Switch-like Arp2/3 activation upon WASP and WIP recruitment to an apparent threshold level by multivalent linker proteins in vivo

Actin-related protein 2/3 (Arp2/3) complex activation by nucleation promoting factors (NPFs) such as WASP, plays an important role in many actin-mediated cellular processes. In yeast, Arp2/3-mediated actin filament assembly drives endocytic membrane invagination and vesicle scission. Here we used genetics and quantitative live-cell imaging to probe the mechanisms that concentrate NPFs at endocytic sites, and to investigate how NPFs regulate actin assembly onset. Our results demonstrate that SH3 (Src homology 3) domain-PRM (proline-rich motif) interactions involving multivalent linker proteins play central roles in concentrating NPFs at endocytic sites. Quantitative imaging suggested that productive actin assembly initiation is tightly coupled to accumulation of threshold levels of WASP and WIP, but not to recruitment kinetics or release of autoinhibition. These studies provide evidence that WASP and WIP play central roles in establishment of a robust multivalent SH3 domain-PRM network in vivo, giving actin assembly onset at endocytic sites a switch-like behavior.

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Myosin 1E coordinates actin assembly and cargo trafficking during clathrin-mediated endocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e11-04-0383 ◽

2012 ◽

Vol 23 (15) ◽

pp. 2891-2904 ◽

Cited By ~ 57

Author(s):

Jackie Cheng ◽

Alexandre Grassart ◽

David G. Drubin

Keyword(s):

Mammalian Cells ◽

Actin Polymerization ◽

Type I ◽

Actin Assembly ◽

Significant Delay ◽

Src Homology 3 ◽

Integral Role ◽

Src Homology 3 Domain ◽

Src Homology ◽

Endocytic Vesicles

Myosin 1E (Myo1E) is recruited to sites of clathrin-mediated endocytosis coincident with a burst of actin assembly. The recruitment dynamics and lifetime of Myo1E are similar to those of tagged actin polymerization regulatory proteins. Like inhibition of actin assembly, depletion of Myo1E causes reduced transferrin endocytosis and a significant delay in transferrin trafficking to perinuclear compartments, demonstrating an integral role for Myo1E in these actin-mediated steps. Mistargeting of GFP-Myo1E or its src-homology 3 domain to mitochondria results in appearance of WIP, WIRE, N-WASP, and actin filaments at the mitochondria, providing evidence for Myo1E's role in actin assembly regulation. These results suggest for mammalian cells, similar to budding yeast, interdependence in the recruitment of type I myosins, WIP/WIRE, and N-WASP to endocytic sites for Arp2/3 complex activation to assemble F-actin as endocytic vesicles are being formed.

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Abelson Interactor 1 (Abi1) and Its Interaction with Wiskott-Aldrich Syndrome Protein (Wasp) Are Critical for Proper Eye Formation in Xenopus Embryos

Journal of Biological Chemistry ◽

10.1074/jbc.m112.445643 ◽

2013 ◽

Vol 288 (20) ◽

pp. 14135-14146 ◽

Cited By ~ 4

Author(s):

Arvinder Singh ◽

Emily F. Winterbottom ◽

Yon Ju Ji ◽

Yoo-Seok Hwang ◽

Ira O. Daar

Keyword(s):

Progenitor Cells ◽

Cultured Cells ◽

Scaffold Protein ◽

Wiskott Aldrich Syndrome ◽

Src Homology 3 ◽

Eye Field ◽

Src Homology 3 Domain ◽

Src Homology ◽

Syndrome Protein

Abl interactor 1 (Abi1) is a scaffold protein that plays a central role in the regulation of actin cytoskeleton dynamics as a constituent of several key protein complexes, and homozygous loss of this protein leads to embryonic lethality in mice. Because this scaffold protein has been shown in cultured cells to be a critical component of pathways controlling cell migration and actin regulation at cell-cell contacts, we were interested to investigate the in vivo role of Abi1 in morphogenesis during the development of Xenopus embryos. Using morpholino-mediated translation inhibition, we demonstrate that knockdown of Abi1 in the whole embryo, or specifically in eye field progenitor cells, leads to disruption of eye morphogenesis. Moreover, signaling through the Src homology 3 domain of Abi1 is critical for proper movement of retinal progenitor cells into the eye field and their appropriate differentiation, and this process is dependent upon an interaction with the nucleation-promoting factor Wasp (Wiskott-Aldrich syndrome protein). Collectively, our data demonstrate that the Abi1 scaffold protein is an essential regulator of cell movement processes required for normal eye development in Xenopus embryos and specifically requires an Src homology 3 domain-dependent interaction with Wasp to regulate this complex morphogenetic process.

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Mutations inDrosophilaEnabled and Rescue by Human Vasodilator-stimulated Phosphoprotein (VASP) Indicate Important Functional Roles for Ena/VASP Homology Domain 1 (EVH1) and EVH2 Domains

Molecular Biology of the Cell ◽

10.1091/mbc.9.8.2157 ◽

1998 ◽

Vol 9 (8) ◽

pp. 2157-2171 ◽

Cited By ~ 86

Author(s):

Shawn M. Ahern-Djamali ◽

Allen R. Comer ◽

Christiane Bachmann ◽

Andrew S. Kastenmeier ◽

Srinevas K. Reddy ◽

...

Keyword(s):

Cultured Cells ◽

Physiological Activity ◽

Focal Adhesions ◽

Functional Roles ◽

Src Homology 3 ◽

Abelson Tyrosine Kinase ◽

Src Homology 3 Domain ◽

Src Homology

Drosophila Enabled (Ena) was initially identified as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase and, more recently, as a member of the Ena/human vasodilator-stimulated phosphoprotein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. In addition, we have defined the roles of Ena domains identified as essential for its activity in vivo. We have demonstrated that VASP rescues the embryonic lethality associated with loss of Ena function inDrosophila and have shown that Ena, like VASP, is associated with actin filaments and focal adhesions when expressed in cultured cells. To define sequences that are central to Ena function, we have characterized the molecular lesions present in two lethalena mutant alleles that affected the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that resulted in an amino acid substitution in the EVH1 domain eliminated in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. A nonsense mutation that resulted in a C-terminally truncated Ena protein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 domain. Our analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena.

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Faculty Opinions recommendation of The Src homology 3 domain of the beta-subunit of voltage-gated calcium channels promotes endocytosis via dynamin interaction.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1064854.517791 ◽

2007 ◽

Author(s):

Brett Adams

Keyword(s):

Calcium Channels ◽

Beta Subunit ◽

Voltage Gated ◽

Src Homology 3 ◽

Voltage Gated Calcium Channels ◽

Src Homology 3 Domain ◽

Src Homology

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Faculty Opinions recommendation of Identification of a link between the SAMP repeats of adenomatous polyposis coli tumor suppressor and the Src homology 3 domain of DDEF.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1141847.598934 ◽

2009 ◽

Author(s):

Chloe Bulinski ◽

Andy Tran

Keyword(s):

Tumor Suppressor ◽

Adenomatous Polyposis Coli ◽

Adenomatous Polyposis ◽

Polyposis Coli ◽

Src Homology 3 ◽

Src Homology 3 Domain ◽

Src Homology

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Four proline-rich sequences of the guanine-nucleotide exchange factor C3G bind with unique specificity to the first Src homology 3 domain of Crk

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)30059-4 ◽

1994 ◽

Vol 269 (52) ◽

pp. 32781-32787

Author(s):

B S Knudsen ◽

S M Feller ◽

H Hanafusa

Keyword(s):

Guanine Nucleotide Exchange Factor ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Exchange Factor ◽

Src Homology 3 ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Src Homology 3 Domain ◽

Src Homology ◽

Unique Specificity

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Group I PAKs function downstream of Rac to promote podosome invasion during myoblast fusion in vivo

The Journal of Cell Biology ◽

10.1083/jcb.201204065 ◽

2012 ◽

Vol 199 (1) ◽

pp. 169-185 ◽

Cited By ~ 33

Author(s):

Rui Duan ◽

Peng Jin ◽

Fengbao Luo ◽

Guofeng Zhang ◽

Nathan Anderson ◽

...

Keyword(s):

Cell Shape ◽

Myoblast Fusion ◽

Small Gtpase ◽

Fusion Pore ◽

Cellular Processes ◽

Filament Assembly ◽

Group I ◽

Redundant Functions ◽

Shape Changes

The p21-activated kinases (PAKs) play essential roles in diverse cellular processes and are required for cell proliferation, apoptosis, polarity establishment, migration, and cell shape changes. Here, we have identified a novel function for the group I PAKs in cell–cell fusion. We show that the two Drosophila group I PAKs, DPak3 and DPak1, have partially redundant functions in myoblast fusion in vivo, with DPak3 playing a major role. DPak3 is enriched at the site of fusion colocalizing with the F-actin focus within a podosome-like structure (PLS), and promotes actin filament assembly during PLS invasion. Although the small GTPase Rac is involved in DPak3 activation and recruitment to the PLS, the kinase activity of DPak3 is required for effective PLS invasion. We propose a model whereby group I PAKs act downstream of Rac to organize the actin filaments within the PLS into a dense focus, which in turn promotes PLS invasion and fusion pore initiation during myoblast fusion.

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