Clathrin Research Articles

Characterization of Mayaro virus (strain BeAn343102) biology in vertebrate and invertebrate cellular backgrounds.

Mayaro virus (MAYV) is an emerging New World alphavirus (genus Alphavirus, family Togaviridae) that causes acute multiphasic febrile illness, skin rash, polyarthritis and occasional severe clinical phenotypes. The virus lifecycle alternates between invertebrate and vertebrate hosts. Here we characterize the replication features, cell entry, lifecycle and virus-related cell pathology of MAYV using vertebrate and invertebrate in vitro models. Electron-dense clathrin-coated pits in infected cells and reduced viral production in the presence of dynasore, ammonium chloride and bafilomycin indicate that viral entry occurs through pH-dependent endocytosis. Increase in FITC-dextran uptake (an indicator of macropinocytosis) in MAYV-infected cells, and dose-dependent infection inhibition by 5-(N-ethyl-N-isopropyl) amiloride (a macropinocytosis inhibitor), indicated that macropinocytosis is an additional entry mechanism of MAYV in vertebrate cells. Acutely infected vertebrate and invertebrate cells formed cytoplasmic or membrane-associated extracytoplasmic replication complexes. Mosquito cells showed modified hybrid cytoplasmic vesicles that supported virus replication, nucleocapsid production and maturation. Mature virus particles were released from cells by both exocytosis and budding from the cell membrane. MAYV replication was cytopathic and associated with induction of apoptosis by the intrinsic pathway, and later by the extrinsic pathway in infected vertebrate cells. Given that MAYV is expanding its geographical existence as a potential public health problem, this study lays the foundation for biological understanding that will be valuable for therapeutic and preventive interventions.

Gp35/50 mucin molecules of Trypanosoma cruzi metacyclic forms that mediate host cell invasion interact with annexin A2.

Host cell invasion is a critical step for infection by Trypanosoma cruzi, the agent of Chagas disease. In natural infection, T. cruzi metacyclic trypomastigote (MT) forms establish the first interaction with host cells. The gp35/50 mucin molecules expressed in MT have been implicated in cell invasion process, but the mechanisms involved are not well understood. We performed a series of experiments to elucidate the mode of gp35/50-mediated MT internalization. Comparing two parasite strains from genetically divergent groups, G strain (TcI) and CL strain (TcVI), expressing variant forms of mucins, we demonstrated that G strain mucins participate in MT invasion. Only G strain-derived mucins bound to HeLa cells in a receptor-dependent manner and significantly inhibited G strain MT invasion. CL strain MT internalization was not affected by mucins from either strain. HeLa cell invasion by G strain MT was associated with actin recruitment and did not rely on lysosome mobilization. To examine the involvement of annexin A2, which plays a role in actin dynamic, annexin A2-depleted HeLa cells were generated. Annexin A2-deficient cell lines were significantly more resistant than wild type controls to G strain MT invasion. In a co-immunoprecipitation assay, to check whether annexin A2 might be the receptor for mucins, protein A/G magnetic beads crosslinked with monoclonal antibody to G strain mucins were incubated with detergent extracts of MT and HeLa cells. Binding of gp35/50 mucins to annexin A2 was detected. Both G strain MT and purified mucins induced focal adhesion kinase activation in HeLa cells. By confocal immunofluorescence microscopy, colocalization of invading G strain MT with clathrin was visualized. Inhibition of clathrin-coated vesicle formation reduced parasite internalization. Taken together, our data indicate that gp35/50-mediated MT invasion is accomplished through interaction with host cell annexin A2 and clathrin-dependent endocytosis.

Endocytosis at extremes: Formation and internalization of giant clathrin-coated pits under elevated membrane tension.

Internalization of clathrin-coated vesicles from the plasma membrane constitutes the major endocytic route for receptors and their ligands. Dynamic and structural properties of endocytic clathrin coats are regulated by the mechanical properties of the plasma membrane. Here, we used conventional fluorescence imaging and multiple modes of structured illumination microscopy (SIM) to image formation of endocytic clathrin coats within live cells and tissues of developing fruit fly embryos. High resolution in both spatial and temporal domains allowed us to detect and characterize distinct classes of clathrin-coated structures. Aside from the clathrin pits and plaques detected in distinct embryonic tissues, we report, for the first time, formation of giant coated pits (GCPs) that can be up to two orders of magnitude larger than the canonical pits. In cultured cells, we show that GCP formation is induced by increased membrane tension. GCPs take longer to grow but their mechanism of curvature generation is the same as the canonical pits. We also demonstrate that GCPs split into smaller fragments during internalization. Considering the supporting roles played by actin filament dynamics under mechanically stringent conditions that slow down completion of clathrin coats, we suggest that local changes in the coat curvature driven by actin machinery can drive splitting and internalization of GCPs.

Three-dimensional visualization of planta clathrin-coated vesicles at ultrastructural resolution

Biological systems are the sum of their dynamic three-dimensional (3D) parts. Therefore, it is critical to study biological structures in 3D and at high resolution to gain insights into their physiological functions. Electron microscopy of metal replicas of unroofed cells and isolated organelles has been a key technique to visualize intracellular structures at nanometer resolution. However, many of these methods require specialized equipment and personnel to complete them. Here, we present novel accessible methods to analyze biological structures in unroofed cells and biochemically isolated organelles in 3D and at nanometer resolution, focusing on Arabidopsis clathrin-coated vesicles (CCVs). While CCVs are essential trafficking organelles, their detailed structural information is lacking due to their poor preservation when observed via classical electron microscopy protocols experiments. First, we establish a method to visualize CCVs in unroofed cells using scanning transmission electron microscopy tomography, providing sufficient resolution to define the clathrin coat arrangements. Critically, the samples are prepared directly on electron microscopy grids, removing the requirement to use extremely corrosive acids, thereby enabling the use of this method in any electron microscopy lab. Secondly, we demonstrate that this standardized sample preparation allows the direct comparison of isolated CCV samples with those visualized in cells. Finally, to facilitate the high-throughput and robust screening of metal replicated samples, we provide a deep learning analysis method to screen the “pseudo 3D” morphologies of CCVs imaged with 2D modalities. Collectively, our work establishes accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs.

VAMP4 regulates insulin levels by targeting secretory granules to lysosomes.

Insulin levels are essential for the maintenance of glucose homeostasis, and deviations lead to pathoglycemia or diabetes. However, the metabolic mechanism controlling insulin quantity and quality is poorly understood. In pancreatic β cells, insulin homeostasis and release are tightly governed by insulin secretory granule (ISG) trafficking, but the required regulators and mechanisms are largely unknown. Here, we identified that VAMP4 controlled the insulin levels in response to glucose challenge. VAMP4 deficiency led to increased blood insulin levels and hyperresponsiveness to glucose. In β cells, VAMP4 is packaged into immature ISGs (iISGs) at trans-Golgi networks and subsequently resorted to clathrin-coated vesicles during granule maturation. VAMP4-positive iISGs and resorted vesicles then fuse with lysosomes facilitated by a SNARE complex consisting of VAMP4, STX7, STX8, and VTI1B, which ensures the breakdown of excess (pro)insulin and obsolete materials and thus maintenance of intracellular insulin homeostasis. Thus, VAMP4 is a key factor regulating the insulin levels and a potential target for the treatment of diabetes.

Structure Composition and Intracellular Transport of Clathrin-Mediated Intestinal Transmembrane Tight Junction Protein.

Tight junctions (TJs) are located in the apical region of the junctions between epithelial cells and are widely found in organs such as the brain, retina, intestinal epithelium, and endothelial system. As a mechanical barrier of the intestinal mucosa, TJs can not only maintain the integrity of intestinal epithelial cells but also maintain intestinal mucosal permeability by regulating the entry of ions and molecules into paracellular channels. Therefore, the formation disorder or integrity destruction of TJs can induce damage to the intestinal epithelial barrier, ultimately leading to the occurrence of various gastrointestinal diseases, such as inflammatory bowel disease (IBD), gastroesophageal reflux disease (GERD), and irritable bowel syndrome (IBS). However, a large number of studies have shown that TJs protein transport disorder from the endoplasmic reticulum to the apical membrane can lead to TJs formation disorder, in addition to disruption of TJs integrity caused by external pathological factors and reduction of TJs protein synthesis. In this review, we focus on the structural composition of TJs, the formation of clathrin-coated vesicles containing transmembrane TJs from the Golgi apparatus, and the transport process from the Golgi apparatus to the plasma membrane via microtubules and finally fusion with the plasma membrane. At present, the mechanism of the intracellular transport of TJ proteins remains unclear. More studies are needed in the future to focus on the sorting of TJs protein vesicles, regulation of transport processes, and recycling of TJ proteins, etc.

Eps15/Pan1p is a master regulator of the late stages of the endocytic pathway.

Endocytosis is a multistep process involving the sequential recruitment and action of numerous proteins. This process can be divided into two phases: an early phase, in which sites of endocytosis are formed, and a late phase in which clathrin-coated vesicles are formed and internalized into the cytosol, but how these phases link to each other remains unclear. In this study, we demonstrate that anchoring the yeast Eps15-like protein Pan1p to the peroxisome triggers most of the events occurring during the late phase at the peroxisome. At this ectopic location, Pan1p recruits most proteins that function in the late phases-including actin nucleation promoting factors-and then initiates actin polymerization. Pan1p also recruited Prk1 kinase and actin depolymerizing factors, thereby triggering disassembly immediately after actin assembly and inducing dissociation of endocytic proteins from the peroxisome. These observations suggest that Pan1p is a key regulator for initiating, processing, and completing the late phase of endocytosis.

Regulation of membrane scission in yeast endocytosis.

During clathrin-mediated endocytosis, a flat plasma membrane is shaped into an invagination that undergoes scission to form a vesicle. In mammalian cells, the force that drives the transition from invagination to vesicle is primarily provided by the GTPase dynamin that acts in concert with crescent-shaped BAR domain proteins. In yeast cells, the mechanism of endocytic scission is unclear. The yeast BAR domain protein complex Rvs161/167 (Rvs) nevertheless plays an important role in this process: deletion of Rvs dramatically reduces scission efficiency. A mechanistic understanding of the influence of Rvs on scission, however, remains incomplete. We used quantitative live-cell imaging and genetic manipulation to understand the recruitment and function of Rvs and other late-stage proteins at yeast endocytic sites. We found that arrival of Rvs at endocytic sites is timed by interaction of its BAR domain with specific membrane curvature. A second domain of Rvs167-the SH3 domain-affects localization efficiency of Rvs. We show that Myo3, one of the two type-I myosins in Saccharomyces cerevisiae, has a role in recruiting Rvs167 via the SH3 domain. Removal of the SH3 domain also affects assembly and disassembly of actin and impedes membrane invagination. Our results indicate that both BAR and SH3 domains are important for the role of Rvs as a regulator of scission. We tested other proteins implicated in vesicle formation in S. cerevisiae and found that neither synaptojanins nor dynamin contribute directly to membrane scission. We propose that recruitment of Rvs BAR domains delays scission and allows invaginations to grow by stabilizing them. We also propose that vesicle formation is dependent on the force exerted by the actin network.

Basolateral Sorting of the Sodium/Iodide Symporter Is Mediated by Adaptor Protein 1 Clathrin Adaptor Complexes.

Background: The sodium/iodide symporter (NIS) is a transmembrane protein located on the basolateral membrane of thyrocytes. Despite its physiological and clinical relevance, little is known about the mechanisms that mediate NIS subcellular sorting. In the present study, we examined NIS basolateral trafficking in vitro using non-thyroid and thyroid epithelial cells. Methods: Immunofluorescence and Western blotting were performed to analyze NIS subcellular location and function in cells grown in monolayers under unpolarized and/or polarized conditions. Strategic NIS residues were mutated, and binding of NIS to clathrin adaptor complexes was determined by immunoprecipitation. Results: We show that NIS reaches the plasma membrane (PM) through a thyrotropin-dependent mechanism 24 hours after treatment with the hormone. We demonstrate that NIS basolateral trafficking is a clathrin-mediated mechanism, in which the clathrin adaptor complexes AP-1 (A and B) sort NIS from the trans-Golgi network (TGN) and recycling endosomes (REs). Specifically, we show that the AP-1B μ1 subunit controls NIS basolateral sorting through common REs. In its absence, NIS is apically missorted but remains functional. Additionally, direct NIS basolateral transport from the TGN to the basolateral membrane is mediated by AP-1A through clathrin-coated vesicles that also carry the transferrin receptor. Loss of the μ1 subunit of AP-1A is functionally compensated by AP-1B. Furthermore, loss of both subunits diminishes NIS trafficking to the PM. Finally, we demonstrate that AP-1A binds to the L121 and LL562/563 residues on NIS, whereas AP-1B binds to L583. Conclusions: Our findings highlight the novel involvement of the clathrin-coated machinery in basolateral NIS trafficking. Given that AP-1A expression is reduced in tumors, and its expression correlates with that of NIS, these findings will help uncover new targets in thyroid cancer treatment.

Chlorpromazine cooperatively induces apoptosis with tyrosine kinase inhibitors in EGFR-mutated lung cancer cell lines and restores the sensitivity to gefitinib in T790M-harboring resistant cells

We previously reported that the antipsychotic drug chlorpromazine (CPZ), which inhibits the formation of clathrin-coated vesicles (CCVs) essential for endocytosis and intracellular transport of receptor tyrosine kinase (RTK), inhibits the growth/survival of acute myeloid leukemia cells with mutated RTK (KIT D816V or FLT3-ITD) by perturbing the intracellular localization of these molecules. Here, we examined whether these findings are applicable to epidermal growth factor receptor (EGFR). CPZ dose-dependently inhibited the growth/survival of the non-small cell lung cancer (NSCLC) cell line, PC9 harboring an EGFR-activating (EGFR exon 19 deletion). In addition, CPZ not only suppressed the growth/survival of gefitinib (GEF)-resistant PC9ZD cells harboring T790 M, but also restored their sensitivities to GEF. Furthermore, CPZ overcame GEF resistance caused by Met amplification in HCC827GR cells. As for the mechanism of CPZ-induced growth suppression, we found that although CPZ hardly influenced the phosphorylation of EGFR, it effectively reduced the phosphorylation of ERK and AKT. When utilized in combination with trametinib (a MEK inhibitor), dabrafenib (an RAF inhibitor), and everolimus (an mTOR inhibitor), CPZ suppressed the growth of PC9ZD cells cooperatively with everolimus but not with trametinib or dabrafenib. Immunofluorescent staining revealed that EGFR shows a perinuclear pattern and was intensely colocalized with the late endosome marker, Rab11. However, after CPZ treatment, EGFR was unevenly distributed in the cells, and colocalization with the early endosome marker Rab5 and EEA1 became more apparent, indicating that CPZ disrupted the intracellular transport of EGFR. These results suggest that CPZ has therapeutic potential for NSCLC with mutated EGFR by a novel mechanism different from conventional TKIs alone or in combination with other agents.

Generation of nanoscopic membrane curvature for membrane trafficking.

Curved membranes are key features of intracellular organelles, and their generation involves dynamic protein complexes. Here we describe the fundamental mechanisms such as the hydrophobic insertion, scaffolding and crowding mechanisms these proteins use to produce membrane curvatures and complex shapes required to form intracellular organelles and vesicular structures involved in endocytosis and secretion. For each mechanism, we discuss its cellular functions as well as the underlying physical principles and the specific membrane properties required for the mechanism to be feasible. We propose that the integration of individual mechanisms into a highly controlled, robust process of curvature generation often relies on the assembly of proteins into coats. How cells unify and organize the curvature-generating factors at the nanoscale is presented for three ubiquitous coats central for membrane trafficking in eukaryotes: clathrin-coated pits, caveolae, and COPI and COPII coats. The emerging theme is that these coats arrange and coordinate curvature-generating factors in time and space to dynamically shape membranes to accomplish membrane trafficking within cells.

Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation.

The formation of a clathrin-coated vesicle (CCV) is a major membrane remodeling process that is crucial for membrane traffic in cells. Besides clathrin, these vesicles contain at least 100 different proteins although it is unclear how many are essential for the formation of the vesicle. Here, we show that intracellular clathrin-coated formation can be induced in living cells using minimal machinery and that it can be achieved on various membranes, including the mitochondrial outer membrane. Chemical heterodimerization was used to inducibly attach a clathrin-binding fragment 'hook' to an 'anchor' protein targeted to a specific membrane. Endogenous clathrin assembled to form coated pits on the mitochondria, termed MitoPits, within seconds of induction. MitoPits are double-membraned invaginations that form preferentially on high curvature regions of the mitochondrion. Upon induction, all stages of CCV formation - initiation, invagination, and even fission - were faithfully reconstituted. We found no evidence for the functional involvement of accessory proteins in this process. In addition, fission of MitoPit-derived vesicles was independent of known scission factors including dynamins and dynamin-related protein 1 (Drp1), suggesting that the clathrin cage generates sufficient force to bud intracellular vesicles. Our results suggest that, following its recruitment, clathrin is sufficient for intracellular CCV formation.

Advances in structural, spatial, and temporal mechanics of plant endocytosis.

Endocytic trafficking underlies processes essential for plant growth and development, including the perception of and response to abiotic and extracellular stimuli, post-Golgi and exocytic trafficking, and cytokinesis. Protein adaptors and regulatory factors of clathrin-mediated endocytosis that contribute to the formation of endocytic clathrin-coated vesicles are evolutionarily conserved. Yet, work of the last ten years has identified differences between the endocytic mechanisms of plants and Opisthokonts involving the endocytic adaptor TPLATE complex, the requirement of actin during CME, and the function of clathrin-independent endocytosis in the uptake of plant-specific plasma membrane proteins. Here, we review clathrin-mediated and -independent pathways in plants and describe recent advances enabled by new proteomic and imaging methods, and conditional perturbation of endocytosis. In addition, we summarize the formation and trafficking of clathrin-coated vesicles based on temporal and structural data garnered from high-resolution quantitative imaging studies. Finally, new information about the cross-talk between endocytosis and other endomembrane trafficking pathways and organelles will also be discussed.

Abstract LB032: CXCR7 drives prostate cancer growth through AURKA activation

Abstract Prostate cancer (PCa) is the most commonly diagnosed cancer among males in the United States. The mainstay treatment for metastatic PCa patients is androgen-deprivation therapies, including high-affinity androgen receptor antagonists, such as enzalutamide. Unfortunately, most patients develop resistance and relapse with castration-resistant PCa (CRPC). We have previously reported up-regulation of CXCR7, an atypical chemokine receptor, in CRPC. CXCR7 interacts with beta-arrestin (ARRB2), a cytoplasmic scaffold protein, and translocates to the cytoplasm to induce signal transduction and PCa progression. However, much remains to be known regarding CXCR7-driven downstream signaling and its therapeutic targeting in advanced PCa. To unveil CXCR7-downstream signaling, we utilized RNA-seq to profile gene expression. Gene ontology analyses revealed remarkable enrichment of CXCR7-regulated genes in G2/M cell cycle progression. Comprehensive phospho-proteomics analysis of CXCR7-knockdown PCa cells showed markedly reduced phosphorylation of AURKA substrates. AURKA, a serine/threonine-protein kinase, is a key regulator of mitosis and G2/M progression and is frequently upregulated in CRPC and the late-stage neuroendocrine PCa (NEPC). Activated AURKA experiences autophosphorylation and phosphorylates downstream substrates, e.g., TACC3, that mediate proper cell division. Immunoblotting confirmed that CXCR7 depletion reduces phosphorylation of AURKA and TACC3. Co-immunoprecipitation revealed protein-protein interaction between CXCR7 and AURKA, which is mediated by ARRB2. Accordingly, ARRB2-knockdown similarly inhibits AURKA phosphorylation and arrests cell proliferation. Immunofluorescence coupled with confocal imaging demonstrated that CXCR7 is packed into clathrin-coated vesicles and locates in the plasma membrane, cytoplasm, and perinuclear Golgi complex, which surrounds the centrosomal AURKA and ARRB2. We found that CXCR7 translocates to the Golgi complex along microtubule-dependent trafficking. Finally, we found that pharmacological inhibitors of AURKA abolish CXCR7-driven PCa cell proliferation in vitro and tumor growth in vivo. Conclusions: Our results describe for the first time that the CXCR7-ARRB2 complex interacts with AURKA and promotes its activation. Our data suggest that AURKA-targeting is a promising therapeutic strategy for CRPC and NEPC with CXCR7 up-regulation. Citation Format: Galina Gritsina, Zhuoyuan Lin, Ka Wing Fong, Xiaodong Lu, Changsheng Zhao, Jindan Yu. CXCR7 drives prostate cancer growth through AURKA activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB032.

Abstract 1079: Anti-cancer efficacy of TH1902, a SORT1 docetaxel peptide-drug conjugate, against ovarian and endometrial cancers xenografts alone or in combination with carboplatin

Abstract Effective, safe chemotherapy treatments for gynecological cancers remain a daunting challenge. One strategy to achieve greater selectivity and better anticancer drug delivery into cancer cells is to conjugate cytotoxic agents to specific peptide ligands that selectively target receptors abundantly expressed on these cells. Increased expression of sortilin (SORT1), a scavenging receptor, has been shown to be highly expressed in many cancers, including ovarian and endometrial cancers. The peptide-drug conjugate TH1902 (a peptide which targets SORT1 and is bis-linked to two docetaxel molecules) was investigated to determine whether it could be efficacious against SORT1-positive ovarian (ES-2, A-2780 and SKOV-3) and endometrial (AN3-CA) cancer models. The Alexa488-fluorescent peptide used to generate TH1902 was transported into ES-2 and SKOV-3 cells by a process requiring clathrin-coated vesicles, and which was strongly diminished by siRNA-mediated SORT1 silencing or by competition with SORT1 ligands. In vitro, TH1902 enabled an increase in apoptosis of over 2-fold compared to that of unconjugated docetaxel. The effects of TH1902 administration were also examined in multiple subcutaneous xenograft tumor models (ES-2, SKOV-3, A-2780 or AN3-CA cells). Following two weeks treatment at equivalent docetaxel doses, the ES-2 xenograft sizes in mice treated with vehicle or with docetaxel were statistically indistinguishable but there was a significant decrease in tumor size by 78% for mice treated with TH1902. In SKOV-3 xenografts, treatment with either drug both stopped tumor growth and induced similar tumor regressions. In AN3-CA xenografts, treatments administered at equivalent docetaxel maximum tolerated dose (MTD) induced similar initial tumor regressions. However, TH1902-treated mice showed prolonged tumors regression whereas tumors started to regrow upon docetaxel treatment interruption. At one quarter of the docetaxel MTD, TH1902 significantly inhibited AN3-CA tumor growth whereas docetaxel had minimal effect (-73% vs -11%, respectively). Mice bearing A-2780 xenograft tumors were treated with TH1902, paclitaxel or docetaxel alone, as well as with each in combination with carboplatin. Comparison of the tumor sizes between the various treatments showed that TH1902 alone caused stronger A-2780 tumor growth inhibition than did either of the unconjugated taxanes or carboplatin. Moreover, TH1902 combined with carboplatin also demonstrated better efficacy than did either of the taxane-carboplatin combinations. Overall, the results indicate that TH1902 possesses an in vivo efficacy superior to those of docetaxel against ovarian and endometrial cancers in the animal models tested, and that TH1902 could be safely combined with carboplatin to reach optimal inhibition of tumor growth. Citation Format: Christian Marsolais, Michel Demeule, Cyndia Charfi, Jean-Christophe Currie, Alain Larocque, Alain Zgheib, Richard Béliveau, Borhane Annabi. Anti-cancer efficacy of TH1902, a SORT1 docetaxel peptide-drug conjugate, against ovarian and endometrial cancers xenografts alone or in combination with carboplatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1079.

Ca 2+ Regulates Dimerization of the BAR Domain Protein PICK1 and Consequent Membrane Curvature.

Bin-Amphiphysin-Rvs (BAR) domain proteins are critical regulators of membrane geometry. They induce and stabilize membrane curvature for processes, such as clathrin-coated pit formation and endosomal membrane tubulation. BAR domains form their characteristic crescent-shaped structure in the dimeric form, indicating that the formation of the dimer is critical to their function of inducing membrane curvature and suggesting that a dynamic monomer–dimer equilibrium regulated by cellular signaling would be a powerful mechanism for controlling BAR domain protein function. However, to the best of our knowledge, cellular mechanisms for regulating BAR domain dimerization remain unexplored. PICK1 is a Ca2+-binding BAR domain protein involved in the endocytosis and endosomal recycling of neuronal AMPA receptors and other transmembrane proteins. In this study, we demonstrated that PICK1 dimerization is regulated by a direct effect of Ca2+ ions via acidic regions in the BAR domain and at the N-terminus. While the cellular membrane tubulating activity of PICK1 is absent under basal conditions, Ca2+ influx causes the generation of membrane tubules that originate from the cell surface. Furthermore, in neurons, PICK1 dimerization increases transiently following NMDA receptor stimulation. We believe that this novel mechanism for regulating BAR domain dimerization and function represents a significant conceptual advance in our knowledge about the regulation of cellular membrane curvature.

Expansion microscopy with ninefold swelling (NIFS) hydrogel permits cellular ultrastructure imaging on conventional microscope.

Superresolution microscopy enables probing of cellular ultrastructures. However, its widespread applications are limited by the need for expensive machinery, specific hardware, and sophisticated data processing. Expansion microscopy (ExM) improves the resolution of conventional microscopy by physically expanding biological specimens before imaging and currently provides ~70-nm resolution, which still lags behind that of modern superresolution microscopy (~30 nm). Here, we demonstrate a ninefold swelling (NIFS) hydrogel, that can reduce ExM resolution to 31 nm when using regular traditional microscopy. We also design a detachable chip that integrates all the experimental operations to facilitate the maximal reproducibility of this high-resolution imaging technology. We demonstrate this technique on the superimaging of nuclear pore complex and clathrin-coated pits, whose structures can hardly be resolved by conventional microscopy. The method presented here offers a universal platform with superresolution imaging to unveil cellular ultrastructural details using standard conventional laboratory microscopes.

Abstract 117: A Genome-wide CRISPR/Cas9 Knockout Screen Reveals New Targets To Increase Cellular LDL Uptake.

Identifying new genetic factors involved in lipoprotein metabolism will provide new targets to lower plasma lipids and prevent cardiovascular disease; however, genetic epidemiological studies failed to recognize all the contributors to plasma lipids levels. Recently, using a CRISPR-Cas9 knockout screen in HepG2 cells, we found that knocking out transgelin, an actin-binding protein, reduces LDL uptake in hepatoma cells by interfering with the actin-dependent phase of the clathrin-mediated endocytosis (CME). An ideal therapeutic target to reduce plasma LDL would be one like PCSK9 in which, once knocked out or inhibited, cellular LDL uptake would increase. Aim: To identify genetic factors that, by knocking them out, increase LDL uptake in hepatoma cells. Methods and Results: We performed a whole-genome CRISPR-Cas9 knockout screen (targeting 19,144 genes) on HepG2 cells, sorting for cells with increased LDL uptake. Gene enrichment was analyzed with MAGeCK software. As expected, MYLIP , a mediator of LDL receptor ubiquitination and degradation, was among the most enriched genes ( ranked #9, log fold change: 4.82 ). The 200 top-enriched genes (1%) were analyzed for associations with plasma lipid traits using the NHLBI-GRASP (v2.0.0.0). 35 out of 200 genes had associations with p<2.5x10-4 (0.05/200) and were considered for further in vitro analysis. Stable knockout HepG2 cells were generated for a subset of these candidate genes and were tested for LDL uptake. Increased in LDL uptake was observed for: MYLIP , +66%; HUNK , +38%; PACSIN1 , +41%; PACSIN3 , +55%; RAI14 , +75%; PSPH , +134% (% to control, p<0.01 for all). Interestingly, these genes showed interactions with CME-related genes in a protein-protein interactome analysis, revealing interactions with genes related to clathrin-coated pit and actin filament dynamics. Conclusion: We found novel genetic factors associated with LDL internalization by applying a validated whole-genome gene editing tool. Our data suggest that these factors may be involved in different steps of LDL endocytosis and may function as possible therapeutic targets to increase hepatic LDL clearance. Further studies will be needed to elucidate the mechanisms underlying these associations.

FCHO controls AP2's initiating role in endocytosis through a PtdIns(4,5)P2-dependent switch.

Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane-localized Fer/Cip4 homology domain-only proteins (FCHO). Here, live-cell enhanced total internal reflection fluorescence–structured illumination microscopy shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform-sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Epidermal growth factor potential receptor substrate number 15 (Eps15) ring. We dissect the network of interactions between the FCHO interdomain linker and AP2, which concentrates, orients, tethers, and partially destabilizes closed AP2 at the plasma membrane. AP2’s subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with phosphatidylinositol 4,5-bisphosphate, clathrin, cargo, and CME accessory factors. FCHO can now relocate toward a CCP’s outer edge to engage and activate further AP2s to drive CCP growth/maturation.

Load adaptation by endocytic actin networks.

Clathrin-mediated endocytosis (CME) robustness under elevated membrane tension is maintained by actin assembly–mediated force generation. However, whether more actin assembles at endocytic sites in response to increased load has not previously been investigated. Here actin network ultrastructure at CME sites was examined under low and high membrane tension. Actin and N-WASP spatial organization indicate that actin polymerization initiates at the base of clathrin-coated pits and that the network then grows away from the plasma membrane. Actin network height at individual CME sites was not coupled to coat shape, raising the possibility that local differences in mechanical load feed back on assembly. By manipulating membrane tension and Arp2/3 complex activity, we tested the hypothesis that actin assembly at CME sites increases in response to elevated load. Indeed, in response to elevated membrane tension, actin grew higher, resulting in greater coverage of the clathrin coat, and CME slowed. When membrane tension was elevated and the Arp2/3 complex was inhibited, shallow clathrin-coated pits accumulated, indicating that this adaptive mechanism is especially crucial for coat curvature generation. We propose that actin assembly increases in response to increased load to ensure CME robustness over a range of plasma membrane tensions.