Formin and capping protein together embrace the actin filament in a ménage à trois

Shekhar, Shashank; Kerleau, Mikaël; Kühn, Sonja; Pernier, Julien; Romet-Lemonne, Guillaume; Jégou, Antoine; Carlier, Marie-France

doi:10.1038/ncomms9730

Cited by 91 publications

(129 citation statements)

References 51 publications

Supporting

Mentioning

118

Contrasting

Order By: Relevance

“…Formins and VASP, conversely, consider CP a "pest" and use physical mechanisms to keep it at bay as they build linear actin structures such as filopodia (48,49). Importantly, although formins are fairly effective at physically shielding the barbed end from CP, it is likely that their robustness as filopodia generators in vivo would be increased by a reduction in CP levels (13,14). Given all this information, interventions that impair CP function, such as CP knockdown or V-1 overexpression, would be predicted to reduce the formation of cortical actin structures built by the Arp2/3 complex (lamellipodia and pseudopodia) and promote the formation of cortical actin structures built by formins and VASP (filopodia).…”

Section: Discussionmentioning

confidence: 99%

“…The extension of filopodia likely involves the action of formins and/or VASP (9)(10)(11)(12), two actin polymerization machines that operate at the growing barbed end as processive polymerases to create the linear actin filaments that fill filopodia. Although both proteins are fairly effective at physically shielding the barbed end from CP (10,13,14), it is likely that their robustness as filopodia generators in vivo would be increased by a reduction in CP levels. Given the recent work demonstrating that formins and the Arp2/3 complex compete for G-actin in vivo (15)(16)(17), the increase in filopodia number seen upon CP knockdown may also be due in part to an increase in the amount of monomer available for formin/VASP after the reduction in Arp2/ 3-dependent nucleation caused by CP knockdown.…”

mentioning

confidence: 99%

See 1 more Smart Citation

V-1 regulates capping protein activity in vivo

Jung

Alexander

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Capping Protein (CP) plays a central role in the creation of the Arp2/ 3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."T he addition of Capping Protein (CP) to seed-initiated actin polymerization assays results in the rapid cessation of polymerization because CP binds with very high affinity to the fastgrowing barbed end of the actin filament to block further monomer addition (1). Direct extrapolation of this simple, potent biochemical property would suggest that the cell's content of F-actin should rise and fall as its content of CP is artificially forced to fall and rise, respectively. Indeed, this finding was reported many years ago in Dictyostelium amoeba (2). This simple view of CP's role in regulating actin assembly in vivo falls short of the whole story, however. The additional complexity arises from the critical relationship between CP and the Arp2/3 complex, the major actin nucleating machine that generates the branched actin networks comprising lamellipodia and pseudopodia (3). At the heart of this relationship is the fact that CP increases the rate of Arp2/3-dependent filament nucleation and promotes optimal branching by rapidly capping filaments (4). As a result, CP promotes actin-related proteins 2 and 3 (Arp2/3)-driven actin assembly and motility (4, 5). This effect was evident from early solution experiments focused on defining the function of the Arp2/3 complex (6), verified by in vitro reconstitution of the Arp2/3-dependent motility of Listeria (5), and explained mecha...

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

V-1 regulates capping protein activity in vivo

Jung

Alexander

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…In this transient ternary complex, each of the two ligands displays a lower affinity for the barbed end than when it binds individually. The WH2 domain present in VopF, an effector of the pathogen Vibrio cholerae, and the FH2 domain of formins, have been identified as such 'uncappers' (Pernier et al, 2013;Shekhar et al, 2015). VopF, owing to its structural organization with multimerized WH2 repeats, has been proposed as a functional homolog model for Ena or VASP family proteins (Pernier et al, 2013).…”

Section: Barbed-end Regulation In the Cytoplasmmentioning

confidence: 99%

“…In addition to the simple mechanism of a mutually exclusive binding of two proteins to barbed ends, two different barbed-end-binding proteins can also bind simultaneously to barbed ends with mutually reduced affinities and enhanced dissociation rates (Bombardier et al, 2015;Shekhar and Carlier, 2016;Shekhar et al, 2015). Thus, fast changes in reactivity of the barbed ends, from being blocked to full speed growth are elicited.…”

Section: Introductionmentioning

confidence: 99%

Regulators of actin filament barbed ends at a glance

Shekhar

Pernier

Carlier

2016

Journal of Cell Science

Self Cite

View full text Add to dashboard Cite

Cells respond to external stimuli by rapidly remodeling their actin cytoskeleton. At the heart of this function lies the intricately controlled regulation of individual filaments. The barbed end of an actin filament is the hotspot for the majority of the biochemical reactions that control filament assembly. Assays performed in bulk solution and with single filaments have enabled characterization of a plethora of barbed-endregulating proteins. Interestingly, many of these regulators work in tandem with other proteins, which increase or decrease their affinity for the barbed end in a spatially and temporally controlled manner, often through simultaneous binding of two regulators at the barbed ends, in addition to standard mutually exclusive binding schemes. In this Cell Science at a Glance and the accompanying poster, we discuss key barbed-end-interacting proteins and the kinetic mechanisms by which they regulate actin filament assembly. We take F-actin capping protein, gelsolin, profilin and barbed-endtracking polymerases, including formins and WH2-domaincontaining proteins, as examples, and illustrate how their activity and competition for the barbed end regulate filament dynamics.

show abstract

“…Spire and Fmn2 stand as the second example of two proteins regulating filament assembly by binding together at the barbed end in a transient ternary complex. Other examples include the multiple WH2 domain protein VopF and Capping Protein (22) and more recently formin and Capping Protein (23,24).…”

mentioning

confidence: 99%

Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments

Montaville¹,

Kühn²,

Compper

et al. 2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Formin 2 (Fmn2), a member of the FMN family of formins, plays an important role in early development. This formin cooperates with profilin and Spire, a WASP homology domain 2 (WH2) repeat protein, to stimulate assembly of a dynamic cytoplasmic actin meshwork that facilitates translocation of the meiotic spindle in asymmetric division of mouse oocytes. The kinase-like non-catalytic domain (KIND) of Spire directly interacts with the C-terminal extension of the formin homology domain 2 (FH2) domain of Fmn2, called FSI. This direct interaction is required for the synergy between the two proteins in actin assembly. We have recently demonstrated how Spire, which caps barbed ends via its WH2 domains, activates Fmn2. Fmn2 by itself associates very poorly to filament barbed ends but is rapidly recruited to Spire-capped barbed ends via the KIND domain, and it subsequently displaces Spire from the barbed end to elicit rapid processive assembly from profilin⅐actin. Here, we address the mechanism by which Spire and Fmn2 compete at barbed ends and the role of FSI in orchestrating this competition as well as in the processivity of Fmn2. We have combined microcalorimetric, fluorescence, and hydrodynamic binding assays, as well as bulk solution and single filament measurements of actin assembly, to show that removal of FSI converts Fmn2 into a Capping Protein. This activity is mimicked by association of KIND to Fmn2. In addition, FSI binds actin at filament barbed ends as a weak capper and plays a role in displacing the WH2 domains of Spire from actin, thus allowing the association of actin-binding regions of FH2 to the barbed end.Polarized assembly of actin filaments is pivotal in motile processes. It is precisely regulated by proteins that bind the barbed ends of actin filaments and either block or assist filament assembly using various mechanisms (1). Among these regulators, formins are acknowledged to nucleate and track filament barbed ends, mediating rapid processive elongation of filaments (2, 3). In contrast, WASP homology 2 (WH2) 3 repeatcontaining proteins display versatile barbed end capping or tracking and filament severing activities (4). In early oogenesis of Drosophila as well as in mammalian meiosis, a formin (Fmn2/Cappuccino) and a WH2-repeat protein (Spire) synergize in an intriguing fashion to stimulate massive assembly of a dynamic cytoplasmic actin meshwork, required for completion of crucial steps in axis patterning or asymmetric division (5-12). This complexity adds to the known need of profilin for rapid processive assembly of all formins, including Cappuccino (9). Like most formins, Fmn2 harbors conserved C-terminal formin homology 1 (FH1) and formin homology 2 (FH2) domains. The C-terminal FH1-FH2 domain of Fmn2 represents a constitutive active module in which the FH1 domain binds profilin and the FH2 domain to the barbed end of F-actin. The head-to-tail FH2 dimer of formins is thought, from structural data, to encircle the terminal and subterminal actin subunits that constitute the barbed end of the fil...

show abstract

Formin and capping protein together embrace the actin filament in a ménage à trois

Cited by 91 publications

References 51 publications

V-1 regulates capping protein activity in vivo

V-1 regulates capping protein activity in vivo

Regulators of actin filament barbed ends at a glance

Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments

Contact Info

Product

Resources

About