Friction Mediates Scission of Tubular Membranes Scaffolded by BAR Proteins

International audienceMembrane 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

Efficient ER exit and vacuole targeting of yeast Sna2p require two tyrosine-based sorting motifs : the proteome of lipid bodies

SNA proteins (Sensitive to Na+) form a membrane protein family, which, in the yeast Saccharomyces cerevisiae, is composed of four members : Sna1p/Pmp3p, Sna2p, Sna3p and Sna4p. In this study, we focused on the 79 residue Sna2p protein. We found that Sna2p is localized in the vacuolar membrane. Directed mutagenesis showed that two functional tyrosine motifs YXXØ are present in the C-terminal region. Each of these is involved in a different Golgi-to-vacuole targeting pathway : the tyrosine 65 motif is involved in adaptor protein 1 (AP-1)-dependent targeting, whereas the tyrosine 75 motif is involved in AP-3-dependent targeting. Moreover, our data suggest that these motifs also play a crucial role in the exit of Sna2p from the endoplasmic reticulum (ER). Directed mutagenesis of these tyrosines led to a partial redirection of Sna2p to lipid bodies (LBs), probably because of a decrease in ER exit efficiency. Sna2p is the first yeast protein in which two YXXØ motifs have been identified and both were demonstrated to be functional at two different steps of the secretory pathway, ER exit and Golgi-to-vacuole transport. Furthermore, the LB localization of Sna2p mutants led us to study further those subcellular structures. Different proteomic studies on LBs from yeast have been published previously. However, all of them were using a SDS-PAGE approach : LB proteins were separated on a gel and analyzed by mass spectrometry. Here, we used a gel-free approach. Proteins from purified LBs were digested by trypsin and the peptides separated by reverse phase liquid chromatography before to be submitted to mass spectrometry analysis. This technique allowed us to find all the known LB proteins identified in previous mass spectromotry studies, but also new candidates.(AGRO 3) -- UCL, 201

Unconventional endocytic mechanisms

Endocytosis mediates the uptake of extracellular proteins, micronutrients and transmembrane cell surface proteins. Importantly, many viruses, toxins and bacteria hijack endocytosis to infect cells. The canonical pathway is clathrin-mediated endocytosis (CME) and is active in all eukaryotic cells to support critical house-keeping functions. Unconventional mechanisms of endocytosis exit in parallel of CME, to internalize specific cargoes and support various cellular functions. These clathrin-independent endocytic (CIE) routes use three distinct mechanisms: acute signaling-induced membrane remodeling drives macropinocytosis, activity-dependent bulk endocytosis (ADBE), massive endocytosis (MEND) and EGFR non-clathrin endocytosis (EGFR-NCE). Cargo capture and local membrane deformation by cytosolic proteins is used by fast endophilin-mediated endocytosis (FEME), IL-2Rβ endocytosis and ultrafast endocytosis at synapses. Finally, the formation of endocytic pits by clustering of extracellular lipids or cargoes according to the Glycolipid-Lectin (GL-Lect) hypothesis mediates the uptake of SV40 virus, Shiga and cholera toxins, and galectin-clustered receptors by the CLIC/GEEC and the endophilin-A3-mediated CIE

Rac1, actin cytoskeleton and microtubules are key players in clathrin-independent endophilin-A3-mediated endocytosis

Endocytic mechanisms actively regulate plasma membrane composition and sustain fundamental cellular functions. Recently, we identified a clathrin-independent endocytic (CIE) modality mediated by the BAR domain protein endophilin-A3 (endoA3), which controls the cell surface homeostasis of the tumor marker CD166/ALCAM. Deciphering the molecular machinery of endoA3-dependent CIE should therefore contribute to a better understanding of its pathophysiological role, which remains so far unknown. Here,we investigate the role in this mechanism of actin, Rho GTPases and microtubules, which are major actors of CIE processes. We show that the actin cytoskeleton is dynamically associated with endoA3- and CD166-positive endocytic carriers and that its perturbation strongly inhibits the uptake process of CD166. We also reveal that the Rho GTPase Rac1, but not Cdc42, is a master regulator of this endocytic route. Finally, we provide evidence that microtubules and kinesin molecular motors are required to potentiate endoA3-dependent endocytosis. Of note, our study also highlights potential compensation phenomena between endoA3-dependent CIE and macropinocytosis. Altogether, our data deepen our understanding of this CIE modality and further differentiate it from other unconventional endocytic mechanisms

Increasing Diversity of Biological Membrane Fission Mechanisms.

Membrane fission is essential to life. It is required for many fundamental cellular processes, as diverse as cyto- and karyokinesis, organelle division, membrane repair, and membrane trafficking and endocytosis. While membrane fission was originally seen as resulting from the action of mechanoenzymes such as dynamin, it is clear that the reality is more complex. In this review, we propose an updated overview of fission mechanisms, and try to extract essential requirements for each. We also present examples of cellular processes that involve these fission mechanisms. Finally, we list pending questions, whether they are specific to a peculiar fission mechanism or more general to the field

Novel clathrin-independent endocytic routes: role of curvature sensing/inducing BAR domain proteins

Endocytosis is an essential cellular process required for uptake of nutrients from cell environment and turnover of plasma membrane components. Clathrin-mediated endocytosis is by far the best characterized endocytic process. Since the mid-90's, the existence of endocytic routes independent of clathrin emerged. Therefore, the most challenging question in membrane biology rose: how could the plasma membrane be deformed in the absence of an organized clathrin coat? Until today, this process is not fully understood. First attempts to shed light into this topic demonstrated the requirement of glycosphingolipids for membrane deformation in lectin-driven endocytosis. Recently, BAR domain proteins (BAR stands for Bin/Amphiphysin/Rvs) have been described to be crucial for clathrin-independent endocytic routes. BAR domain proteins interact with membranes and act as curvature sensors/inducers. The function of BAR domain proteins remains unclear in the landscape of clathrin-independent endocytosis, and especially the endocytic pit formation. We hypothesize that this protein family constitute a module, which defines cargo specificity and is able to deform plasma membrane in clathrin-independent endocytic processes. To verify this hypothesis, we will perform a knock-down screen of various BAR domain proteins in mammalian cells and analyse the abundance of plasma membrane proteins via quantitative proteomics. Identified BAR domain proteins/potential plasma membrane cargoes couples will be selected, and deeply characterized using advanced cell biology techniques and model membranes: Do they constitute new clathrin-independent endocytic routes? What is the function of the BAR domain proteins in the new endocytic processesses

Efficient ER exit and vacuole targeting of yeast Sna2p require two tyrosine-based sorting motifs.

SNA proteins (Sensitive to Na+) form a membrane protein family, which, in the yeast Saccharomyces cerevisiae, is composed of four members, Sna1p/Pmp3p, Sna2p, Sna3p and Sna4p. In this study, we focused on the 79 residue Sna2p protein. We found that Sna2p is localized in the vacuolar membrane. Directed mutagenesis showed that two functional tyrosine motifs YXXØ are present in the C-terminal region. Each of these is involved in a different Golgi-to-vacuole targeting pathway : the tyrosine 65 motif is involved in AP-1-dependent targeting, while the tyrosine 75 motif is involved in AP-3-dependent targeting. Moreover, our data suggest that these motifs also play a crucial role in the exit of Sna2p from the ER. Directed mutagenesis of these tyrosines led to a partial redirection of Sna2p to lipid bodies, probably due to a decrease in ER exit efficiency. Sna2p is the first yeast protein in which two YXXØ motifs have been identified and both were demonstrated to be functional at two different steps of the secretory pathway, ER exit and Golgi-to-vacuole transport

Novel clathrin-independent endocytic routes: role of curvature sensing/inducing BAR domain proteins

Endocytosis is an essential cellular process required for uptake of nutrients from cell environment and turnover of plasma membrane components. Clathrin-mediated endocytosis is by far the best characterized endocytic process. Since the mid-90's, the existence of endocytic routes independent of clathrin emerged. Therefore, the most challenging question in membrane biology rose: how could the plasma membrane be deformed in the absence of an organized clathrin coat? Until today, this process is not fully understood. First attempts to shed light into this topic demonstrated the requirement of glycosphingolipids for membrane deformation in lectin-driven endocytosis. Recently, BAR domain proteins (BAR stands for Bin/Amphiphysin/Rvs) have been described to be crucial for clathrin-independent endocytic routes. BAR domain proteins interact with membranes and act as curvature sensors/inducers. The function of BAR domain proteins remains unclear in the landscape of clathrin-independent endocytosis, and especially the endocytic pit formation. We hypothesize that this protein family constitute a module, which defines cargo specificity and is able to deform plasma membrane in clathrin-independent endocytic processes. To verify this hypothesis, we will perform a knock-down screen of various BAR domain proteins in mammalian cells and analyse the abundance of plasma membrane proteins via quantitative proteomics. Identified BAR domain proteins/potential plasma membrane cargoes couples will be selected, and deeply characterized using advanced cell biology techniques and model membranes: Do they constitute new clathrin-independent endocytic routes? What is the function of the BAR domain proteins in the new endocytic processesses