Article

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: 4.55). 03/2005; 16(2):964-75. DOI: 10.1091/mbc.E04-09-0774
Source: PubMed

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.

Download full-text

Full-text

Available from: Clare M Waterman-Storer, Aug 25, 2014
0 Followers
 · 
215 Views
  • Source
    • "However, by a number of tests, foci of polymerization were not closely associated with clathrin puncta within the spine, and the velocity of actin monomers on filaments near the EZ was not different than the surrounding spine milieu. This is particularly surprising given that the endocytic zone contains numerous actin-binding molecules which likely regulate endocytosis (Engqvist-Goldstein and Drubin, 2003; Rocca et al., 2008; Yarar et al., 2005). However, consistent with a limited tonic role of actin polymerization at the spine EZ, clathrin puncta are not disassembled or disrupted during latrunculin application (Blanpied et al., 2002), whereas AMPA receptors and PSD scaffold proteins are quickly lost (Kuriu et al., 2006; Zhou et al., 2001). "
    [Show abstract] [Hide abstract]
    ABSTRACT: In the brain, the strength of synaptic transmission between neurons is principally set by the organization of proteins within the receptive, postsynaptic cell. Synaptic strength at an individual site of contact can remain remarkably stable for months or years. However, it also can undergo diverse forms of plasticity which change the strength at that contact independent of changes to neighboring synapses. Such activity-triggered neural plasticity underlies memory storage and cognitive development, and is disrupted in pathological physiology such as addiction and schizophrenia. Much of the short-term regulation of synaptic plasticity occurs within the postsynaptic cell, in small subcompartments surrounding the synaptic contact. Biochemical subcompartmentalization necessary for synapse-specific plasticity is achieved in part by segregation of synapses to micron-sized protrusions from the cell called dendritic spines. Dendritic spines are heavily enriched in the actin cytoskeleton, and regulation of actin polymerization within dendritic spines controls both basal synaptic strength and many forms of synaptic plasticity. However, understanding the mechanism of this control has been difficult because the submicron dimensions of spines limit examination of actin dynamics in the spine interior by conventional confocal microscopy. To overcome this, we developed single-molecule tracking photoactivated localization microscopy (smtPALM) to measure the movement of individual actin molecules within living spines. This revealed inward actin flow from broad areas of the spine plasma membrane, as well as a dense central core of heterogeneous filament orientation. The velocity of single actin molecules along filaments was elevated in discrete regions within the spine, notably near the postsynaptic density but surprisingly not at the endocytic zone which is involved in some forms of plasticity. We conclude that actin polymerization is initiated at many well-separated foci within spines, an organization that may be necessary for the finely tuned adjustment of synaptic molecular content that underlies functional plasticity. Indeed, further single-molecule mapping studies confirm that actin polymerization drives reorganization of molecular organization at the synapse itself.
  • Source
    • "Coupled with the above studies on the various dystonin-a isoforms was the finding that the plakin domain of dystonin-a interacts with the protein clathrin in brain tissues (Bhanot et al., 2011). Clathrin is a protein involved in the coating of newly developed vesicles during both endocytosis and Golgi-exocytosis, and utilizes the actin cytoskeleton in these processes (Galletta et al., 2010; Yarar et al., 2005). This suggests that dystonin may play a role in linking clathrin-coated vesicles with the cytoskeleton. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Neuronal dystonin isoforms are giant cytoskeletal cross-linking proteins capable of interacting with actin and microtubule networks, protein complexes, membrane-bound organelles and cellular membranes. In the neuromuscular system, dystonin proteins are involved in maintaining cytoarchitecture integrity and have more recently been ascribed roles in other cellular processes such as organelle structure and intracellular transport. Loss of dystonin expression in mice results in a profound sensory ataxia termed dystonia musculorum (dt), which is attributed to the degeneration of sensory nerves. This chapter provides a comprehensive overview of the dystonin gene, the structure of encoded proteins, biological functions of neuronal dystonin isoforms, and known roles of dystonin in dt pathogenesis and human disease.
    International review of cell and molecular biology 01/2013; 300C:85-120. DOI:10.1016/B978-0-12-405210-9.00003-5 · 4.52 Impact Factor
  • Source
    • "We previously found that inactive forms of PAK1 locally sequester these GTPases and prevent their action in catalysing actin polymerization (Bisson et al., 2007). Since, regulation of the actin cytoskeleton has been implicated in endocytosis (Boulant et al., 2011; Liu et al., 2010; Taylor et al., 2011; Yarar et al., 2005). Our data suggest a possible role for E- Syt2 binding of PAK1 in this process. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Fibroblast growth factor (FGF) signalling plays an essential role in early vertebrate development. However, the response to FGF requires endocytosis of the activated FGF receptor (FGFR) that is in part dependent on remodelling of the actin cytoskeleton. Recently we showed that the extended synaptotagmin family plasma membrane protein, E-Syt2, is an essential endocytic adapter for FGFR1. Here we show E-Syt2 is also an interaction partner for the p21-GTPase Activated Kinase PAK1. The phospholipid binding C2C domain of E-Syt2 specifically binds a site adjacent to the CRIB/GBD of PAK1. PAK1 and E-Syt2 selectively complex with FGFR1 and functionally cooperate in the FGF signalling. E-Syt2 binding suppresses actin polymerization and inhibits the activation of PAK1 by the GTPases Cdc42 and Rac. Interestingly, the E-Syt2 binding site on PAK1 extensively overlaps a site recently suggested to bind phospholipids. Our data suggest that PAK1 interacts with phospholipid membrane domains via E-Syt2, where it may cooperate in the E-Syt2-dependent endocytosis of activated FGFR1 by modulating cortical actin stability.
    08/2012; 1(8):731-8. DOI:10.1242/bio.2012968
Show more