SUMMARY Formins stimulate actin filament assembly for fundamental cellular processes including division, adhesion, establishing polarity and motility. A formin inhibitor would be useful because most cells express multiple formins whose functions are not known, and because metastatic tumor formation depends upon the deregulation of formin-dependent processes. We identified a general small molecule inhibitor of formin homology 2 domains (SMIFH2) by screening compounds for the ability to prevent formin-mediated actin assembly in vitro. SMIFH2 targets formins from evolutionarily diverse organisms including yeast, nematode worm and mice, with a half-maximal inhibitor concentration of ~5 to 15 μM. SMIFH2 prevents both formin nucleation and processive barbed-end elongation, and decreases formin’s affinity for the barbed end. Furthermore, low micromolar concentrations of SMIFH2 disrupt formin-dependent, but not Arp2/3 complex-dependent, actin cytoskeletal structures in fission yeast and mammalian NIH 3T3 fibroblasts.

The three fission yeast formins (Cdc12, For3, and Fus1) all nucleate actin assembly and remain continuously associated with the elongating actin filament barbed end, while incorporating thousands of actin monomers before dissociating. However, the specific rates for these reactions vary significantly and may therefore be functionally important.

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...

Like animal cells, fission yeast divides by assembling actin filaments into a contractile ring. In addition to formin Cdc12p and profilin, the single tropomyosin isoform SpTm is required for contractile ring assembly. Cdc12p nucleates actin filaments and remains processively associated with the elongating barbed end while driving the addition of profilin-actin. SpTm is thought to stabilize mature filaments, but it is not known how SpTm localizes to the contractile ring and whether SpTm plays a direct role in Cdc12p-mediated actin polymerization. Using "bulk" and single actin filament assays, we discovered that Cdc12p can recruit SpTm to actin filaments and that SpTm has diverse effects on Cdc12p-mediated actin assembly. On its own, SpTm inhibits actin filament elongation and depolymerization. However, Cdc12p completely overcomes the combined inhibition of actin nucleation and barbed end elongation by profilin and SpTm. Furthermore, SpTm increases the length of Cdc12p-nucleated actin filaments by enhancing the elongation rate twofold and by allowing them to anneal end to end. In contrast, SpTm ultimately turns off Cdc12p-mediated elongation by "trapping" Cdc12p within annealed filaments or by dissociating Cdc12p from the barbed end. Therefore, SpTm makes multiple contributions to contractile ring assembly during and after actin polymerization.

Cells contain multiple formin isoforms that drive the assembly of profilin-actin for diverse processes. Given that many organisms also contain several profilin isoforms, specific formin/ profilin pairs might be matched to optimally stimulate actin polymerization. We utilized a combination of bulk actin polymerization and single filament total internal reflection fluorescence microscopy assays to measure the effect of different profilin isoforms on the actin assembly properties of the cytokinesis formins from fission yeast (Cdc12p) and the nematode worm (CYK-1). We discovered that Cdc12p only effectively utilizes the single fission yeast profilin isoform SpPRF. Conversely, CYK-1 prefers the essential worm cytokinesis profilin CePFN-1 to the two non-essential worm profilin isoforms (SpPRF ‫؍‬ CePFN-1 > CePFN-2 > CePFN-3). Chimeras containing the profilin-binding formin homology 1 (FH1) domain from one formin and the barbed-end associated FH2 domain from the other formin, revealed that both the FH1 and FH2 domains help confer profilin isoform specialization. Although the Cdc12p and CYK-1 FH1 domains cannot differentiate between profilin isoforms in the absence of actin, formin FH1 domains appear to preferentially select specific isoforms of profilin-actin. Surprisingly, analysis of profilin point mutants revealed that differences in highly conserved residues in both the poly-L-proline and actin binding regions of profilin do not explain their differential utilization by formin. Therefore, rapid formin-mediated elongation of profilin-actin depends upon favorable interactions of profilin-actin with the FH1 domain as well as the barbed-end associated FH2 domain. Specific formin FH1FH2 domains are tailored to optimally utilize actin bound to particular profilin isoforms.Formin proteins assemble actin filaments for various cellular processes such as division, motility, establishing polarity and adhesion (1-3). The formin family is diverse with at least 20 formin genes in plants, 18 in mammals, six each in Drosophila melanogaster and Caenorhabditis elegans, three in fission yeast and two in budding yeast (4). A prevalent model is that multiple formin isoforms are necessary because they are required for disparate cellular processes. Although we do not yet know the cellular role(s) of many formins, the nematode worm C. elegans formin CYK-1 and the fission yeast Schizosaccharomyces pombe formin Cdc12p are required for cytokinesis (5-7).The molecular basis for the functional specificity of formins is not entirely clear, but likely involves a combination of regulatory and actin assembly properties. Formins are large multidomain proteins that contain a highly conserved actin assembly formin homology 2 (FH2) 2 domain and associated proline-rich profilin-binding FH1 domain, which are flanked on either side by regulatory domains. Some formins are regulated by auto-inhibition through association of their N-and C-terminal regulatory domains (1,2,8). Activated RhoGTPase binds to the N-terminal regulatory region and dissociates the C...