A Dynamic Actin Cytoskeleton Functions at Multiple Stages of Clathrin-mediated Endocytosis

Abstract

Clathrin-mediated endocytosis in mammalian cells is critical for a variety of cellular processes including nutrient uptake and cell surface receptor down-regulation. Despite the findings that numerous endocytic accessory proteins directly or indirectly regulate actin dynamics and that actin assembly is spatially and temporally coordinated with endocytosis, direct functional evidence for a role of actin during clathrin-coated vesicle formation is lacking. Here, we take parallel biochemical and microscopic approaches to address the contribution of actin polymerization/depolymerization dynamics to clathrin-mediated endocytosis. When measured using live-cell fluorescence microscopy, disruption of the F-actin assembly and disassembly cycle with latrunculin A or jasplakinolide results in near complete cessation of all aspects of clathrin-coated structure (CCS) dynamics. Stage-specific biochemical assays and quantitative fluorescence and electron microscopic analyses establish that F-actin dynamics are required for multiple distinct stages of clathrin-coated vesicle formation, including coated pit formation, constriction, and internalization. In addition, F-actin dynamics are required for observed diverse CCS behaviors, including splitting of CCSs from larger CCSs, merging of CCSs, and lateral mobility on the cell surface. Our results demonstrate a key role for actin during clathrin-mediated endocytosis in mammalian cells. INTRODUCTIONClathrin-mediated endocytosis 1 (CME) is critical for a variety of biological processes such as nutrient uptake, cell surface receptor down-regulation, and synaptic vesicle recycling. CME involves the spatial and temporal coordination of numerous lipid components and multiple accessory proteins (Slepnev and De Camilli, 2000). Structural proteins, including clathrin and adaptor proteins, are recruited from the cell cytosol and assemble on the plasma membrane forming a ϳ200-nm clathrin-coated pit (CCP). The CCP becomes invaginated and narrows at the neck, forming a constricted coated pit which subsequently pinches off from the plasma membrane, generating a nascent clathrin-coated vesicle (CCV). Finally, the cycle is completed by disassembly of the clathrin coat, which is utilized for subsequent rounds of endocytosis.Growing evidence has suggested a role for the actin cytoskeleton during clathrin-mediated endocytosis (Qualmann et al., 2000;Merrifield, 2004). In budding yeast, actin assembly and disassembly dynamics are tightly coupled to and essential for endocytosis (Engqvist-Goldstein and Drubin, 2003). Thus, disruption of the yeast cortical actin cytoskeleton by genetic or chemical means inhibits endocytosis. Similarly, several lines of evidence have suggested a close association between the endocytic machinery in mammalian cells and the actin cytoskeleton. First, the GTPase dynamin, a central player in CME, and several dynaminbinding partners, including cortactin, syndapin, mAbp1, intersectin, and profilin, have been shown to directly or indirectly regulate F-actin dynamics (Qualmann et ...

SNX9 Regulates Dynamin Assembly and Is Required for Efficient Clathrin-mediated Endocytosis

“…In addition EGFP-SNX9 fluorescence was detected, in some cases, off-centered from clathrin. This spatial and temporal heterogeneity, which has also been observed for actin's association with CCPs (Yarar et al, 2005), may reflect a dual role for SNX9 in endocytosis. In this regard, it is interesting that SNX9 has been shown to interact with WASP in Drosophila (Worby et al, 2001).…”

“…For EGFP-SNX9 and dynamin 1-mRFP experiments, Swiss 3T3 cells were plated onto glass coverslips and allowed to adhere overnight. Cells were microinjected with plasmids encoding EGFP-Snx9 (0.05 mg/ml) and Dynamin1-mRFP (0.05 mg/ml) into the nucleus 3-5 h before filming.Nearly simultaneous total internal reflection fluorescence (TIRF) and widefield epifluorescence (WF-EF) microscopy was performed using an inverted microscope (Nikon TE2000U, Melville, NY) custom-modified to allow for through-the-objective multispectral total-internal reflection fluorescence microscopy and wide-field epifluorescence (Adams et al, 2004) using a 100ϫ, 1.45 NA objective (Nikon), as previously described (Yarar et al, 2005). Pairs of WF-EF and TIRF images were taken for 10 min at 2-3-s intervals, with 100 -300-ms exposure time, depending on the intensity of the signal.…”

Endosomal clathrin drives actin accumulation at the immunological synapse

“…It is well established that clathrin-mediated endocytosis in yeast requires actin rearrangement (Engqvist-Goldstein and Drubin, 2003). However, the situation in mammalian cells is less clear: whereas many authors show data involving actin in clathrin-dependent endocytosis (Chen and Brodsky, 2005;Engqvist-Goldstein et al, 2004;Ferguson et al, 2009;Toshima et al, 2005;Yarar et al, 2005), others show that actin depletion has no effect on the internalization dynamics of clathrin-coated vesicles (Boucrot et al, 2006). Studies with bacterial and fungal pathogens show that clathrin and actin both participate in pathogen internalization (Moreno-Ruiz et al, 2009;Veiga and Cossart, 2005;Veiga et al, 2007), and it has been proposed that mammalian cells support internalization of large membrane plaques in a process involving clathrin and actin that is very similar to 'classical' clathrin-mediated endocytosis (Saffarian et al, 2009).…”

“…These studies suggest that membranes coated with clathrin might serve as a molecular platform for recruitment of the protein machinery necessary to trigger localized actin polymerization during pathogen invasion. This hypothesis, together with the work of others (Yarar et al, 2005) challenged the classical view of clathrin-mediated endocytosis, which in mammalian cells was assumed to occur independently of actin (Conner and Schmid, 2003). Clathrin functions by binding to cell membranes through interaction with proteins called clathrin adaptors (Owen et al, 2004).…”

Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis

“…Together, these domains cooperate to stimulate Arp2/3 complex activity and to promote actin polymerization (Duncan et al, 2001;Toshima et al, 2005). The later events in internalization, possibly including vesicle scission, involve actin polymerization (Yarar et al, 2005) and Arp2/3 complex recruitment (Merrifield et al, 2005). The central region of Pan1 mediates the formation of Pan1-Pan1 complexes (Miliaras et al, 2004), and also seems to link the N-terminal and C-terminal functions of Pan1 into a single protein complex (Miliaras et al, 2004;Toshima et al, 2005).…”