Lipid droplets are found in all cell types. Normally present at low levels in the brain, they accumulate in tumours and are associated with neurodegenerative diseases. However, little is known about the mechanisms controlling their homeostasis in the brain. We found that GRAF1a, the longest GRAF1 isoform (GRAF1 is also known as ARHGAP26), was enriched in the brains of neonates. Endogenous GRAF1a was found on lipid droplets in oleic-acid-fed primary glial cells. Exclusive localization required a GRAF1a-specific hydrophobic segment and two membrane-binding regions, a BAR and a PH domain. Overexpression of GRAF1a promoted lipid droplet clustering, inhibited droplet mobility and severely perturbed lipolysis following the chase of cells overloaded with fatty acids. Under these conditions, GRAF1a concentrated at the interface between lipid droplets. Although GRAF1-knockout mice did not show any gross abnormal phenotype, the total lipid droplet volume that accumulated in GRAF1(-/-) primary glia upon incubation with fatty acids was reduced compared to GRAF1(+/+) cells. These results provide additional insights into the mechanisms contributing to lipid droplet growth in non-adipocyte cells, and suggest that proteins with membrane sculpting BAR domains play a role in droplet homeostasis.

The ErbB2 receptor is a clinically validated cancer target whose internalization and trafficking mechanisms remain poorly understood. HSP90 inhibitors, such as geldanamycin (GA), have been developed to target the receptor to degradation or to modulate downstream signaling. Despite intense investigations, the entry route and postendocytic sorting of ErbB2 upon GA stimulation have remained controversial. We report that ErbB2 levels inversely impact cell clathrin-mediated endocytosis (CME) capacity. Indeed, the high levels of the receptor are responsible for its own low internalization rate. GA treatment does not directly modulate ErbB2 CME rate but it affects ErbB2 recycling fate, routing the receptor to modified multivesicular endosomes (MVBs) and lysosomal compartments, by perturbing early/recycling endosome structure and sorting capacity. This activity occurs irrespective of the cargo interaction with HSP90, as both ErbB2 and the constitutively recycled, HSP90-independent, transferrin receptor are found within modified endosomes, and within aberrant, elongated recycling tubules, leading to modified MVBs/lysosomes. We propose that GA, as part of its anticancer activity, perturbs early/recycling endosome sorting, routing recycling cargoes toward mixed endosomal compartments.

Cell-to-cell fusion plays an important role in normal physiology and in different pathological conditions. Early fusion stages mediated by specialized proteins and yielding fusion pores are followed by a pore expansion stage that is dependent on cell metabolism and yet unidentified machinery. Because of a similarity of membrane bending in the fusion pore rim and in highly curved intracellular membrane compartments, in the present study we explored whether changes in the activity of the proteins that generate these compartments affect cell fusion initiated by protein fusogens of influenza virus and baculovirus. We raised the intracellular concentration of curvature-generating proteins in cells by either expressing or microinjecting the ENTH (epsin N-terminal homology) domain of epsin or by expressing the GRAF1 (GTPase regulator associated with focal adhesion kinase 1) BAR (Bin/amphiphysin/Rvs) domain or the FCHo2 (FCH domain-only protein 2) F-BAR domain. Each of these treatments promoted syncytium formation. Cell fusion extents were also influenced by treatments targeting the function of another curvature-generating protein, dynamin. Cell-membrane-permeant inhibitors of dynamin GTPase blocked expansion of fusion pores and dominant-negative mutants of dynamin influenced the syncytium formation extents. We also report that syncytium formation is inhibited by reagents lowering the content and accessibility of PtdIns(4,5)P(2), an important regulator of intracellular membrane remodelling. Our findings indicate that fusion pore expansion at late stages of cell-to-cell fusion is mediated, directly or indirectly, by intracellular membrane-shaping proteins.

Adipose tissue plays a major role in regulating whole-body insulin sensitivity and energy metabolism. To accommodate surplus energy, the tissue rapidly expands by increasing adipose cell size (hypertrophy) and cell number (hyperplasia). Previous studies have shown that enlarged, hypertrophic adipocytes are less responsive to insulin, and that adipocyte size could serve as a predictor for the development of type 2 diabetes. In the present study, we demonstrate that changes in adipocyte size correlate with a drastic remodeling of the actin cytoskeleton. Expansion of primary adipocytes following 2 weeks of high-fat diet (HFD)-feeding in C57BL6/J mice was associated with a drastic increase in filamentous (F)-actin as assessed by fluorescence microscopy, increased Rho-kinase activity, and changed expression of actin-regulating proteins, favoring actin polymerization. At the same time, increased cell size was associated with impaired insulin response, while the interaction between the cytoskeletal scaffolding protein IQGAP1 and insulin receptor substrate (IRS)-1 remained intact. Reversed feeding from HFD to chow restored cell size, insulin response, expression of actin-regulatory proteins and decreased the amount of F-actin filaments. Together, we report a drastic cytoskeletal remodeling during adipocyte expansion, a process which could contribute to deteriorating adipocyte function.

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor ACE2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors may reduce but not abolish viral spread.

Diets enriched in sucrose severely impair metabolic regulation and are associated with obesity, insulin resistance and glucose intolerance. In the current study, we investigated the effect of 4 weeks high-sucrose diet (HSD) feeding in C57BL6/J mice, with specific focus on adipocyte function. Mice fed HSD had slightly increased adipose tissue mass but displayed similar hepatic triglycerides, glucose and insulin levels, and glucose clearance capacity as chow-fed mice. Interestingly, we found adipose depot-specific differences, where both the non- and insulin-stimulated glucose transports were markedly impaired in primary adipocytes isolated from the inguinal fat depot from HSD-fed mice. This was accompanied by decreased protein levels of both GLUT4 and AS160. A similar but much less pronounced trend was observed in the retroperitoneal depot. In contrast, both GLUT4 expression and insulin-stimulated glucose uptake were preserved in adipocytes isolated from epididymal adipose tissue with HSD. Further, we found a slight shift in cell size distribution towards larger cells with HSD and a significant decrease of ACC and PGC-1α expression in the inguinal adipose tissue depot. Moreover, fructose alone was sufficient to decrease GLUT4 expression in cultured, mature adipocytes. Altogether, we demonstrate that short-term HSD feeding has deleterious impact on insulin response and glucose transport in the inguinal adipose tissue depot, specifically. These changes occur before the onset of systemic glucose dysmetabolism and therefore could provide a mechanistic link to overall impaired energy metabolism reported after prolonged HSD feeding, alone or in combination with HFD.

Caveolae are small membrane invaginations that generally are stably attached to the plasma membrane. Their release is believed to depend on the GTPase dynamin 2 (Dyn2), in analogy with its role in fission of clathrin-coated vesicles. The mechanistic understanding of caveola fission is, however, sparse. Here, we used microscopy-based tracking of individual caveolae in living cells to determine the role of Dyn2 in caveola dynamics. We report that Dyn2 stably associated with the bulb of a subset of caveolae, but was not required for formation or fission of caveolae. Dyn2-positive caveolae displayed longer plasma membrane duration times, whereas depletion of Dyn2 resulted in shorter duration times and increased caveola fission. The stabilizing role of Dyn2 was independent of its GTPase activity and the caveola stabilizing protein EHD2. Thus, we propose that, in contrast to the current view, Dyn2 is not a core component of the caveolae machinery, but rather functions as an accessory protein that restrains caveola internalization.

Dysregulation of autophagy has been observed in obesity and type 2 diabetes. Salt-inducible kinase 2 (SIK2), a member of the AMPK-related kinase family, is downregulated in adipocytes from obese or insulin resistant individuals and was previously demonstrated to regulate autophagy in cancer and normal cell lines. The aim of this study was thus to investigate a potential role of SIK2 in the regulation of adipocyte autophagy. To do so, SIK2 siRNA silencing or SIKs pharmacological inhibition of SIK2 was employed in murine differentiated 3T3-L1 adipocytes and autophagic flux was monitored. Our data indicate that SIK2 is required for both autophagic flux and expression of TFEB, the transcription factor that regulates autophagy, in adipocytes. The effect of SIK2 on autophagic flux occurs before the regulation of TFEB protein levels, suggesting different mechanisms whereby SIK2 stimulates autophagy. This study broadens the current knowledge on autophagy regulation and SIK2 function in adipocytes.

Membrane scission is essential for intracellular trafficking. While BAR domain proteins such as endophilin have been reported in dynamin-independent scission of tubular membrane necks, the cutting mechanism has yet to be deciphered. Here, we combine a theoretical model, in vitro, and in vivo experiments revealing how protein scaffolds may cut tubular membranes. We demonstrate that the protein scaffold bound to the underlying tube creates a frictional barrier for lipid diffusion; tube elongation thus builds local membrane tension until the membrane undergoes scission through lysis. We call this mechanism friction-driven scission (FDS). In cells, motors pull tubes, particularly during endocytosis. Through reconstitution, we show that motors not only can pull out and extend protein-scaffolded tubes but also can cut them by FDS. FDS is generic, operating even in the absence of amphipathic helices in the BAR domain, and could in principle apply to any high-friction protein and membrane assembly.

Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.

In addition to the classical pathway of secretion, some transmembrane proteins reach the plasma membrane through alternative routes. Several proteins transit through endosomes and are exported in a Rab8-, Rab10-, and/or Rab11-dependent manner. GRAFs are membrane-binding proteins associated with tubules and vesicles. We found extensive colocalization of GRAF1b/2 with Rab8a/b and partial with Rab10. We identified MICAL1 and WDR44 as direct GRAF-binding partners. MICAL1 links GRAF1b/2 to Rab8a/b and Rab10, and WDR44 binds Rab11. Endogenous WDR44 labels a subset of tubular endosomes, which are closely aligned with the ER via binding to VAPA/B. With its BAR domain, GRAF2 can tubulate membranes, and in its absence WDR44 tubules are not observed. We show that GRAF2 and WDR44 are essential for the export of neosynthesized E-cadherin, MMP14, and CFTR ΔF508, three proteins whose exocytosis is sensitive to ER stress. Overexpression of dominant negative mutants of GRAF1/2, WDR44, and MICAL1 also interferes with it, facilitating future studies of Rab8/10/11-dependent exocytic pathways of central importance in biology.

Plague, one of the most devastating diseases in human history, is caused by the bacterium Yersinia pestis. The bacteria use a syringe-like macromolecular assembly to secrete various toxins directly into the host cells they infect. One such Yersinia outer protein, YopJ, performs the task of dampening innate immune responses in the host by simultaneously inhibiting the MAPK and NFkappaB signaling pathways. YopJ catalyzes the transfer of acetyl groups to serine, threonine, and lysine residues on target proteins. Acetylation of serine and threonine residues prevents them from being phosphorylated thereby preventing the activation of signaling molecules on which they are located. In this study, we describe the requirement of a host-cell factor for full activation of the acetyltransferase activity of YopJ and identify this activating factor to be inositol hexakisphosphate (IP(6)). We extend the applicability of our results to show that IP(6) also stimulates the acetyltransferase activity of AvrA, the YopJ homologue from Salmonella typhimurium. Furthermore, an IP(6)-induced conformational change in AvrA suggests that IP(6) acts as an allosteric activator of enzyme activity. Our results suggest that YopJ-family enzymes are quiescent in the bacterium where they are synthesized, because bacteria lack IP(6); once injected into mammalian cells by the pathogen these toxins bind host cell IP(6), are activated, and deregulate the MAPK and NFkappaB signaling pathways thereby subverting innate immunity.

The rho GTPase-activating protein GTPase regulator associated with focal adhesion kinase-1 (GRAF1) remodels membranes into tubulovesicular clathrin-independent carriers (CLICs) mediating lipid-anchored receptor endocytosis. However, the cell biological functions of this highly prevalent endocytic pathway are unclear. In this article, we present biochemical and cell biological evidence that GRAF1 interacted with a network of endocytic and adhesion proteins and was found enriched at podosome-like adhesions and src-induced podosomes. We further demonstrate that these sites comprise microdomains of highly ordered lipid enriched in GRAF1 endocytic cargo. GRAF1 activity was upregulated in spreading cells and uptake via CLICs was concentrated at the leading edge of migrating cells. Depletion of GRAF1, which inhibits CLIC generation, resulted in profound defects in cell spreading and migration. We propose that GRAF1 remodels membrane microdomains at adhesion sites into endocytic carriers, facilitating membrane turnover during cell morphological changes.

Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission.

Eps15 homology domain-containing 2 (EHD2) belongs to the EHD-containing protein family of dynamin-related ATPases involved in membrane remodeling in the endosomal system. EHD2 dimers oligomerize into rings on highly curved membranes, resulting in stimulation of the intrinsic ATPase activity. In this paper, we report that EHD2 is specifically and stably associated with caveolae at the plasma membrane and not involved in clathrin-mediated endocytosis or endosomal recycling, as previously suggested. EHD2 interacts with pacsin2 and cavin1, and ordered membrane assembly of EHD2 is dependent on cavin1 and caveolar integrity. While the EHD of EHD2 is dispensable for targeting, we identified a loop in the nucleotide-binding domain that, together with ATP binding, is required for caveolar localization. EHD2 was not essential for the formation or shaping of caveolae, but high levels of EHD2 caused distortion and loss of endogenous caveolae. Assembly of EHD2 stabilized and constrained caveolae to the plasma membrane to control turnover, and depletion of EHD2, resulting in endocytic and more dynamic and short-lived caveolae. Thus, following the identification of caveolin and cavins, EHD2 constitutes a third structural component of caveolae involved in controlling the stability and turnover of this organelle.

The present work addressed the hypothesis that NG2/CSPG4, CD146/MCAM, and VAP1/AOC3 are target genes of myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MKL1, MRTF-B/MKL2) and serum response factor (SRF). Using a bioinformatics approach, we found that CSPG4, MCAM, and AOC3 correlate with MYOCD, MRTF-A/MKL1, and SRF across human tissues. No other transcription factor correlated as strongly with these transcripts as SRF. Overexpression of MRTFs increased both mRNA and protein levels of CSPG4, MCAM, and AOC3 in cultured human smooth muscle cells (SMCs). Imaging confirmed increased staining for CSPG4, MCAM, and AOC3 in MRTF-A/MKL1-transduced cells. MRTFs exert their effects through SRF, and the MCAM and AOC3 gene loci contained binding sites for SRF. SRF silencing reduced the transcript levels of these genes, and time-courses of induction paralleled the direct target ACTA2. MRTF-A/MKL1 increased the activity of promoter reporters for MCAM and AOC3, and transcriptional activation further depended on the chromatin remodeling enzyme KDM3A. CSPG4, MCAM, and AOC3 responded to the MRTF-SRF inhibitor CCG-1423, to actin dynamics, and to ternary complex factors. Coincidental detection of these proteins should reflect MRTF-SRF activity, and beyond SMCs, we observed co-expression of CD146/MCAM, NG2/CSPG4, and VAP1/AOC3 in pericytes and endothelial cells in the human brain. This work identifies highly responsive vascular target genes of MRTF-SRF signaling that are regulated via a mechanism involving KDM3A.

To accommodate surplus energy, the adipose tissue expands by increasing adipocyte size (hypertrophy) and number (hyperplasia). The presence of hypertrophic adipocytes is a key characteristic of adipose tissue dysfunction. High-fat diet (HFD) fed C57BL/6J mice are a commonly used model to study obesity and obesity-related complications. In the present study, we have characterized adipose plasticity, at both the cellular and tissue level, by examining the temporal development of systemic insulin resistance and adiposity in response to HFD-feeding for 4, 8, and 12 weeks (4w, 8w, and 12w). Within the same time frame, we examined systemic metabolic flexibility and adipose plasticity when switching from HFD- to chow-diet during the last 2 weeks of diet intervention (referred to as the reverse (REV) group: 4wREV (2w HFD+2w chow), 8wREV (6w HFD+2w chow), 12wREV (10w HFD+2w chow)). In response to HFD-feeding over time, the 12w group had impaired systemic insulin sensitivity compared to both the 4w and 8w groups, accompanied by an increase in hypertrophic inguinal adipocytes and liver triglycerides. After reversing from HFD- to chow-feeding, most parameters were completely restored to chow control levels for 4wREV and 8wREV groups. In contrast, the 12wREV group had a significantly increased number of hypertrophic adipocytes, liver triglycerides accumulation, and impaired systemic insulin sensitivity compared to chow-fed mice. Further, image analysis at the single-cell level revealed a cell-size dependent organization of actin filaments for all feeding conditions. Indeed, the impaired adipocyte size plasticity in the 12wREV group was accompanied by increased actin filamentation and reduced insulin-stimulated glucose uptake compared with chow-fed mice. In summary, these results demonstrate that the C57BL/6J HFD-feeding model has a large capacity to restore adipocyte cell size and systemic insulin sensitivity, and that a metabolic tipping point occurs between 8 and 12w of HFD-feeding where this plasticity deteriorates. We believe these findings provide substantial understanding of C57BL/6J mice as an obesity model, and that an increased pool of hypertrophic ING adipocytes could contribute to aggravated insulin resistance.

Myocardin related transcription factors (MRTFs: MYOCD/myocardin, MRTF-A, and MRTF-B) play a key role in smooth muscle cell differentiation by activating contractile genes. In atherosclerosis, MRTF levels change, and most notable is a fall of MYOCD. Previous work described anti-inflammatory properties of MRTF-A and MYOCD, occurring through RelA binding, suggesting that MYOCD reduction could contribute to vascular inflammation. Recent studies have muddled this picture showing that MRTFs may show both anti- and pro-inflammatory properties, but the basis of these discrepancies remain unclear. Moreover, the impact of MRTFs on inflammatory signaling pathways in tissues relevant to human arterial disease is uncertain. The current work aimed to address these issues. RNA-sequencing after forced expression of myocardin in human coronary artery smooth muscle cells (hCASMCs) showed reduction of pro-inflammatory transcripts, including CCL2, CXCL8, IL6, and IL1B. Side-by-side comparison of MYOCD, MRTF-A, and MRTF-B in hCASMCs, showed that the anti-inflammatory impact was shared among MRTFs. Correlation analyses using human arterial transcriptomic datasets revealed negative correlations between MYOCD, MRTFA, and SRF, on the one hand, and the inflammatory transcripts, on the other. A pro-inflammatory drive from lipopolysaccharide, did not change the size of the suppressive effect of MRTF-A in hCASMCs on either mRNA or protein levels. To examine cell type-dependence, we compared the anti-inflammatory impact in hCASMCs, with that in human bladder SMCs, in endothelial cells, and in monocytes (THP-1 cells). Surprisingly, little anti-inflammatory activity was seen in endothelial cells and monocytes, and in bladder SMCs, MRTF-A was pro-inflammatory. CXCL8, IL6, and IL1B were increased by the MRTF-SRF inhibitor CCG-1423 and by MRTF-A silencing in hCASMCs, but depolymerization of actin, known to inhibit MRTF activity, had no stimulatory effect, an exception being IL1B. Co-immunoprecipitation supported binding of MRTF-A to RelA, supporting sequestration of this important pro-inflammatory mediator as a mechanism. Dexamethasone treatment and silencing of RelA (by 76 ± 1%) however only eliminated a fraction of the MRTF-A effect (≈25%), suggesting mechanisms beyond RelA binding. Indeed, SRF silencing suggested that MRTF-A suppression of IL1B and CXCL8 depends on SRF. This work thus supports an anti-inflammatory impact of MRTF-SRF signaling in hCASMCs and in intact human arteries, but not in several other cell types.

Adipocytes play a central role in energy balance, and dysfunctional adipose tissue severely affects systemic energy homeostasis. The ATPase EH domain-containing 2 (EHD2) has previously been shown to regulate caveolae, plasma membrane-specific domains that are involved in lipid uptake and signal transduction. Here, we investigated the role of EHD2 in adipocyte function. We demonstrate that EHD2 protein expression is highly up-regulated at the onset of triglyceride accumulation during adipocyte differentiation. Small interfering RNA-mediated EHD2 silencing affected the differentiation process and impaired insulin sensitivity, lipid storage capacity, and lipolysis. Fluorescence imaging revealed localization of EHD2 to caveolae, close to cell surface-associated lipid droplets in primary human adipocytes. These lipid droplets stained positive for glycerol transporter aquaporin 7 and phosphorylated perilipin-1 following adrenergic stimulation. Further, EHD2 overexpression in human adipocytes increased the lipolytic signaling and suppressed the activity of transcription factor PPARγ. Overall, these data suggest that EHD2 plays a key role for adipocyte function.

Expression of Eph receptors and their ligands, the ephrins, have important functions in boundary formation and morphogenesis in both adult and embryonic tissue. The EphB receptors and ephrinB ligands are transmembrane proteins that are expressed in different cells and their interaction drives cell repulsion. For cell repulsion to occur, trans-endocytosis of the inter-cellular receptor-ligand EphB-ephrinB complex is required. The molecular mechanism underlying trans-endocytosis is poorly defined. Here we show that the process is clathrin- and Eps15R-mediated using Co115 colorectal cell lines stably expressing EphB2 and ephrinB1. Cell repulsion in co-cultures of EphB2- and ephrinB1-expressing cells is significantly reduced by knockdown of Eps15R but not Eps15. A novel interaction motif in Eps15R, DPFxxLDPF, is shown to bind directly to the clathrin terminal domain in vitro. Moreover, the interaction between Eps15R and clathrin is required for EphB2-mediated cell repulsion as shown in a rescue experiment in the EphB2 co-culture assay where wild type Eps15R but not the clathrin-binding mutant rescues cell repulsion. These results provide the first evidence that Eps15R together with clathrin control EphB/ephrinB trans-endocytosis and thereby cell repulsion.