A Novel Actin-Binding Motif in Las17/WASP Nucleates Actin Filaments Independently of Arp2/3

Urbanek, Agnieszka; Smith, Adam P.; Allwood, Ellen G.; Booth, Wesley I.; Ayscough, Kathryn R.

doi:10.1016/j.cub.2012.12.024

Cited by 34 publications

(70 citation statements)

References 34 publications

(45 reference statements)

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“…Our data show that the basic residues are required for the interaction of LGM with actin but the ability of flanking sequences to form PxxP motifs is only marginally important (Figure 1). LGM is also distinct from a filamentous actin-binding site present in a region of Las17 located over 100 residues distal to LGM and that even though it has not been fully mapped depends on proline residues (Las17 amino acids 506 and 507) (33). Structural analysis will be required to understand how LGM interacts with the actin monomer at the molecular level and its relationship with Tβ, WH2 and other actin-binding motifs.…”

Section: Discussionmentioning

confidence: 99%

“…Although the sequences in ActA are involved in binding and regulation of the host cell actin pool, they lack some of the key functional residues conserved in WH2 domains (32). Recently, studies performed on yeast have described novel actin-binding sequences with important roles in cell polarity, bundling of actin filaments and de novo actin nucleation processes independent of the Arp2/3 complex (29,33). The variable nature of actin-binding proteins in terms of their sequence and function suggests that there may be actin-binding modules that have yet to be discovered and could be involved in fundamental cellular processes such as CME.…”

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confidence: 99%

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A Second Las17 Monomeric Actin‐Binding Motif Functions in Arp2/3‐Dependent Actin Polymerization During Endocytosis

Feliciano

Tolsma

Farrell

et al. 2015

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During clathrin-mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott–Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G-actin) and a central-acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3-dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G-actin-binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G-actin-binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two-hybrid system, GST-pulldown, fluorescence polarization and pyrene-actin polymerization assays, we show that LGM binds G-actin and is necessary for normal Arp2/3-mediated actin polymerization in vitro. Live-cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G-actin-binding motif, WH2. These results establish a second G-actin-binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

A Second Las17 Monomeric Actin‐Binding Motif Functions in Arp2/3‐Dependent Actin Polymerization During Endocytosis

Feliciano

Tolsma

Farrell

et al. 2015

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“…This hypothesis offers one feasible explanation for the source of mother filaments that are absolutely required for Arp2/3 complex to nucleate polymerization [9] but appear to be absent from the cortex of yeast [11, 42]. We recognize other potential sources of mother filaments including the possibility that Wsp1p/Las17p may nucleate actin filaments in patches [43]. …”

Section: Discussionmentioning

confidence: 99%

Actin Filament Severing by Cofilin Dismantles Actin Patches and Produces Mother Filaments for New Patches

Chen

Pollard

2013

Current Biology

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Summary Background Yeast cells depend on Arp2/3 complex to assemble actin filaments at sites of endocytosis, but the source of the initial filaments required to activate Arp2/3 complex is not known. Results We tested the proposal that cofilin severs actin filaments during endocytosis in fission yeast cells using a mutant cofilin defective in severing. We used quantitative fluorescence microscopy to track mGFP-tagged proteins, including early endocytic adaptor proteins, activators of Arp2/3 complex and actin filaments. Consistent with the hypothesis, actin patches disassembled far slower in cells depending on severing-deficient cofilin than wild type cells. Even more interesting, actin patches assembled slowly in these cofilin mutant cells. Adaptor proteins End4p and Pan1p accumulated and persisted at endocytic sites more than 10 times longer than in wild type cells, followed by slow put persistent recruitment of activators of Arp2/3 complex, including WASP and myosin-I. Mutations revealed that actin filament binding sites on adaptor proteins Pan1p and End4p contribute to initiating actin polymerization in actin patches. Conclusions We propose a “sever, diffuse and trigger” model for the nucleation of actin filaments at sites of endocytosis whereby cofilin generates actin filament fragments that diffuse through the cytoplasm, bind adapter proteins at nascent sites of endocytosis and serve as mother filaments to initiate the autocatalytic assembly of the branched actin filament network of each new patch. This hypothesis explains the source of the “mother filaments” that are absolutely required for Arp2/3 complex to nucleate polymerization.

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“…It is also possible that there are growing actin filaments that were not detected in the previous filament counts based on Arp2/3 complex-mediated branching (20,23). These could, for example, be generated by domains on the actin nucleator Las-17 that act independently of the Arp2/3 complex (47).…”

Section: Alternative Mechanismsmentioning

confidence: 88%

Local Turgor Pressure Reduction via Channel Clustering

Scher-Zagier

2017

Biophysical Journal

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The primary drivers of yeast endocytosis are actin polymerization and curvature-generating proteins, such as clathrin and BAR domain proteins. Previous work has indicated that these factors may not be capable of generating the forces necessary to overcome turgor pressure. Thus local reduction of the turgor pressure, via localized accumulation or activation of solute channels, might facilitate endocytosis. The possible reduction in turgor pressure was calculated numerically, by solving the diffusion equation through a Legendre polynomial expansion. It was found that for a region of increased permeability having radius 45 nm, as few as 60 channels with a spacing of 10 nm could locally decrease the turgor pressure by 50%. We identified a key dimensionless parameter, p ¼ P 1 a/D, where P 1 is the increased permeability, a is the radius of the permeable region, and D is the solute diffusion coefficient. When p > 0.44, the turgor pressure is locally reduced by >50%. An approximate analytic theory was used to generate explicit formulas for the turgor pressure reduction in terms of key parameters. These findings may also be relevant to plants, where the mechanisms that allow endocytosis to proceed despite high turgor pressure are largely unknown.

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A Novel Actin-Binding Motif in Las17/WASP Nucleates Actin Filaments Independently of Arp2/3

Abstract: Our data demonstrate a new actin-binding and nucleation mechanism in Las17/WASP that is required for its function in actin regulation during endocytosis.

Cited by 34 publications

References 34 publications

A Second Las17 Monomeric Actin‐Binding Motif Functions in Arp2/3‐Dependent Actin Polymerization During Endocytosis

A Second Las17 Monomeric Actin‐Binding Motif Functions in Arp2/3‐Dependent Actin Polymerization During Endocytosis

Actin Filament Severing by Cofilin Dismantles Actin Patches and Produces Mother Filaments for New Patches

Local Turgor Pressure Reduction via Channel Clustering

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