Bar domain proteins: a role in tubulation, scission and actin assembly in clathrin-mediated endocytosis (original) (raw)
Related papers
2017
Numerous endocytic pathways operate simultaneously at the cell surface. Here we focus on the molecular machinery involved in the generation of endocytic vesicles of the clathrin and dynamin-independent CLIC/GEEC (CG) pathway. This pathway internalises many GPI-anchored proteins and a large fraction of the fluid-phase in different cell types. We developed a real-time TIRF assay using pH-sensitive GFP-GPI to identify nascent CG endocytic sites. The temporal profile of known CG pathway modulators showed that ARF1/GBF1 (GTPase/GEF pair) and CDC42 (RhoGTPase) are recruited sequentially to CG endocytic sites, ∼60s and ∼9s prior to scission. Using a limited RNAi screen, we found several BAR domain proteins affecting CG endocytosis and focused on IRSp53 and PICK1 that have interactions with CDC42 and ARF1 respectively. IRSp53, an I-BAR domain containing protein, was recruited to the plasma membrane at the site of forming CG endocytic vesicles and in its absence, nascent endocytic CLICs, did...
A Modular Design for the Clathrin- and Actin-Mediated Endocytosis Machinery
Cell, 2005
Endocytosis depends on an extensive network of interacting proteins that execute a series of distinct subprocesses. Previously, we used live-cell imaging of six budding-yeast proteins to define a pathway for association of receptors, adaptors, and actin during endocytic internalization. Here, we analyzed the effects of 61 deletion mutants on the dynamics of this pathway, revealing functions for 15 proteins, and we analyzed the dynamics of 8 of these proteins. Our studies provide evidence for four protein modules that cooperate to drive coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission during clathrin/actin-mediated endocytosis. We found that clathrin facilitates the initiation of endocytic-site assembly but is not needed for membrane invagination or vesicle formation. Finally, we present evidence that the actin-meshwork assembly that drives membrane invagination is nucleated proximally to the plasma membrane, opposite to the orientation observed for previously studied actin-assembly-driven motility processes.
The Journal of Cell Biology, 2006
The conserved FER-CIP4 homology (FCH) domain is found in the pombe Cdc15 homology (PCH) protein family members, including formin-binding protein 17 (FBP17). However, the amino acid sequence homology extends beyond the FCH domain. We have termed this region the extended FC (EFC) domain. We found that FBP17 coordinated membrane deformation with actin cytoskeleton reorganization during endocytosis. The EFC domains of FBP17, CIP4, and other PCH protein family members show weak homology to the Bin-amphiphysin-Rvs (BAR) domain. The EFC domains bound strongly to phosphatidylserine and phosphatidylinositol 4,5-bisphosphate and deformed the plasma membrane and liposomes into narrow tubules. Most PCH proteins possess an SH3 domain that is known to bind to dynamin and that recruited and activated neural Wiskott-Aldrich syndrome protein (N-WASP) at the plasma membrane. FBP17 and/or CIP4 contributed to the formation of the protein complex, including N-WASP and dynamin-2, in the early stage of en...
Harnessing actin dynamics for clathrin-mediated endocytosis
Nature Reviews Molecular Cell Biology, 2006
Sheet-like plasma-membrane protrusions at the leading edge of motile cells that are formed by actin polymerization. www.nature.com/reviews/molcellbio Lamellipodia/filopodia Macropinocytosis Caveolae-mediated endocytosis Clathrin-mediated endocytosis Clathrin Clathrin-mediated endocytosis (actin patch) Nucleus Phagocytosis Actin cable Caveolin-1 Vesicle Saccharomyces cerevisiae cell Mammalian cell Actin Endosome Caveolae-mediated endocytosis A form of uptake at the plasma membrane that involves the protein caveolin. Cofilin A conserved actin-binding protein that is thought to be involved in actin-filament severing and disassembly.
F1000 - Post-publication peer review of the biomedical literature, 2000
The GTPase dynamin, a key player in endocytic membrane fission, interacts with numerous proteins that regulate actin dynamics and generate/sense membrane curvature. To determine the functional relationship between these proteins and dynamin, we have analyzed endocytic intermediates that accumulate in cells that lack dynamin (derived from dynamin 1 and 2 double conditional knockout mice). In these cells, actin nucleating proteins, actin and BAR domain proteins accumulate at the base of arrested endocytic clathrin-coated pits where they support the growth of dynamic long tubular necks. These results, which we show reflect the sequence of events in wildtype cells, demonstrate a concerted action of these proteins prior to, and independent of, dynamin, and emphasize similarities between clathrin-mediated endocytosis in yeast and higher eukaryotes. Our data also demonstrate that the relationship between dynamin and actin is intimately connected to dynamin's endocytic role and that dynamin terminates a powerful actin-and BAR protein-dependent tubulating activity.
Adaptive actin organization counteracts elevated membrane tension to ensure robust endocytosis
Clathrin-mediated endocytosis (CME) remains robust despite variations in plasma membrane tension. Actin assembly-mediated force generation becomes essential for CME under high membrane tension, but the underlying mechanisms are not understood. We investigated actin network ultrastructure at each stage of CME by super-resolution imaging. Actin and N-WASP spatial organization indicate that polymerization initiates at the base of clathrin-coated pits and that the actin network then grows away from the plasma membrane. Actin network organization is not tightly coupled to endocytic clathrin coat growth and deformation. Membrane tension-dependent changes in actin organization explain this uncoupling. Under elevated membrane tension, CME dynamics slow down and the actin network grows higher, resulting in greater coverage of the clathrin coat. This adaptive mechanism is especially crucial during the initial membrane curvature-generating stages of CME. Our findings reveal that adaptive force...
SUMMARYForce generation due to actin assembly is a fundamental aspect of membrane sculpting for many essential processes. In this work, we use a multiscale computational model constrained by experimental measurements to show that a minimal branched actin network is sufficient to internalize endocytic pits against physiological membrane tension. A parameter sweep identified the number of Arp2/3 complexes as particularly important for robust internalization, which prompted the development of a molecule-counting method in live mammalian cells. Using this method, we found that ~200 Arp2/3 complexes assemble at sites of clathrin-mediated endocytosis in human cells. Our simulations also revealed that actin networks self-organize in a radial branched array with barbed filament ends oriented to grow toward the base of the pit, and that the distribution of linker proteins around the endocytic pit is critical for this organization. Surprisingly, our model predicted that long actin filaments b...
Nature Communications
Actin assembly facilitates vesicle formation in several trafficking pathways, including clathrin-mediated endocytosis (CME). Interestingly, actin does not assemble at all CME sites in mammalian cells. How actin networks are organized with respect to mammalian CME sites and how assembly forces are harnessed, are not fully understood. Here, branched actin network geometry at CME sites was analyzed using three different advanced imaging approaches. When endocytic dynamics of unperturbed CME sites are compared, sites with actin assembly show a distinct signature, a delay between completion of coat expansion and vesicle scission, indicating that actin assembly occurs preferentially at stalled CME sites. In addition, N-WASP and the Arp2/3 complex are recruited to one side of CME sites, where they are positioned to stimulate asymmetric actin assembly and force production. We propose that actin assembles preferentially at stalled CME sites where it pulls vesicles into the cell asymmetricall...