Regulated Interactions between Dynamin and the Actin-Binding Protein Cortactin Modulate Cell Shape

The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes. We have observed that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods we demonstrate that the cortactin–SH3 domain associates with the proline-rich domain (PRD) of dynamin. Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortΔSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)ΔPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.

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Mammalian Abp1, a Signal-Responsive F-Actin–Binding Protein, Links the Actin Cytoskeleton to Endocytosis via the Gtpase Dynamin

The Journal of Cell Biology ◽

10.1083/jcb.153.2.351 ◽

2001 ◽

Vol 153 (2) ◽

pp. 351-366 ◽

Cited By ~ 156

Author(s):

Michael M. Kessels ◽

Åsa E.Y. Engqvist-Goldstein ◽

David G. Drubin ◽

Britta Qualmann

Keyword(s):

Actin Cytoskeleton ◽

Binding Protein ◽

Sh3 Domain ◽

Actin Binding ◽

Actin Binding Protein ◽

Domain Interactions ◽

Endocytic Machinery ◽

Transferrin Uptake

The actin cytoskeleton has been implicated in endocytosis, yet few molecular links to the endocytic machinery have been established. Here we show that the mammalian F-actin–binding protein Abp1 (SH3P7/HIP-55) can functionally link the actin cytoskeleton to dynamin, a GTPase that functions in endocytosis. Abp1 binds directly to dynamin in vitro through its SH3 domain. Coimmunoprecipitation and colocalization studies demonstrated the in vivo relevance of this interaction. In neurons, mammalian Abp1 and dynamin colocalized at actin-rich sites proximal to the cell body during synaptogenesis. In fibroblasts, mAbp1 appeared at dynamin-rich sites of endocytosis upon growth factor stimulation. To test whether Abp1 functions in endocytosis, we overexpressed several Abp1 constructs in Cos-7 cells and assayed receptor-mediated endocytosis. While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake. This implicates the Abp1/dynamin interaction in endocytic function. The endocytosis block was rescued by cooverexpression of dynamin. Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.

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Clathrin Hub Expression Dissociates the Actin-Binding Protein Hip1R from Coated Pits and Disrupts Their Alignment with the Actin Cytoskeleton

Traffic ◽

10.1034/j.1600-0854.2001.21114.x ◽

2001 ◽

Vol 2 (11) ◽

pp. 851-858 ◽

Cited By ~ 31

Author(s):

Elizabeth M. Bennett ◽

Chih-Ying Chen ◽

Asa E. Y. Engqvist-Goldstein ◽

David G. Drubin ◽

Frances M. Brodsky

Keyword(s):

Actin Cytoskeleton ◽

Binding Protein ◽

Actin Binding ◽

Coated Pits ◽

Actin Binding Protein

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Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

The Journal of Cell Biology ◽

10.1083/jcb.200206113 ◽

2002 ◽

Vol 159 (6) ◽

pp. 993-1004 ◽

Cited By ~ 126

Author(s):

Christine L. Humphries ◽

Heath I. Balcer ◽

Jessica L. D'Agostino ◽

Barbara Winsor ◽

David G. Drubin ◽

...

Keyword(s):

Binding Protein ◽

Coiled Coil ◽

Actin Binding ◽

Complex Activity ◽

Actin Binding Protein ◽

Coiled Coil Domain ◽

Allele Specific ◽

And Function

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.

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Identification of a Novel Cortactin SH3 Domain-Binding Protein and Its Localization to Growth Cones of Cultured Neurons

Molecular and Cellular Biology ◽

10.1128/mcb.18.10.5838 ◽

1998 ◽

Vol 18 (10) ◽

pp. 5838-5851 ◽

Cited By ~ 164

Author(s):

Yunrui Du ◽

Scott A. Weed ◽

Wen-Cheng Xiong ◽

Trudy D. Marshall ◽

J. Thomas Parsons

Keyword(s):

Hippocampal Neurons ◽

Binding Protein ◽

Consensus Sequence ◽

Growth Cones ◽

Sh3 Domain ◽

Actin Binding ◽

Sequence Motifs ◽

Cortical Actin ◽

Cytoskeleton Reorganization

ABSTRACT Cortactin is an actin-binding protein that contains several potential signaling motifs including a Src homology 3 (SH3) domain at the distal C terminus. Translocation of cortactin to specific cortical actin structures and hyperphosphorylation of cortactin on tyrosine have been associated with the cortical cytoskeleton reorganization induced by a variety of cellular stimuli. The function of cortactin in these processes is largely unknown in part due to the lack of information about cellular binding partners for cortactin. Here we report the identification of a novel cortactin-binding protein of approximately 180 kDa by yeast two-hybrid interaction screening. The interaction of cortactin with this 180-kDa protein was confirmed by both in vitro and in vivo methods, and the SH3 domain of cortactin was found to direct this interaction. Since this protein represents the first reported natural ligand for the cortactin SH3 domain, we designated it CortBP1 for cortactin-binding protein 1. CortBP1 contains two recognizable sequence motifs within its C-terminal region, including a consensus sequence for cortactin SH3 domain-binding peptides and a sterile alpha motif. Northern and Western blot analysis indicated that CortBP1 is expressed predominately in brain tissue. Immunofluorescence studies revealed colocalization of CortBP1 with cortactin and cortical actin filaments in lamellipodia and membrane ruffles in fibroblasts expressing CortBP1. Colocalization of endogenous CortBP1 and cortactin was also observed in growth cones of developing hippocampal neurons, implicating CortBP1 and cortactin in cytoskeleton reorganization during neurite outgrowth.

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Proteomic profiling reveals that EhPC4 transcription factor induces cell migration through up-regulation of the 16-kDa actin-binding protein EhABP16 in Entamoeba histolytica

Journal of Proteomics ◽

10.1016/j.jprot.2014.03.041 ◽

2014 ◽

Vol 111 ◽

pp. 46-58 ◽

Cited By ~ 16

Author(s):

Olga Hernández de la Cruz ◽

Marcos Muñiz-Lino ◽

Nancy Guillén ◽

Christian Weber ◽

Laurence A. Marchat ◽

...

Keyword(s):

Transcription Factor ◽

Cell Migration ◽

Entamoeba Histolytica ◽

Binding Protein ◽

Actin Binding ◽

Proteomic Profiling ◽

Actin Binding Protein

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AIM1 is an actin-binding protein that suppresses cell migration and micrometastatic dissemination

Nature Communications ◽

10.1038/s41467-017-00084-8 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 13

Author(s):

Michael C. Haffner ◽

David M. Esopi ◽

Alcides Chaux ◽

Meltem Gürel ◽

Susmita Ghosh ◽

...

Keyword(s):

Cell Migration ◽

Binding Protein ◽

Actin Binding ◽

Actin Binding Protein

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α-Catenin-independent Recruitment of ZO-1 to Nectin-based Cell-Cell Adhesion Sites through Afadin

Molecular Biology of the Cell ◽

10.1091/mbc.12.6.1595 ◽

2001 ◽

Vol 12 (6) ◽

pp. 1595-1609 ◽

Cited By ~ 67

Author(s):

Shigekazu Yokoyama ◽

Kouichi Tachibana ◽

Hiroyuki Nakanishi ◽

Yasunori Yamamoto ◽

Kenji Irie ◽

...

Keyword(s):

Cell Adhesion ◽

Actin Cytoskeleton ◽

Tight Junctions ◽

Cell Lines ◽

Adhesion Molecule ◽

Binding Protein ◽

Actin Binding ◽

Actin Binding Protein ◽

Adhesion Sites ◽

Cell Cell

ZO-1 is an actin filament (F-actin)–binding protein that localizes to tight junctions and connects claudin to the actin cytoskeleton in epithelial cells. In nonepithelial cells that have no tight junctions, ZO-1 localizes to adherens junctions (AJs) and may connect cadherin to the actin cytoskeleton indirectly through β- and α-catenins as one of many F-actin–binding proteins. Nectin is an immunoglobulin-like adhesion molecule that localizes to AJs and is associated with the actin cytoskeleton through afadin, an F-actin–binding protein. Ponsin is an afadin- and vinculin-binding protein that also localizes to AJs. The nectin-afadin complex has a potency to recruit the E-cadherin–β-catenin complex through α-catenin in a manner independent of ponsin. By the use of cadherin-deficient L cell lines stably expressing various components of the cadherin-catenin and nectin-afadin systems, and α-catenin–deficient F9 cell lines, we examined here whether nectin recruits ZO-1 to nectin-based cell-cell adhesion sites. Nectin showed a potency to recruit not only α-catenin but also ZO-1 to nectin-based cell-cell adhesion sites. This recruitment of ZO-1 was dependent on afadin but independent of α-catenin and ponsin. These results indicate that ZO-1 localizes to cadherin-based AJs through interactions not only with α-catenin but also with the nectin-afadin system.

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Actin cytoskeleton remodelling in the anterior pituitary folliculostellate cell line TtT/GF: participation of the actin-binding protein cortactin

Journal of Molecular Histology ◽

10.1007/s10735-006-9021-1 ◽

2006 ◽

Vol 36 (8-9) ◽

pp. 461-474 ◽

Cited By ~ 5

Author(s):

Guifu Zheng ◽

Sara Solinet ◽

R.-Marc. Pelletier ◽

María Leiza Vitale

Keyword(s):

Actin Cytoskeleton ◽

Cell Line ◽

Anterior Pituitary ◽

Binding Protein ◽

Actin Binding ◽

Actin Binding Protein ◽

Folliculostellate Cell

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Actin-binding protein G (AbpG) participates in modulating the actin cytoskeleton and cell migration in Dictyostelium discoideum

Molecular Biology of the Cell ◽

10.1091/mbc.e14-05-0972 ◽

2015 ◽

Vol 26 (6) ◽

pp. 1084-1097 ◽

Cited By ~ 3

Author(s):

Wei-Chi Lin ◽

Liang-Chen Wang ◽

Te-Ling Pang ◽

Mei-Yu Chen

Keyword(s):

Cell Migration ◽

Dictyostelium Discoideum ◽

Molecular Mechanisms ◽

Binding Protein ◽

Protein Domain ◽

Actin Dynamics ◽

Actin Binding ◽

Protein G ◽

Wild Type ◽

Actin Binding Protein

Cell migration is involved in various physiological and pathogenic events, and the complex underlying molecular mechanisms have not been fully elucidated. The simple eukaryote Dictyostelium discoideum displays chemotactic locomotion in stages of its life cycle. By characterizing a Dictyostelium mutant defective in chemotactic responses, we identified a novel actin-binding protein serving to modulate cell migration and named it actin-binding protein G (AbpG); this 971–amino acid (aa) protein contains an N-terminal type 2 calponin homology (CH2) domain followed by two large coiled-coil regions. In chemoattractant gradients, abpG− cells display normal directional persistence but migrate significantly more slowly than wild-type cells; expressing Flag-AbpG in mutant cells eliminates the motility defect. AbpG is enriched in cortical/lamellipodial regions and colocalizes well with F-actin; aa 401–600 and aa 501–550 fragments of AbpG show the same distribution as full-length AbpG. The aa 501–550 region of AbpG, which is essential for AbpG to localize to lamellipodia and to rescue the phenotype of abpG− cells, is sufficient for binding to F-actin and represents a novel actin-binding protein domain. Compared with wild-type cells, abpG− cells have significantly higher F-actin levels. Collectively our results suggest that AbpG may participate in modulating actin dynamics to optimize cell locomotion.

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