The Role of the FH1 Domain and Profilin in Formin-Mediated Actin-Filament Elongation and Nucleation

Formins assemble non-branched actin filaments and modulate microtubule dynamics during cell migration and cell division. At the end of mitosis formins contribute to the generation of actin filaments that form the contractile ring. Rho small GTPbinding proteins activate mammalian diaphanous-related (mDia) formins by directly binding and disrupting an intramolecular autoinhibitory mechanism. Although the Rho-regulated activation mechanism is well characterized, little is known about how formins are switched off. Here we reveal a novel mechanism of formin regulation during cytokinesis based on the following observations; 1) mDia2 is degraded at the end of mitosis, 2) mDia2 is targeted for disposal by post-translational ubiquitin modification, 3) forced expression of activated mDia2 yields binucleate cells due to failed cytokinesis, and 4) the cytokinesis block is dependent upon mDia2-mediated actin assembly as versions of mDia2 incapable of nucleating actin but that still stabilize microtubules have no effect on cytokinesis. We propose that the tight control of mDia2 expression and ubiquitin-mediated degradation is essential for the completion of cell division. Because of the many roles for formins in cell morphology, we discuss the relevance of mDia protein turnover in other processes where ubiquitin-mediated proteolysis is an essential component.

Section: Discussionmentioning

confidence: 99%

Ubiquitin-mediated Degradation of the Formin mDia2 upon Completion of Cell Division

DeWard

Alberts

2009

“…This binding reaction is rate-limiting for elongation of actin filaments by formins such as Cdc12p that strongly inhibit actin filament elongation in the absence of profilin (16,47). The second difference is the presence of wider wings on Hs profilin formed by 10 extra residues not present is fission yeast profilin (Fig.…”

Section: Tablementioning

confidence: 99%

Incompatibility with Formin Cdc12p Prevents Human Profilin from Substituting for Fission Yeast Profilin

Ezezika

Younger

et al. 2009

The small protein profilin not only helps to maintain a cytoplasmic pool of actin monomers ready to elongate actin filament barbed ends (2), but it also binds to type II poly-L-proline helices (3, 4). The actin (5) and poly-L-proline (6 -8) binding sites are on opposite sides of the profilin molecule, so profilin can link actin to proline-rich targets. Viability of fission yeast depends independently on profilin binding to both actin and poly-L-proline, although cells survive Ͼ10-fold reductions in affinity for either ligand (1).Fission yeast Schizosaccharomyces pombe depend on formin Cdc12p (9, 10) and profilin (11) to assemble actin filaments for the cytokinetic contractile ring. Formins are multidomain proteins that nucleate and assemble unbranched actin filaments (12). Formin FH2 domains form homodimers that can associate processively with the barbed ends of growing actin filaments (13,14). FH2 dimers slow the elongation of barbed ends (15). Most formin proteins have an FH1 domain linked to the FH2 domain. Binding profilin-actin to multiple polyproline sites in an FH1 domain concentrates actin near the barbed end of an actin filament associated with a formin FH2 homodimer. Actin transfers very rapidly from the FH1 domains onto the filament end (16) allowing profilin to stimulate elongation of the filament (15, 17).We tested the ability of human (Homo sapiens, Hs) 7 profilin-I to complement the temperature-sensitive cdc3-124 mutation (11) in the single fission yeast profilin gene with the aim of using yeast to characterize human profilin mutations. The failure of expression of Hs profilin-I to complement the cdc3-124 mutation prompted us to compare human and fission yeast profilins more carefully. We report here a surprising incompatibility of Hs profilin-I with fission yeast formin Cdc12p, a crystal structure of fission yeast profilin, which allowed a detailed comparison with Hs profilin, and mutations that revealed how overexpression of Hs profilin-I compromises the viability of wild-type fission yeast. EXPERIMENTAL PROCEDURESStrains, Media, and Chemicals-Kathleen Gould of Vanderbilt University provided strains of S. pombe: KYG491, a haploid temperature-sensitive strain with a point mutation in the * This work was supported, in whole or in part, by National Institutes of Health Grants GM-026338 and GM-066311 (to T. D. P.) and GM-079265 (to D. R. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5. The atomic coordinates and structure factors (codes 3D9Y and 3DAV)

“…Formins increase the elongation rate of profilin-actin through a combination of increasing the local actin concentration and steering profilin-actin monomers onto the barbed end (22,25,26,40). Various formins increase the elongation rate of profilin-actin by different extents.…”

Section: Actin Assembly Properties Of the Cytokinesis Formins Cdc12p mentioning

confidence: 99%

“…Additionally, the rates of actin assembly may also be important. Several properties differ significantly between diverse formins, such as the actin filament elongation rate, which can vary almost 10-fold (22)(23)(24)(25)(26).…”

mentioning

confidence: 99%

The Cytokinesis Formins from the Nematode Worm and Fission Yeast Differentially Mediate Actin Filament Assembly

Neidt

Skau

Kovar

2008

Formins drive actin filament assembly for diverse cellular processes including motility, establishing polarity, and cell division. To investigate the mechanism of contractile ring assembly in animal cells, we directly compared the actin assembly properties of formins required for cytokinesis in the nematode worm early embryo (CYK-1) and fission yeast (Cdc12p). Like Cdc12p and most other formins, CYK-1 nucleates actin filament assembly and remains processively associated with the elongating barbed end while facilitating the addition of profilin-actin above the theoretical diffusion-limited rate. However, specific properties differ significantly between Cdc12p and CYK-1. Cdc12p efficiently nucleates filaments that in the presence of profilin elongate at approximately the same rate as control filaments without formin (ϳ10.0 subunits/s). CYK-1 is an inefficient nucleator but allows filaments to elongate profilin-actin 6-fold faster than Cdc12p (ϳ60 subunits/s). Both Cdc12p and CYK-1 bind to pre-assembled actin filaments with low nanomolar affinity, but CYK-1 dissociates 2 orders of magnitude more quickly. However, CYK-1 rapidly re-associates with free barbed ends. Cdc12p allows barbed ends to elongate in the presence of excess capping protein, whereas capping protein inhibits CYK-1-mediated actin assembly. Therefore, these evolutionarily diverse formins can drive contractile ring assembly by a generally similar mechanism, but cells with unique dimensions and physical parameters might require proteins with carefully tuned actin assembly properties.The final step of cell division is cytokinesis, the physical separation of a mother cell into two daughter cells (1, 2). Animal cells spatially and temporally coordinate cleavage site positioning through both astral and spindle microtubules (3, 4). Upon choosing a division site, far less is known about how actin and myosin II motor filaments assemble into the contractile ring. The mechanism is clearer in the unicellular fission yeast Schizosaccharomyces pombe (see Fig. 7A) (5). The fission yeast contractile ring is constructed from ϳ60 "pre-ring" nodes through the coordinated activities of numerous proteins including the anillin-like protein Mid1p (6 -8), the type II myosin motor Myo2p (9 -11), the actin monomer binding protein profilin Cdc3p/SpPRF (12), and the actin filament nucleator formin Cdc12p (13).A major question is whether animal cells assemble the contractile ring by a similar mechanism as fission yeast. Many of the proteins required for cytokinesis are conserved between fission yeast and animal cells including anillin, type II myosin, profilin, and formin (1, 2). Formins are large multidomain proteins that, in addition to cytokinesis, assemble actin filaments for multiple cellular processes including motility and establishing polarity (14 -16). Formins contain a highly conserved actin assembly FH2 2 domain, and associated proline-rich profilin binding FH1 domain, which are flanked on either side by regulatory domains.The formin family is large and diverse with a...