Formation Of Endocytic Vesicles Research Articles

The autotaxin-LPA axis promotes membrane trafficking and secretion in yolk sac visceral endoderm cells.

Autotaxin, encoded by the Enpp2 gene, is an exoenzyme that produces lysophosphatidic acid, thereby regulating many biologic functions. We previously reported that Enpp2 mRNA was abundantly expressed in yolk sac visceral endoderm (VE) cells and that Enpp2-/- mice were lethal at embryonic day 9.5 owing to angiogenic defects in the yolk sac. Enpp2-/- mice showed lysosome fragmentation in VE cells and embryonic abnormalities including allantois malformation, neural tube defects, no axial turning, and head cavity formation. However, whether the defects in endocytic vesicle formation affect membrane trafficking in VE cells remained to be directly examined. In this study, we found that pinocytosis, transcytosis, and secretion of angiogenic factors such as vascular endothelial growth factor and transforming growth factor β1 were impaired in Enpp2-/- VE cells. Moreover, pharmacologic inhibition of membrane trafficking phenocopied the defects of Enpp2-/- mice. These findings demonstrate that Enpp2 spromotes endocytosis and secretion of angiogenic factors in VE cells, thereby regulating angiogenesis/vasculogenesis and embryonic development.

A mechanism-based theory of cellular and tissue plasticity.

Plastic deformation in cells and tissues has been found to play crucial roles in collective cell migration, cancer metastasis, and morphogenesis. However, the fundamental question of how plasticity is initiated in individual cells and then propagates within the tissue remains elusive. Here, we develop a mechanism-based theory of cellular and tissue plasticity that accounts for all key processes involved, including the activation and development of active contraction at different scales as well as the formation of endocytic vesicles on cell junctions and show that this theory achieves quantitative agreement with all existing experiments. Specifically, it reveals that, in response to optical or mechanical stimuli, the myosin contraction and thermal fluctuation-assisted formation and pinching of endocytic vesicles could lead to permanent shortening of cell junctions and that such plastic constriction can stretch neighboring cells and trigger their active contraction through mechanochemical feedbacks and eventually their plastic deformations as well. Our theory predicts that endocytic vesicles with a size around 1 to 2 µm will most likely be formed and a higher irreversible shortening of cell junctions could be achieved if a long stimulation is split into multiple short ones, all in quantitative agreement with experiments. Our analysis also shows that constriction of cells in tissue can undergo elastic/unratcheted to plastic/ratcheted transition as the magnitude and duration of active contraction increases, ultimately resulting in the propagation of plastic deformation waves within the monolayer with a constant speed which again is consistent with experimental observations.

Protein phase separation in plant membrane biology: more than just a compartmentalization strategy.

The formation of biomolecular condensates through phase separation is an important strategy to compartmentalize cellular functions. While it is now well established that condensates exist throughout eukaryotic cells, how condensates assemble and function on lipid membranes is only beginning to be understood. In this perspective, we highlight work from plant, animal, and yeast model systems showing that condensates assemble on many endomembrane surfaces to carry out diverse functions. In vesicle trafficking, condensation has reported roles in the formation of endocytic vesicles and autophagosomes, and in the inactivation of secretory COPII vesicles. We briefly discuss how membranes and membrane lipids regulate the formation and function of membrane-associated condensates. This includes how membranes act as surfaces for condensate assembly, with lipids mediating the nucleation of condensates during endocytosis and other processes. Additionally, membrane-condensate interactions give rise to the biophysical property of 'wetting', which has functional importance in shaping autophagosomal and vacuolar membranes. We also speculate on the existence of membrane-associated condensates during cell polarity in plants, and discuss how condensation may help to establish functional plasma membrane domains. Lastly, we provide advice on relevant in vitro and in vivo approaches and techniques to study membrane-associated phase separation.

A Plethora of Functions Condensed into Tiny Phospholipids: The Story of PI4P and PI(4,5)P2.

Phosphoinositides (PIs) are small, phosphorylated lipids that serve many functions in the cell. They regulate endo- and exocytosis, vesicular trafficking, actin reorganization, and cell mobility, and they act as signaling molecules. The most abundant PIs in the cell are phosphatidylinositol-4-monophosphate (PI4P) and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. PI4P is mostly localized at the Golgi apparatus where it regulates the anterograde trafficking from the Golgi apparatus to the plasma membrane (PM), but it also localizes at the PM. On the other hand, the main localization site of PI(4,5)P2 is the PM where it regulates the formation of endocytic vesicles. The levels of PIs are regulated by many kinases and phosphatases. Four main kinases phosphorylate the precursor molecule phosphatidylinositol into PI4P, divided into two classes (PI4KIIα, PI4KIIβ, PI4KIIIα, and PI4KIIIβ), and three main kinases phosphorylate PI4P to form PI(4,5)P2 (PI4P5KIα, PI4P5KIβ, and PI4P5KIγ). In this review, we discuss the localization and function of the kinases that produce PI4P and PI(4,5)P2, as well as the localization and function of their product molecules with an overview of tools for the detection of these PIs.

Fluorescent visualization and evaluation of NPC1L1-mediated vesicular endocytosis during intestinal cholesterol absorption in mice

Abstract Excessive cholesterol absorption from intestinal lumen contributes to the pathogenesis of hypercholesterolemia, which is an independent risk factor for atherosclerotic cardiovascular disease. Niemann-Pick C1-like 1 (NPC1L1) is a major membrane protein responsible for cholesterol absorption, in which the physiological role of vesicular endocytosis is still controversial, and it lacks a feasible tool to visualize and evaluate the endocytosis of NPC1L1 vesicles in vivo. Here, we genetically labeled endogenous NPC1L1 protein with EGFP in a knock-in mouse model, and demonstrated fluorescent visualization and evaluation of the endocytic vesicles of NPC1L1-cago during intestinal cholesterol absorption. The homozygous NPC1L1-EGFP mice have normal NPC1L1 expression pattern as well as cholesterol homeostasis on chow or high-cholesterol diets. The fluorescence of NPC1L1-EGFP fusion protein localizes at the brush border membrane of small intestine, and EGFP-positive vesicles is visualized beneath the membrane as early as 5 min post oral gavage of cholesterol. Of note, the vesicles colocalize with the early endosomal marker early endosome antigen 1 (EEA1) and the filipin-stained free cholesterol. Pretreatment with NPC1L1 inhibitor ezetimibe inhibits the formation of these cholesterol-induced endocytic vesicles. Our data support the notion that NPC1L1-mediated cholesterol absorption is a vesicular endocytic process. NPC1L1-EGFP mice are a useful model for visualizing cellular NPC1L1-cargo vesicle itineraries and for evaluating NPC1L1 activity in vivo in response to diverse pharmacological agents and nutrients.

Abstract TMP33: Obesity And Hyperinsulinemia Impair Insulin Receptor Transport In The Brain Microvascular Endothelium

Introduction: Obesity afflicts over 650 million individuals worldwide and is a strong risk factor for both stroke and dementia. Hyperinsulinemia-induced insulin resistance, a finding commonly seen in obesity, is linked to brain insulin deficiency. As insulin is neuroprotective, impaired transport from the peripheral circulation into the brain may underlie the enhanced risk of vascular cognitive impairment in obese individuals. Thus, the focus of this study is to understand the role of aberrant insulin receptor endocytosis in impaired brain insulin transport associated with diet-induced obesity. Methods: C57Bl/6J mice were placed on a control or high fat diet beginning at 6 weeks of age for 24 weeks (n=3-5) and hippocampi collected to image microvascular receptor transport. To isolate the effects of insulin resistance, primary murine brain microvascular endothelial cells (MBEC) were exposed to hyperinsulinemia (HI; 20nM insulin for 12 hours). Naïve and HI cells were stimulated for 15 minutes with 20nM insulin and processed for immunoblotting, microscopy, and flow cytometry (n=3-12). Results: High fat diet fed mice exhibited reduced internalization of insulin receptor-positive vesicles in the vascular endothelium as compared to controls (0.27+/-0.04 v. 0.37+/-0.02). Correspondingly, HI MBECs displayed reduced insulin receptor internalization, disrupted actin architecture, and a decreased abundance of early endosomes as compared to naïve cells (0.90+/-0.12 v. 2.07+/-0.29). HI cells further demonstrated a shift towards increased endocytic vesicle formation and swelling relative to naïve cells (1.90+/-0.21 v. 1.04+/-0.08), the result of which may be due to an observed increase in endoplasmic reticulum stress and impaired nitric oxide synthase capacity. Conclusion: Diet-induced obesity impairs insulin receptor endocytosis into the brain vascular endothelium. These data suggest that hyperinsulinemia induces a compensatory shift in endocytic machinery, with receptors potentially becoming trapped within endocytic buds as opposed to trafficking into the cell. By uncovering the mechanisms linking obesity to cognitive decline, this study will help identify novel therapeutic targets for vascular cognitive impairment and dementia.

Computational modeling of endocytic vesicle formation imaged by simultaneous two-wavelength axial ratiometry.

Clathrin-mediated endocytosis (CME) facilitates the internalization of extracellular cargoes. However, how clathrin-coated vesicles (CCVs) form remains unclear due to the limited resolution of live-cell fluorescence microscopy and the need for sample fixation in electron microscopy. To bridge this gap, our lab developed Simultaneous Two-wavelength Axial Ratiometry (STAR) microscopy that leverages the wavelength-dependent properties of Total Internal Reflection Fluorescence (TIRF) microscopy. Dual-tagging a protein of interest with two spectrally separated fluorophores allows STAR to measure the intensity ratio and retrieve the z-position of the protein. Although the exponential decay of the evanescent wave is critical for STAR, it results in an uneven excitation of fluorescently tagged proteins. This will bias STAR measurements when dual-tagged proteins are distributed on a 3D object such as CCVs. To understand the accuracy of STAR for studying vesicle formation, we used mathematical modeling. We represented the CCV as a monodisperse sphere and used the STAR equations to calculate vesicle height (∆z) and compared it to the “ground truth” center of mass (CM). We investigated the influence of vesicle formation, radius, the number and distribution of proteins, the distance of the vesicle from the plasma membrane, and Poisson noise on STAR. This mathematical model of STAR microscopy assesses how the ∆z measured by STAR compares to the ground truth. The theoretical findings will be used to relate experimental ∆z results to vesicle morphology and eventually to develop a 3D dynamic model of CCV formation from our experimental data.

Sucrose targets clathrin-mediated endocytosis kinetics supporting cell elongation in Arabidopsis thaliana.

Sucrose is a central regulator of plant growth and development, coordinating cell division and cell elongation according to the energy status of plants. Sucrose is known to stimulate bulk endocytosis in cultured cells; however, its physiological role has not been described to date. Our work shows that sucrose supplementation induces root cell elongation and endocytosis. Sucrose targets clathrin-mediated endocytosis (CME) in epidermal cells. Its presence decreases the abundance of both the clathrin coating complex and phosphatidylinositol 4,5-biphosphate at the plasma membrane, while increasing clathrin complex abundance in intracellular spaces. Sucrose decreases the plasma membrane residence time of the clathrin complex, indicating that it controls the kinetics of endocytic vesicle formation and internalization. CME regulation by sucrose is inducible and reversible; this on/off mechanism reveals an endocytosis-mediated mechanism for sensing plant energy status and signaling root elongation. The sucrose monosaccharide fructose also induces CME, while glucose and mannitol have no effect, demonstrating the specificity of the process. Overall, our data show that sucrose can mediate CME, which demonstrates that sucrose signaling for plant growth and development is dependent on endomembrane trafficking.

Exosomes in osteoarthritis: Updated insights on pathogenesis, diagnosis, and treatment.

Osteoarthritis (OA) has remained a prevalent public health problem worldwide over the past decades. OA is a global challenge because its specific pathogenesis is unclear, and no effective disease-modifying drugs are currently available. Exosomes are small and single-membrane vesicles secreted via the formation of endocytic vesicles and multivesicular bodies (MVBs), which are eventually released when MVBs fuse with the plasma membrane. Exosomes contain various integral surface proteins derived from cells, intercellular proteins, DNAs, RNAs, amino acids, and metabolites. By transferring complex constituents and promoting macrophages to generate chemokines and proinflammatory cytokines, exosomes function in pathophysiological processes in OA, including local inflammation, cartilage calcification and degradation of osteoarthritic joints. Exosomes are also detected in synovial fluid and plasma, and their levels continuously change with OA progression. Thus, exosomes, specifically exosomal miRNAs and lncRNAs, potentially represent multicomponent diagnostic biomarkers for OA. Exosomes derived from various types of mesenchymal stem cells and other cell or tissue types affect angiogenesis, inflammation, and bone remodeling. These exosomes exhibit promising capabilities to restore OA cartilage, attenuate inflammation, and balance cartilage matrix formation and degradation, thus demonstrating therapeutic potential in OA. In combination with biocompatible and highly adhesive materials, such as hydrogels and cryogels, exosomes may facilitate cartilage tissue engineering therapies for OA. Based on numerous recent studies, we summarized the latent mechanisms and clinical value of exosomes in OA in this review.

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.

Dynamin 1 controls vesicle size and endocytosis at hippocampal synapses

Following calcium-triggered vesicle exocytosis, endocytosis regenerates vesicles to maintain exocytosis and thus synaptic transmission, which underlies neuronal circuit activities. Although most molecules involved in endocytosis have been identified, it remains rather poorly understood how endocytic machinery regulates vesicle size. Vesicle size, together with the transmitter concentration inside the vesicle, determines the amount of transmitter the vesicle can release, the quantal size, that may control the strength of synaptic transmission. Here, we report that, surprisingly, knockout of the GTPase dynamin 1, the most abundant brain dynamin isoform known to catalyze fission of the membrane pit's neck (the last step of endocytosis), not only significantly slowed endocytosis but also increased the synaptic vesicle diameter by as much as ∼40–64% at cultured hippocampal synapses. Furthermore, dynamin 1 knockout increased the size of membrane pits, the precursor for endocytic vesicle formation. These results suggest an important function of dynamin other than its well-known fission function – control of vesicle size at the pit formation stage.

Candida albicans ENT2 Contributes to Efficient Endocytosis, Cell Wall Integrity, Filamentation, and Virulence.

ABSTRACTEpsins play a pivotal role in the formation of endocytic vesicles and potentially provide a linkage between endocytic and other trafficking pathways. We identified a Candida albicans epsin, ENT2, that bears homology to the Saccharomyces cerevisiae early endocytosis genes ENT1 and ENT2 and studied its functions by a reverse genetic approach utilizing CRISPR-Cas9-mediated gene deletion. The C. albicans ent2Δ/Δ null mutant displayed cell wall defects and altered antifungal drug sensitivity. To define the role of C. albicans ENT2 in endocytosis, we performed assays with the lipophilic dye FM4-64 that revealed greatly reduced uptake in the ent2Δ/Δ mutant. Next, we showed that the C. albicans ent2Δ/Δ mutant was unable to form hyphae and biofilms. Assays for virulence properties in an in vitro keratinocyte infection model demonstrated reduced damage of mammalian adhesion zippers and host cell death from the ent2Δ/Δ mutant. We conclude that C. albicans ENT2 has a role in efficient endocytosis, a process that is required for maintaining cell wall integrity, hyphal formation, and virulence-defining traits.IMPORTANCE The opportunistic fungal pathogen Candida albicans is an important cause of invasive infections in hospitalized patients and a source of considerable morbidity and mortality. Despite its clinical importance, we still need to improve our ability to diagnose and treat this common pathogen. In order to support these advancements, a greater understanding of the biology of C. albicans is needed. In these studies, we are focused on the fundamental biological process of endocytosis, of which little is directly known in C. albicans. In addition to studying the function of a key gene in this process, we are examining the role of endocytosis in the virulence-related processes of filamentation, biofilm formation, and tissue invasion. These studies will provide greater insight into the role of endocytosis in causing invasive fungal infections.

Cooperative regulation of endocytic vesicle transport by yeast Eps15-like protein Pan1p and epsins

Dynamic actin filaments are required for the formation and internalization of endocytic vesicles. Yeast actin cables serve as a track for the translocation of endocytic vesicles to early endosomes, but the molecular mechanisms regulating the interaction between vesicles and the actin cables remain ambiguous. Previous studies have demonstrated that the yeast Eps15-like protein Pan1p plays an important role in this interaction, and that interaction is not completely lost even after deletion of the Pan1p actin-binding domain, suggesting that additional proteins mediate association of the vesicle with the actin cable. Other candidates for mediating the interaction are endocytic coat proteins Sla2p (yeast Hip1R) and Ent1p/2p (yeast epsins), as these proteins can bind to both the plasma membrane and the actin filament. Here, we investigated the degree of redundancy in the actin-binding activities of Pan1p, Sla2p, and Ent1p/2p involved in the internalization and transport of endocytic vesicles. Expression of the nonphosphorylatable form of Pan1p, Pan1-18TA, caused abnormal accumulation of both actin cables and endocytic vesicles, and this accumulation was additively suppressed by deletion of the actin-binding domains of both Pan1p and Ent1p. Interestingly, deletion of the actin-binding domains of Pan1p and Ent1p in cells lacking the ENT2 gene resulted in severely defective internalization of endocytic vesicles and recruitment of actin cables to the site of endocytosis. These results suggest that Pan1p and Ent1p/2p cooperatively regulate the interaction between the endocytic vesicle and the actin cable.

An online gathering about the latest on molecular membrane biology

Gordon Research Conferences (GRC) are among the meetings that are particularly important as they are rather small and foster scientific exchange. Many established researchers participate and meet for a week, often in some remote location. The 2021 molecular membrane biology Gordon conference, usually held at Proctor Academy, was one of the many meetings affected when the SARS-CoV2 pandemic resulted in an immediate shutdown of all public life and world travel. Scientists and scientific organizations reacted immediately and discovered the advantages of online meetings. Several members of the membrane biology community felt that a complete halt of scientific conferences would particularly harm young investigators in their career path as they would not get the chance to present their data in front of the scientific community. As a result, we decided to take the initiative and try to find a format for an online meeting in 2021. Before starting, we sent around a survey to ask for interest and received overwhelming enthusiasm and support—a testament to the vitality of the membrane trafficking community. We agreed on a format that would mimic somehow the in-person meeting: poster sessions, followed by a dense set of talks with sufficient time for discussion, a meet-the-speaker slot, then another set of talks, and eventually another poster session or meeting time. Talks were presented mostly by early career researchers, postdocs, or assistant professors. To foster discussion and presentation of unpublished data, talks were live and not recorded. Gather.town was used as a platform for poster sessions and social interactions. Very importantly, the meeting was completely free as funding was secured via four journals (Journal of Cell Biology, Journal of Cell Science, FEBS Journal, and Traffic Journal) and three German DFG-funded research consortia (SFB 944, 958, and TR 186), which covered the user fees for all participants. How did it work? Maybe we start from the end. “This was the best online meeting I attended during the entire pandemic period” was one of the enthusiastic responses. Why? Because the social platform was as close as one can get to real-life social interactions, and the sessions worked almost perfectly for our needs. Two poster sessions with a total of some 50 posters flanked the talks on each day. Posters were accessible, presenters available, and it felt like being in a real meeting. Poster sessions were well attended and so busy that people had to wait, just like an in-person meeting. But since posters were available throughout the entire meeting, people were also able to view posters in their own spare time. The entire community was present, from PhD students to postdocs, young investigators, and established figures in the field. For the talks, we explicitly asked speakers to keep to their time to 10 min to allow for questions, asked the audience to write their questions into the chat and the chair to act as a mediator between questions and speaker. This made sessions dense, but also to the point. As all speakers kept to their time, and the chairs monitored their sessions so well, enough time was left for questions. We were impressed, how much science can be compressed in a short talk, and how excellent all speakers presented. The more exciting the talk, the earlier the questions appeared in the chat. As the chairs combined questions and condensed the information, the most important open issues were immediately addressed. Breaks were reserved to meet-the-speaker, and by the end of the day—or better the end of the meeting—many of us realized how long they had been sitting fixed in front of their screen without moving much. Throughout the meeting, 200–250 people were listening to the talks, more than 150 were present at the poster sessions. It was as good as it gets. We selected speakers based on their science and tried to balance the field and explicitly focused on postdocs and early career investigators. As the science of the talks was meant to be “off the record” and live, in the spirit of a Gordon Research Conference, we decided to just let the program speak for itself (see supporting information). Liz Miller (MRC Cambridge, United Kingdom) opened the dances of the first ER-focused session with a talk about the role of unstructured regions in assembly of the COPII coat (unpublished and (1Stancheva V.G. Li X.-H. Hutchings J. Gomez-Navarro N. Santhanam B. Babu M.M. Zanetti G. Miller E.A. Combinatorial multivalent interactions drive cooperative assembly of the COPII coat.J. Cell Biol. 2020; 219e202007135Crossref PubMed Google Scholar)). The session featured structural work by Tino Pleiner (Caltech, USA) and Antonio Galindo (MRC, United Kingdom). Tino's work shed light on the mechanisms of membrane protein insertion into the ER and the regulation of the human 9-subunit membrane protein insertase ER membrane protein complex (EMC) via the kinase WNK1 (2Pleiner T. Hazu M. Tomaleri G.P. Januszyk K. Oania R.S. Sweredoski M.J. Moradian A. Guna A. Voorhees R.M. WNK1 is an assembly factor for the human ER membrane protein complex.Mol. Cell. 2021; 81: 2693-2704.e12Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 3Pleiner T. Tomaleri G.P. Januszyk K. Inglis A.J. Hazu M. Voorhees R.M. Structural basis for membrane insertion by the human ER membrane protein complex.Science. 2020; 369: 433-436Crossref PubMed Scopus (38) Google Scholar). Antonio's TRAPPIII structure reveals insights into the mechanisms of Rab1 activation to regulate secretory trafficking (4Galindo A. Planelles-Herrero V.J. Degliesposti G. Munro S. Cryo-EM structure of metazoan TRAPPIII, the multi-subunit complex that activates the GTPase Rab1.EMBO J. 2021; 40e107608Crossref PubMed Scopus (5) Google Scholar). Ishier Raote (CRG, Spain) shared the latest about the function of TANGO1 in creating a tunnel at the ER for export of bulky cargo (5Raote I. Chabanon M. Walani N. Arroyo M. Garcia-Parajo M.F. Malhotra V. Campelo F. A physical mechanism of TANGO1-mediated bulky cargo export.Elife. 2020; 9e59426Crossref PubMed Scopus (8) Google Scholar, 6Raote I. Ernst A.M. Campelo F. Rothman J.E. Pincet F. Malhotra V. TANGO1 membrane helices create a lipid diffusion barrier at curved membranes.Elife. 2020; 9e57822Crossref PubMed Scopus (13) Google Scholar, 7Raote I. Ortega-Bellido M. Santos A.J. Foresti O. Zhang C. Garcia-Parajo M.F. Campelo F. Malhotra V. TANGO1 builds a machine for collagen export by recruiting and spatially organizing COPII, tethers and membranes.Elife. 2018; 7e32723Crossref PubMed Scopus (52) Google Scholar), including some exciting unpublished results about the role of liquid–liquid phase separation (LLPS) in the assembly of TANGO1 at ER exit sites. Sessions 2 and 3, focused on trafficking through the Golgi compartments and secretory pathway, included talks by Ivan Castello Serrano (University of Virginia, USA) about the importance of lipid rafts for export of transmembrane proteins from the Golgi network, and Anup Parchure (Yale University, USA) whose work suggests that secretory granule biogenesis at the TGN is facilitated by LLPS. Lauren Jackson (Vanderbilt University, USA) presented structural work shedding light on the regulation of post-Golgi cargo recycling in yeast via the ArfGAP Glo3 (8Xie B. Jung C. Chandra M. Engel A. Kendall A.K. Jackson L.P. The Glo3 GAP crystal structure supports the molecular niche model for ArfGAPs in COPI coats.Adv. Biol. Regul. 2021; 79: 100781Crossref PubMed Scopus (1) Google Scholar). Mara Duncan (University of Michigan, USA) presented some recent work from her lab where a human iPSC model system combined with proximity-based proteomics was used to identify trafficking machinery responsible for polarized trafficking (9Wang S. Lin C.-W. Carleton A.E. Cortez C.L. Johnson C. Taniguchi L.E. Sekulovski N. Townshend R.F. Basrur V. Nesvizhskii A.I. Zou P. Fu J. Gumucio D.L. Duncan M.C. Taniguchi K. Spatially resolved cell polarity proteomics of a human epiblast model.Sci. Adv. 2021; 7eabd8407Crossref PubMed Scopus (3) Google Scholar). David Murray (University of Dundee, United Kingdom) presented exciting unpublished data about the functional characterization of the exocyst tethering complex and its cross talk with phosphoinositide signaling. The session was closed by Allison Zajac (University of Chicago, USA), whose latest preprint (10Zajac A.L. Horne-Badovinac S. Kinesin-3 and kinesin-1 motors direct basement membrane protein secretion to a basal sub-region of the basolateral plasma membrane in epithelial cells.bioRxiv. 2021; ([preprint])https://doi.org/10.1101/2021.01.31.429062Crossref Scopus (0) Google Scholar) highlights the role of Kinesin-3 and -1 motors in basolateral secretion in epithelial cells. The first day wrapped up with a session focused on mitochondria/peroxisomal membranes packed with excellent unpublished findings. Tim König (McGill University, Canada)'s work reveals the lipid and protein composition and the mechanisms of formation of mitochondrial-derived vesicles (MDVs). Joshua Pemberton (NICHD, USA) developed tools to alter the lipid composition of the outer mitochondrial membrane and showed that localized production of diacylglycerol altered mitochondrial morphology. Cansu Kuey (University of Warwick, United Kingdom) showed that acute redistribution of clathrin mediated endocytosis machinery on mitochondria is sufficient to drive the formation of clathrin-coated vesicles. Noa Dahan (Weizman Institute, Israel) talked about the importance of local mRNA translation for peroxisomal function. The second day kicked off with a session on the endocytic pathway, in which Andreas Mayer (University of Lausanne, Switzerland) discussed his unpublished data on the regulation of membrane fission in the endolysosomal system by CROP, a novel complex consisting of Atg18 and the retromer complex. Staying on the membrane fission theme, Sho Suzuki (Cornell University, USA) revealed how the yeast Mvp1/SNX8 protein and the dynamin-like GTPase Vps1 drive fission and endosomal recycling. While Doris Höglinger (Biochemistry Center Heidelberg, Germany) discussed routes for cholesterol and sphingolipid transport, Florian Fröhlich (University of Osnabrück, Germany) presented his proteomic mapping of the endolysosomal system, which is available as a preprint (11Eising S. Esch B.M. Wälte M. Duarte P.V. Walter S. Ungermann C. Bohnert M. Fröhlich F. A yeast lysosomal biogenesis map uncovers the cargo spectrum of lysosomal protein targeting pathways.bioRxiv. 2021; ([preprint])https://doi.org/10.1101/2021.07.24.453616Crossref Scopus (0) Google Scholar). In the second session of the day, Kamalesh Kumari (Weizmann Institute, Israel) argued that mechanochemical sequestration and vesicle crumpling drive exocytosis while maintaining apical membrane homeostasis. On the theme of membrane compartmentalization, Kasey Day (University of Texas, USA) provided evidence that liquid-like protein droplets are important for endocytic vesicle formation, and Claudia Matthaeus (NIH, USA) discussed the role of lipid uptake in caveolae. Henning Arlt (Harvard University, USA) provided structural and mechanistic insights into lipid droplet biogenesis. Andrés Guillén-Samander (Yale University, USA) revealed how VPS13D together with Miro facilitates interorganelle contacts between ER and mitochondria and peroxisomes (12Guillén-Samander A. Leonzino M. Hanna M.G. Tang N. Shen H. Camilli P.D. VPS13D bridges the ER to mitochondria and peroxisomes via Miro.J. Cell Biol. 2021; 220e202010004Crossref PubMed Scopus (14) Google Scholar). These presentations were followed by two talks on ESCRT-III: Jiwei Liu (Rosalind Franklin Institute, UK) provided compelling evidence for the presence of ESCRT-III membrane remodelers in bacteria, while Joachim Moser von Filseck (University of Geneva, Switzerland) provided molecular details on ESCRT-III membrane remodeling in eukaryotes (13Moser von Filseck J. Barberi L. Talledge N. Johnson I.E. Frost A. Lenz M. Roux A. Anisotropic ESCRT-III architecture governs helical membrane tube formation.Nat. Commun. 2020; 11: 1516Crossref PubMed Scopus (19) Google Scholar). The last three talks of the day were on autophagy. Tara Fisher (NIH, USA) started with a presentation on how STING induces LC3 lipidation (14Fischer T.D. Wang C. Padman B.S. Lazarou M. Youle R.J. STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain.J. Cell Biol. 2020; 219e202009128Crossref PubMed Google Scholar). Rachel Ulferts (Francs Crick Institute, United Kingdom) then discussed the role of the V-ATPase in inflammation and autophagy. Finally, Florian Wilfling (Max-Planck-Institute for Biophysics, Germany) shared his data on intrinsic autophagy receptors and how they function in the disposal of macromolecular machines including stalled endocytic machinery (15Wilfling F. Lee C.-W. Erdmann P.S. Zheng Y. Sherpa D. Jentsch S. Pfander B. Schulman B.A. Baumeister W. A selective autophagy pathway for phase-separated endocytic protein deposits.Mol. Cell. 2020; 80: 764-778.e7Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Virtual molecular membrane biology 2021 was a success mainly because junior scientists were given a platform to present their exciting (and unpublished!) research. Given that many talks at previous meetings were presented by established scientists, who are also one reason to attract others (and some would otherwise also not join the meeting if not invited), the online meeting could make sure that more junior researchers were allowed to present and make their presence to the scientific community. In-person meetings mean frequent get-togethers on all occasions and time to talk and interact beyond listening to the talks. As scientists, we need this type of interaction to process what we learned and develop new ideas and directions. In an online format, travel, organization, and a week away from the lab and family are not happening, with all its pros and cons. Will such online meetings continue to work in the future? Or will we return to what we had before? We believe virtual gatherings cannot completely substitute for in-person meetings, but they can be a powerful way to keep the community connected outside of conference season. It definitely worked for us. This article contains supporting information. Download .pdf (.28 MB) Help with pdf files Supporting information

Actin-generated force applied during endocytosis measured by Sla2-based FRET tension sensors

Mechanical forces are integral to many cellular processes, including clathrin-mediated endocytosis, a principal membrane trafficking route into the cell. During endocytosis, forces provided by endocytic proteins and the polymerizing actin cytoskeleton reshape the plasma membrane into a vesicle. Assessing force requirements of endocytic membrane remodeling is essential for understanding endocytosis. Here, we determined actin-generated force applied during endocytosis using FRET-based tension sensors inserted into the major force-transmitting protein Sla2 in yeast. We measured at least 8 pN force transmitted over Sla2 molecule, hence possibly more than 300-880 pN applied during endocytic vesicle formation. Importantly, decreasing cell turgor pressure and plasma membrane tension reduced force transmitted over the Sla2. The measurements in hypotonic conditions and mutants lacking BAR-domain membrane scaffolds then showed the limits of the endocytic force-transmitting machinery. Our study provides force values and force profiles critical for understanding the mechanics of endocytosis and potentially other key cellular membrane-remodeling processes.

Preformed Ω-profile closure and kiss-and-run mediate endocytosis and diverse endocytic modes in neuroendocrine chromaffin cells

Transformation of flat membrane into round vesicles is generally thought to underlie endocytosis and produce speed-, amount-, and vesicle-size-specific endocytic modes. Visualizing depolarization-induced exocytic and endocytic membrane transformation in live neuroendocrine chromaffin cells, we found that flat membrane is transformed into Λ-shaped, Ω-shaped, and O-shaped vesicles via invagination, Λ-base constriction, and Ω-pore constriction, respectively. Surprisingly, endocytic vesicle formation is predominantly from not flat-membrane-to-round-vesicle transformation but calcium-triggered and dynamin-mediated closure of (1) Ω profiles formed before depolarization and (2) fusion pores (called kiss-and-run). Varying calcium influxes control the speed, number, and vesicle size of these pore closures, resulting in speed-specific slow (more than ∼6 s), fast (less than ∼6 s), or ultrafast (<0.6 s) endocytosis, amount-specific compensatory endocytosis (endocytosis= exocytosis) or overshoot endocytosis (endocytosis > exocytosis), and size-specific bulk endocytosis. These findings reveal major membrane transformation mechanisms underlying endocytosis, diverse endocytic modes, and exocytosis-endocytosis coupling, calling for correction of the half-a-century concept that the flat-to-round transformation predominantly mediates endocytosis after physiological stimulation.

Galectin-3 plays a protective role in Leishmania (Leishmania) amazonensis infection

Leishmania (L.) amazonensis is one of the species responsible for the development of cutaneous leishmaniasis in South America. After entering the vertebrate host, L. (L.) amazonensis invades mainly neutrophils, macrophages and dendritic cells. Studies have shown that gal-3 acts as a pattern recognition receptor. However, the role of this protein in the context of L. (L.) amazonensis infection remains unclear. Here, we investigated the impact of gal-3 expression on experimental infection by L. (L.) amazonensis. Our data showed that gal-3 plays a role in controlling parasite invasion, replication and the formation of endocytic vesicles. Moreover, mice with gal-3 deficiency showed an exacerbated inflammatory response. Taken together, our data shed light to a critical role of gal-3 in the host response to infection by L. (L.) amazonensis.

Brain-specific functions of the endocytic machinery.

Endocytosis is an essential cellular process required for multiple physiological functions, including communication with the extracellular environment, nutrient uptake, and signaling by the cell surface receptors. In a broad sense, endocytosis is accomplished through either constitutive or ligand-induced invagination of the plasma membrane, which results in the formation of the plasma membrane-retrieved endocytic vesicles, which can either be sent for degradation to the lysosomes or recycled back to the PM. This additional function of endocytosis in membrane retrieval has been adopted by excitable cells, such as neurons, for membrane equilibrium maintenance at synapses. The last two decades were especially productive with respect to the identification of brain-specific functions of the endocytic machinery, which additionally include but not limited to regulation of neuronal differentiation and migration, maintenance of neuron morphology and synaptic plasticity, and prevention of neurotoxic aggregates spreading. In this review, we highlight the current knowledge of brain-specific functions of endocytic machinery with a specific focus on three brain cell types, neuronal progenitor cells, neurons, and glial cells.

Liquid-like protein assemblies initiate endocytosis.

Endocytosis is critical for regulating plasma membrane composition and function. The mechanisms that control the assembly and dynamic rearrangement of the endocytic protein network to enable endocytic vesicle formation have remained elusive. Two endocytic initiator proteins are now shown to form liquid-like condensates to initiate endocytosis.

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells.

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by invivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior.