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

Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Molecular Biology of the Cell (Impact Factor: 4.47). 03/2005; 16(2):964-75. DOI: 10.1091/mbc.E04-09-0774
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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.

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Available from: Clare M Waterman-Storer, Aug 25, 2014
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    • "A second type of supramolecular organization involves a branched F-actin network as typically found in lamellipodia at the leading edge of motile cells. More recently, the structural organization of actin assemblies at distinct sites of endocytosis has begun to unfold (Collins et al., 2011; Idrissi et al., 2008; Yarar et al., 2005). Other actin assemblies, for example those in the nucleus, remain enigmatic. "
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    ABSTRACT: Polymerization of actin monomers into filaments requires the initial formation of nuclei composed of a few actin subunits, however, their instability has hindered their detailed study. Therefore we used chemically cross-linked actin oligomers to analyze their effect on actin polymerization. Actin-dimer (upper dimer = UD), -trimer and -tetramer intermolecularly cross-linked by phenylene-bis-maleimide along the genetic helix (between Lys199 and Cys374) were isolated by gelfiltration and found to increasingly stimulate actin polymerization as shown by the pyrene-assay and TIRF microscopy. In contrast, the so-called lower actin-dimer (LD) characterized by a Cys374-Cys374 cross-link stimulated actin polymerization only at low but inhibited at high concentrations. UD and trimer stimulated the repolymerization of actin from complexes with thymosin beta4 (Tß4) or profilin, whereas the LD stimulated repolymerization only from the profilin:actin but not the actin:Tß4 complex. In vivo, actin polymerization is stimulated by nucleation factors. Therefore the interaction and effects of purified LD, UD and trimer on the actin nucleating activity of gelsolin, the formin mDia and Arp2/3 complex were analyzed. Native gel electrophoresis demonstrated binding of LD, UD and trimer to gelsolin and its fragment G1-3, to the FH2-domains of the formins mDia1 and 3, and to Arp2/3 complex. UD and trimer increased the nucleating activity of gelsolin and G1-3, but not of the mDia-FH2-domain nor of Arp2/3 complex. In contrast, LD at equimolar concentration to Arp2/3 complex stimulated its nucleating activity, but inhibited that of mDia-FH2-domains, gelsolin and G1-3 demonstrating differential regulation of their nucleating activity by dimers containing differently oriented actin-subunits. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    FEBS Journal 07/2015; 282(19). DOI:10.1111/febs.13381 · 4.00 Impact Factor
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    • "Implications of these findings for understanding rhomboid proteins and actin regulation in more complex eukaryotes Similar to yeast, actin is also assembled at sites of CME in mammalian cells, in which it facilitates membrane invagination, fission, and vesicle movement (Merrifield et al., 2005; Yarar et al., 2005) and becomes essential under conditions of high membrane tension (Boulant et al., 2011). Both mammalian N-WASP (Galovic et al., 2011) and myosin 1E (Krendel et al., 2007; Cheng et al., 2012) localize to sites of CME in mammalian cells, where they regulate actin assembly. "
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    ABSTRACT: Clathrin-mediated endocytosis (CME) is facilitated by a precisely regulated burst of actin assembly. PtdIns(4,5)P2 is an important signaling lipid with conserved roles in CME and actin assembly regulation. Rhomboid family multi-pass, transmembrane proteins regulate diverse cellular processes; however, rhomboid-mediated CME regulation has not been described. We report that yeast lacking the rhomboid protein, Rbd2, exhibit accelerated endocytic site dynamics and premature actin assembly during CME through a PtdIns(4,5)P2-dependent mechanism. Combined genetic and biochemical studies showed that the cytoplasmic tail of Rbd2 binds directly to PtdIns(4,5)P2 and is sufficient for Rbd2's role in actin regulation. Analysis of an Rbd2 mutant with diminished PtdIns(4,5)P2 binding capacity indicates that this interaction is necessary for the temporal regulation of actin assembly during CME. The cytoplasmic tail of Rbd2 appears to modulate PtdIns(4,5)P2 distribution on the cell cortex. The syndapin-like, F-BAR protein Bzz1 functions in a pathway with Rbd2 to precisely control the timing of type-1 myosin recruitment and actin polymerization onset during CME. This work reveals that the previously unstudied rhomboid protein Rbd2 functions in vivo at the nexus of three highly conserved processes: lipid organization, cytoskeletal function and endocytic regulation. © 2015 by The American Society for Cell Biology.
    Molecular Biology of the Cell 02/2015; 26(8). DOI:10.1091/mbc.E14-11-1540 · 4.47 Impact Factor
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    • "Asa Engqvist-Goldstein et al. reported that the disruption of the cortical actin cytoskeleton by genetic approaches or chemical methods inhibited clathrin-mediated endocytosis in yeast [32]. Similarly, Defne Yarar et al. determined that an actin cytoskeleton-depolymerizing agent, latrunculin A, resulted in an 80% reduction in the formation of clathrin-coated vesicles in mammalian cells [33]. However, the depolymerization of the actin cytoskeleton with latrunculin A triggers the rapid and massive movement of caveolin-positive structures toward the centrosomal region of the cell [34]. "
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    ABSTRACT: Vascular hyperpermeability induced by lipopolysaccharide (LPS) is a common pathogenic process in cases of severe trauma and sepsis. Vascular endothelial cadherin (VE-cad) is a key regulatory molecule involved in this process, although the detailed mechanism through which this molecule acts remains unclear. We assessed the role of clathrin-mediated and caveolae-mediated endocytosis of VE-cad in LPS-induced vascular hyperpermeability in the human vascular endothelial cell line CRL-2922 and determined that vascular permeability and VE-cad localization at the plasma membrane were negatively correlated after LPS treatment. Additionally, the loss of VE-cad at the plasma membrane was caused by both clathrin-mediated and caveolae-mediated endocytosis. Clathrin-mediated endocytosis was dominant early after LPS treatment, and caveolae-mediated endocytosis was dominant hours after LPS treatment. The caveolae-mediated endocytosis of VE-cad was activated through the LPS-Toll-like receptor 4 (TLR4)-Src signaling pathway. Structural changes in the actin cytoskeleton, specifically from polymerization to depolymerization, were important reasons for the switching of the VE-cad endocytosis pathway from clathrin-mediated to caveolae-mediated. Our findings suggest that clathrin-mediated and caveolae-mediated endocytosis of VE-cad contribute to LPS-induced vascular hyperpermeability, although they contribute via different mechanism. The predominant means of endocytosis depends on the time since LPS treatment.
    PLoS ONE 09/2014; 9(9):e106328. DOI:10.1371/journal.pone.0106328 · 3.23 Impact Factor
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