RNAi-mediated Hip1R Silencing Results in Stable Association between the Endocytic Machinery and the Actin Assembly Machinery

Engqvist-Goldstein, Åsa E. Y.; Zhang, Claire X.; Carréno, Sébastien; Barroso, Consuelo; Heuser, John E.; Drubin, David G.

doi:10.1091/mbc.e03-09-0639

Cited by 146 publications

(175 citation statements)

References 54 publications

(76 reference statements)

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“…In fact, real-time light microscopy of sla2 cells reveals aberrant actin "tails" that remain attached to the cortex (Kaksonen et al, 2003). Similar results were obtained in mammalian cells depleted of the Sla2 homologue Hip1R by using RNA interference (Engqvist-Goldstein et al, 2004). Our results provide a higher resolution view of these aberrant actin structures in yeast and suggest that they may consist of large, actin-coated membrane structures rather than Listeria-like actin comet tails, which contain no membrane component.…”

Section: Ultrastructure Of Cortical Actin Patches In Yeastsupporting

confidence: 81%

Actin and Septin Ultrastructures at the Budding Yeast Cell Cortex

Rodal

Kozubowski

Goode

et al. 2005

MBoC

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174

162

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Budding yeast has been a powerful model organism for studies of the roles of actin in endocytosis and septins in cell division and in signaling. However, the depth of mechanistic understanding that can be obtained from such studies has been severely hindered by a lack of ultrastructural information about how actin and septins are organized at the cell cortex. To address this problem, we developed rapid-freeze and deep-etch techniques to image the yeast cell cortex in spheroplasted cells at high resolution. The cortical actin cytoskeleton assembles into conical or mound-like structures composed of short, cross-linked filaments. The Arp2/3 complex localizes near the apex of these structures, suggesting that actin patch assembly may be initiated from the apex. Mutants in cortical actin patch components with defined defects in endocytosis disrupted different stages of cortical actin patch assembly. Based on these results, we propose a model for actin function during endocytosis. In addition to actin structures, we found that septin-containing filaments assemble into two kinds of higher order structures at the cell cortex: rings and ordered gauzes. These images provide the first high-resolution views of septin organization in cells. INTRODUCTIONBudding yeast has been used for over 20 yr as a model organism to study cytoskeletal function because the genes encoding many components of the cytoskeleton are conserved and because yeast cells are readily amenable to parallel genetic, biochemical, and cell biological analyses. Despite rapid advances in defining the components of the yeast cytoskeleton, its ultrastructure has been difficult to image at the resolution of the electron microscope, with the exception of microtubules (O'Toole et al., 1999). This may be due to the low abundance of the cytoskeleton in yeast relative to mammalian cells, to the presence of a cell wall that complicates extraction procedures, and to high ribosome density in the yeast cytoplasm. As a result, there has been a conspicuous deficit in our understanding of the higher order organization of actin and septin polymers in vivo (Pruyne and Bretscher, 2000). Thus, although it is possible to rapidly identify and mutate cytoskeletal proteins in yeast, there is no method by which to examine the ultrastructural consequences of these mutations. This gap could be filled by developing an approach to image the yeast cytoskeleton at high resolution.The yeast actin cytoskeleton is organized into three distinct structures: motile cortical patches that are polarized in the growing bud, actin cables that run along the motherdaughter cell axis, and a contractile cytokinetic ring at the neck of dividing cells (reviewed in Pruyne and Bretscher, 2000). Cortical actin patches contain many of the actin-associated proteins common to all eukaryotes (Goode and Rodal, 2001) and are composed of actin filaments that undergo rapid turnover (Ayscough et al., 1997). Actin is essential for yeast endocytosis (Kubler and Riezman, 1993). A number of recent studies suggest that act...

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Section: Ultrastructure Of Cortical Actin Patches In Yeastsupporting

confidence: 81%

Actin and Septin Ultrastructures at the Budding Yeast Cell Cortex

Rodal

Kozubowski

Goode

et al. 2005

MBoC

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174

162

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“…Hip1R negatively regulates cortactin-stimulated actin polymerization to guarantee a transient and productive interaction between coated pits and actin during vesicle internalization (Engqvist-Goldstein et al, 2004;Le Clainche et al, 2007). In fact, this same proposal could be formulated in our study on CaM/cortactin and vesicles formation in endosomes.…”

Section: Molecular Machinery Involved In the Formation Of The Actin Csupporting

confidence: 55%

“…Briefly, 19-nucleotide RNAs were chemically synthesized (Ambion, Austin, TX) based on the sequence 5Ј-AAGCUGAGGGAGAAUGUCU-UGU-3Ј (small interfering RNA; [siRNA]1) (Helwani et al, 2004) or 5Ј-GACUGGUUUUGGAGGCAAAUUUU-3Ј (siRNA2) (Engqvist-Goldstein et al, 2004). siRNA against GFP was used as a control.…”

Section: Suppression Of Cortactin Expression By Rna Interference (Rnai)mentioning

confidence: 99%

“…To confirm the involvement of cortactin in the regulation of EGFR recycling from early endosomes, cells were transfected with two different cortactin siRNAs (Engqvist-Goldstein et al, 2004;Helwani et al, 2004) to knock down the endogenous protein ( Figure 7B). In both experiments, EGFR recycling was recovered in cells treated with W13 (from 22 to 32%, consistent with the degree of cortactin depletion shown by Western blotting).…”

Section: Cortactin Is Involved In the Regulation Of Egf Recycling Fromentioning

confidence: 99%

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Protein Kinase Cδ and Calmodulin Regulate Epidermal Growth Factor Receptor Recycling from Early Endosomes through Arp2/3 Complex and Cortactin

Lladó

Timpson

Muga

et al. 2008

MBoC

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The intracellular trafficking of the epidermal growth factor receptor (EGFR) is regulated by a cross-talk between calmodulin (CaM) and protein kinase C␦ (PKC␦). On inhibition of CaM, PKC␦ promotes the formation of enlarged early endosomes and blocks EGFR recycling and degradation. Here, we show that PKC␦ impairs EGFR trafficking due to the formation of an F-actin coat surrounding early endosomes. The PKC␦-induced polymerization of actin is orchestrated by the Arp2/3 complex and requires the interaction of cortactin with PKC␦. Accordingly, inhibition of actin polymerization by using cytochalasin D or by overexpression of active cofilin, restored the normal morphology of the organelle and the recycling of EGFR. Similar results were obtained after down-regulation of cortactin and the sequestration of the Arp2/3 complex. Furthermore we demonstrate an interaction of cortactin with CaM and PKC␦, the latter being dependent on CaM inhibition. In summary, this study provides the first evidence that CaM and PKC␦ organize actin dynamics in the early endosomal compartment, thereby regulating the intracellular trafficking of EGFR. INTRODUCTIONCalmodulin (CaM) is a ubiquitous small calcium sensor that regulates a variety of cellular processes in a spatial and temporal manner within the cell (Toutenhoofd and Strehler, 2000). Microenvironment variations in the concentration of CaM may be critical to the control of cellular processes that involve specific and high-affinity CaM-binding proteins (Berridge et al., 2000;Carafoli, 2002;Kahl and Means, 2003).We have previously shown that CaM is crucial to the regulation of the dynamics of endosomes. In particular, in the presence of W13, a highly specific CaM antagonist, trafficking is blocked in early endosomes, thereby interfering with transport toward degradation and recycling pathways. This blockage was associated with a dramatic alteration of the morphology and size of early endosomes (Tebar et al., 2002;Lladó et al., 2004). Furthermore we showed that a cross-talk between CaM and protein kinase C␦ (PKC␦) is involved in the regulation of the budding from the early endocytic compartment (Lladó et al., 2004).Several lines of evidence implicate the actin cytoskeleton in the CaM-and PKC␦-mediated regulation of vesicular trafficking. Because actin contributes to the maintenance of organelle stability (Apodaca, 2001), swelling and morphological changes observed in response to CaM/PKC␦ activity (Lladó et al., 2004) may involve altered actin dynamics. Moreover, several CaM-binding proteins and PKC␦ substrates are also actin-binding proteins, including ␣-actinin, adducin, and myristoylated alanine-rich protein kinase C substrate (MARCKS) (Chakravarthy et al., 1999). Finally, RhoGTPases and CaM-binding proteins responsible for fusion-fission events during vesicular trafficking also depend on actin filaments for pinch-off and vesicular movement (Mayorga et al., 1994;Mu et al., 1995;Colombo et al., 1997;Ridley, 2001;Burgoyne and Clague, 2003;Lawe et al., 2003;Donaldson, 2005).The actin cytosk...

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“…5E). Remarkably, clathrin depletion also abolished IS-directed relocalization of Huntingtininteracting protein-1-related protein (Hip1R), which regulates interactions between clathrin-coated membranes and the actin cytoskeleton (Engqvist-Goldstein et al, 2004;Wilbur et al, 2008) (Fig. 5F).…”

Section: Clathrin Is Necessary For Recruitment Of Key Adaptor and Regmentioning

confidence: 98%

Endosomal clathrin drives actin accumulation at the immunological synapse

Calabia-Linares

Robles‐Valero

Fuente

et al. 2011

Journal of Cell Science

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SummaryAntigen-specific cognate interaction of T lymphocytes with antigen-presenting cells (APCs) drives major morphological and functional changes in T cells, including actin rearrangements at the immune synapse (IS) formed at the cell-cell contact area. Here we show, using cell lines as well as primary cells, that clathrin, a protein involved in endocytic processes, drives actin accumulation at the IS. Clathrin is recruited towards the IS with parallel kinetics to that of actin. Knockdown of clathrin prevents accumulation of actin and proteins involved in actin polymerization, such as dynamin-2, the Arp2/3 complex and CD2AP at the IS. The clathrin pool involved in actin accumulation at the IS is linked to multivesicular bodies that polarize to the cell-cell contact zone, but not to plasma membrane or Golgi complex. These data underscore the role of clathrin as a platform for the recruitment of proteins that promote actin polymerization at the interface of T cells and APCs.

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RNAi-mediated Hip1R Silencing Results in Stable Association between the Endocytic Machinery and the Actin Assembly Machinery

Cited by 146 publications

References 54 publications

Actin and Septin Ultrastructures at the Budding Yeast Cell Cortex

Actin and Septin Ultrastructures at the Budding Yeast Cell Cortex

Protein Kinase Cδ and Calmodulin Regulate Epidermal Growth Factor Receptor Recycling from Early Endosomes through Arp2/3 Complex and Cortactin

Endosomal clathrin drives actin accumulation at the immunological synapse

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