Branched actin networks are organized for asymmetric force production during clathrin-mediated endocytosis in mammalian cells (original) (raw)
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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...
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...
Actin dynamics coupled to clathrin-coated vesicle formation at the trans-Golgi network
The Journal of Cell Biology
In diverse species, actin assembly facilitates clathrin-coated vesicle (CCV) formation during endocytosis. This role might be an adaptation specific to the unique environment at the cell cortex, or it might be fundamental, facilitating CCV formation on different membranes. Proteins of the Sla2p/Hip1R family bind to actin and clathrin at endocytic sites in yeast and mammals. We hypothesized that Hip1R might also coordinate actin assembly with clathrin budding at the trans-Golgi network (TGN). Using deconvolution and time-lapse microscopy, we showed that Hip1R is present on CCVs emerging from the TGN. These vesicles contain the mannose 6-phosphate receptor involved in targeting proteins to the lysosome, and the actin nucleating Arp2/3 complex. Silencing of Hip1R expression by RNAi resulted in disruption of Golgi organization and accumulation of F-actin structures associated with CCVs on the TGN. Hip1R silencing and actin poisons slowed cathepsin D exit from the TGN. These studies esta...
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.
Molecular biology of the cell, 2015
Over 60 highly conserved proteins appear sequentially at sites of clathrin-mediated endocytosis in yeast and mammals. The yeast Eps15-related proteins Pan1 and End3, and the CIN85-related protein Sla1 are known to interact to each other in vitro and they all appear after endocytic site initiation but before endocytic actin assembly, which facilitates membrane invagination/scission. Here, we used live cell imaging, in parallel with genetics and biochemistry, to comprehensively explore the dynamic interactions and functions of Pan1, End3, and Sla1. Our results indicate that Pan1 and End3 associate in a stable manner and appear at endocytic sites prior to Sla1. The End3 C-terminus is necessary and sufficient for its cortical localization via interaction with Pan1, while the End3 N-terminus plays a crucial role in Sla1 recruitment. We systematically examined the dynamic behaviors of endocytic proteins in cells in which Pan1 and End3 were simultaneously eliminated using the auxin-inducib...
Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis
The Journal of cell biology, 2014
Clathrin-mediated endocytosis (CME) involves the recruitment of numerous proteins to sites on the plasma membrane with prescribed timing to mediate specific stages of the process. However, how choreographed recruitment and function of specific proteins during CME is achieved remains unclear. Using genome editing to express fluorescent fusion proteins at native levels and live-cell imaging with single-molecule sensitivity, we explored dynamin2 stoichiometry, dynamics, and functional interdependency with actin. Our quantitative analyses revealed heterogeneity in the timing of the early phase of CME, with transient recruitment of 2-4 molecules of dynamin2. In contrast, considerable regularity characterized the final 20 s of CME, during which ∼26 molecules of dynamin2, sufficient to make one ring around the vesicle neck, were typically recruited. Actin assembly generally preceded dynamin2 recruitment during the late phases of CME, and promoted dynamin recruitment. Collectively, our resu...
Actin force generation in vesicle formation: mechanistic insights from cryo-electron tomography
2021
SummaryActin assembly provides force for a multitude of cellular processes. Compared to actin assembly- based force production during cell migration, relatively little is understood about how actin assembly generates pulling forces for vesicle formation. Here, cryo-electron tomography revealed actin filament number, organization, and orientation during clathrin-mediated endocytosis in human cells, showing that force generation is robust despite variance in network organization. Actin dynamics simulations incorporating a measured branch angle indicate that sufficient force to drive membrane internalization is generated through polymerization, and that assembly is triggered from ∼4 founding “mother” filaments, consistent with tomography data. Hip1R actin filament anchoring points are present along the entire endocytic invagination, where simulations show that it is key to pulling force generation, and along the neck, where it targets filament growth and makes internalization more robu...
Nature Communications
Clathrin lattices at the plasma membrane coat both invaginated and flat regions forming clathrin-coated pits and clathrin plaques, respectively. The function and regulation of clathrin-coated pits in endocytosis are well understood but clathrin plaques remain enigmatic nanodomains. Here we use super-resolution microscopy, molecular genetics and cell biology to show that clathrin plaques contain the machinery for clathrin-mediated endocytosis and cell adhesion, and associate with both clathrin-coated pits and filamentous actin. We also find that actin polymerization promoted by N-WASP through the Arp2/3 complex is crucial for the regulation of plaques but not pits. Clathrin plaques oppose cell migration and undergo actin-and N-WASP-dependent disassembly upon activation of LPA receptor 1, but not EGF receptor. Most importantly, plaque disassembly correlates with the endocytosis of LPA receptor 1 and down-modulation of AKT activity. Thus, clathrin plaques serve as dynamic actin-controlled hubs for clathrin-mediated endocytosis and signalling that exhibit receptor specificity.
The Journal of Cell Biology, 2001
Huntingtin-interacting protein 1 related (Hip1R) is a novel component of clathrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an actin-binding protein important for both actin organization and endocytosis in yeast. Here, we demonstrate that Hip1R binds via its putative central coiled-coil domain to clathrin, and provide evidence that Hip1R and clathrin are associated in vivo at sites of endocytosis. First, real-time analysis of Hip1R–YFP and DsRed–clathrin light chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at the cell cortex. Second, at the ultrastructure level, immunogold labeling of ‘unroofed’ cells showed that Hip1R localizes to clathrin-coated pits. Third, overexpression of Hip1R affected the subcellular distribution of clathrin LC. Consistent with a functional role for Hip1R in endocytosis, we also demonstrated that it promotes clathrin cage assembly in vitro. Finally, we showed that Hip1R is a r...
Spatial regulation of clathrin-mediated endocytosis through position-dependent site maturation
Journal of Cell Biology
During clathrin-mediated endocytosis (CME), over 50 different proteins assemble on the plasma membrane to reshape it into a cargo-laden vesicle. It has long been assumed that cargo triggers local CME site assembly in Saccharomyces cerevisiae based on the discovery that cortical actin patches, which cluster near exocytic sites, are CME sites. Quantitative imaging data reported here lead to a radically different view of which CME steps are regulated and which steps are deterministic. We quantitatively and spatially describe progression through the CME pathway and pinpoint a cargo-sensitive regulatory transition point that governs progression from the initiation phase of CME to the internalization phase. Thus, site maturation, rather than site initiation, accounts for the previously observed polarized distribution of actin patches in this organism. While previous studies suggested that cargo ensures its own internalization by regulating either CME initiation rates or frequency of abort...