorders.