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

Abstract

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...

Actin Cross-Linking Toxin Is a Universal Inhibitor of Tandem-Organized and Oligomeric G-Actin Binding Proteins

“…Dissociation of VASP coincided with the disruption of focal adhesion contacts to less organized, smaller aggregates that were poorly associated with actin stress fibers (Figure 4D). Given the highly promiscuous binding abilities of the oligomers, it is conceivable that in such aggregates VASP is linked by the oligomers to other multivalent actin-binding proteins (e.g., NPFs, formins, and Spire [42, 45-47]) that often function within proximity of each other. Inhibition of Ena by the oligomers and disruption of VASP-containing structures suggests that the previously reported drop in epithelial integrity [13] may be mediated not only by inhibition of formins, but also of Ena/VASP proteins.…”

Regulators of actin filament barbed ends at a glance

“…The association of Fmn2 to Spirebound barbed ends is followed by fast processive assembly, during which Spire is displaced from the barbed end (Montaville et al, 2014). Recent studies indicate that the FSI region also binds to barbed ends in a similar manner to a WH2 domain and is required for the processive activity of Fmn2 (Montaville et al, 2016;Vizcarra et al, 2014). Accordingly, deletion of the FSI region converts Fmn2 into a capping protein that is bound to barbed ends in the 'closed' conformation.…”

“…Accordingly, deletion of the FSI region converts Fmn2 into a capping protein that is bound to barbed ends in the 'closed' conformation. The FSI domain might increase the fraction of time spent by Fmn2 in the 'open' conformation by outcompeting the knob region of FH2 (Montaville et al, 2016).…”

Development of motor neurons and motor activity in zebrafish requires F-actin nucleation by Fmn2b

“…The F-actin nucleation and elongation activity of Fmn2 has been characterized in vitro (Montaville et al, 2014(Montaville et al, , 2016. Abrogation of F-actin nucleation activity of Fmn2 can be achieved by mutating a conserved Isoleucine residue in the FH2 domain to Alanine (Quinlan et al, 2007;Roth-Johnson et al, 2014;Kundu et al, 2020).…”