Loss of Ena/VASP Interferes with Lamellipodium Architecture, Motility and Integrin-Dependent Adhesion

Damiano-Guercio, Julia; Kurzawa, Laëtitia; Mueller, Jan O.; Dimchev, Georgi; Nemethova, Maria; Bruehmann, Stefan; Linkner, Joern; Blanchoin, Laurent; Sixt, Michael; Rottner, Klemens; Faix, Jan

doi:10.2139/ssrn.3454865

Cited by 21 publications

(48 citation statements)

References 94 publications

(157 reference statements)

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“…9E). We should note that additional actin polymerases have previously been implicated in filopodia formation, foremost of all Ena/VASP family members (Dent et al, 2007), although they are certainly not obligatory for these structures (Damiano-Guercio et al, 2020). It is tempting to speculate that during spreading in the presence of Arp2/3 complex, Ena/VASP proteins are more relevant for the formation of the filopodia formed under these conditions than FMNL2/3 proteins.…”

Section: Resultsmentioning

confidence: 98%

Induced Arp2/3 complex depletion increases FMNL2/3 formin expression and filopodia formation

Dimchev

Lahmann

Koestler

et al. 2020

Preprint

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The Arp2/3 complex generates branched actin filament networks operating in cell edge protrusion and vesicle trafficking. Here we employ a novel, conditional knockout mouse model permitting tissue- or cell-type specific deletion of the murine Actr3 gene (encoding Arp3). A functional Actr3 gene appeared essential for fibroblast viability and growth. Thus, we developed cell lines for exploring the consequences of acute, tamoxifen-induced Actr3 deletion causing near-complete loss of Arp/3 complex function as well as abolished lamellipodia formation and membrane ruffling, as expected. However, Arp3-depleted cells displayed enhanced rather than reduced cell spreading, employing numerous filopodia, and showed little defects in individual cell migration. Reduction of collective cell migration as observed for instance in wound healing assays likely derived from defects in maintaining directionality during migration, while the principal ability to chemotax was only moderately affected. Analyses of actin turnover at the cell periphery revealed reduced actin turnover rates in Arp2/3-deficient cells, clearly deviating from previous sequestration approaches. Most surprisingly, induced removal of Arp2/3 complexes reproducibly increased FMNL formin expression, which correlated with the explosive induction of filopodia formation. Our results thus highlight both direct and indirect effects of acute Arp2/3 complex removal on actin cytoskeleton regulation.

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Section: Resultsmentioning

confidence: 98%

Induced Arp2/3 complex depletion increases FMNL2/3 formin expression and filopodia formation

Dimchev

Lahmann

Koestler

et al. 2020

Preprint

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“…One class of filopodia grows from dynamic, lamellipodial actin networks by a process of “convergent elongation.” In this mechanism, the growing barbed ends of several preexisting actin filaments converge to a point in the plasma membrane where they are held together by a self-assembling filopodial “tip complex” ( Lewis and Bridgman, 1992 ; Svitkina et al , 2003 ; Mogilner and Rubinstein, 2005 ). The tip complex contains actin polymerases, such as Ena/VASP-family proteins that accelerate filament growth and inhibit filament capping ( Breitsprecher et al , 2008 ; Hansen and Mullins , 2010 ) and are required for the formation of filopodia in several cell types ( Kwiatkowski et al , 2007 ; Damiano-Guercio et al , 2020 ). The spatial constraint imposed on growing barbed ends of filaments associated with tip complexes causes them to become aligned and subsequently crosslinked by the protein fascin ( Vignjevic et al , 2003 ).…”

Section: Introductionmentioning

confidence: 99%

Initiation and disassembly of filopodia tip complexes containing VASP and lamellipodin

Cheng

Mullins

2020

MBoC

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The shape of many eukaryotic cells depends on the actin cytoskeleton, and changes in actin assembly dynamics underlie many changes in cell shape. Ena/VASP-family actin polymerases, for example, modulate cell shape by locally accelerating actin filament assembly and slowing filament capping. When concentrated into discrete foci at the leading edge, VASP promotes filopodia assembly, and forms part of a poorly understood molecular complex that remains associated with growing filopodia tips. Here we identify precursors of this filopodia tip complex in migrating B16F1 cells: small leading-edge clusters of the adaptor protein lamellipodin (Lpd) which subsequently recruit VASP and initiate filopodia formation. Dimerization, membrane association, and VASP binding are all required for lamellipodin to incorporate into filopodia tip complexes, and over-expression of monomeric, membrane-targeted lamellipodin mutants disrupts tip complex assembly. Once formed, tip complexes containing VASP and lamellipodin grow by fusing with each other, but their growth is limited by a size-dependent, dynamic instability. Our results demonstrate that assembly and disassembly dynamics of filopodia tip complexes are determined, in part, by a network of multivalent interactions between Ena/VASP proteins, EVH1 ligands, and actin filaments. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

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“…To test whether PCARE B could selectively bind to ENAH in cells, we used Ena/VASP-family-deficient cell line MV D7 (40) and expressed the individual paralogs ENAH, EVL, and VASP to evaluate their interactions with PCARE. MV D7 are embryonic fibroblasts derived from ENAH −/− VASP −/− mice, with low EVL expression (41). We used an shRNA against EVL to further decrease residual EVL (Figure S5).…”

Section: Resultsmentioning

confidence: 99%

Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP proteins

Hwang

Grant

Ilunga

et al. 2021

Preprint

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Protein interactions between intrinsically disordered, short linear motifs (SLiMs) and modular recognition domains are critical elements in many signal transduction pathways. Yet for most interactions, it is still unclear how low-complexity SLiMs discriminate between highly conserved but biologically distinct SLiM-binding proteins. The Ena/VASP family of proteins pose one such specificity problem. Paralogs ENAH, VASP, and EVL bind to a 5-residue SLiM prevalent in the proteome and perform distinct cellular functions despite sharing high sequence and structural similarity. To interrogate how the sequence context of SLiMs impacts Ena/VASP interactions, we performed an unbiased proteomic screen against the ENAH EVH1 domain. We discovered unexpected ways in which local and distal sequence elements flanking native SLiMs modulate binding. Particularly notable is a peptide from PCARE that achieves paralog specificity by stabilizing a unique conformation adopted only by ENAH. A PCARE-derived peptide can selectively recruit ENAH and block its activity in cells, establishing it as a valuable reagent to disentangle the roles of Ena/VASP paralogs and a potential agent for modulating ENAH function in breast cancer. Guided by our analyses of native interactions, we designed the tightest known ENAH EVH1 binder with a dissociation constant of 50 nM and 400-600-fold selectivity over EVL and VASP. Our work demonstrates that sequence context plays a prominent role in dictating SLiM interactions and can inform the design of custom molecules that target SLiM-based interactions.

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Loss of Ena/VASP Interferes with Lamellipodium Architecture, Motility and Integrin-Dependent Adhesion

Cited by 21 publications

References 94 publications

Induced Arp2/3 complex depletion increases FMNL2/3 formin expression and filopodia formation

Induced Arp2/3 complex depletion increases FMNL2/3 formin expression and filopodia formation

Initiation and disassembly of filopodia tip complexes containing VASP and lamellipodin

Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP proteins

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