Christopher J. Gould

Rocket launcher mechanism of collaborative actin assembly defined by single-molecule imaging

See How They Grow Controlled assembly and disassembly of the actin cytoskeleton is essential for processes such as cell motility, cytokinesis, and tumor metastasis. The formation of new actin filaments appears to involve the protein formin paired with another actin assembly-promoting factor. Breitsprecher et al. (p. 1164) used triplecolor single-molecule fluorescence microscopy to visualize actin assembly promoted by the formin, mDia1, and the tumor-suppressor, adenomatous polyposis coli (APC). The two assembly factors interacted directly to initiate filament assembly, after which mDia1 moved with the growing barbed ends while APC remained at the site of nucleation. Triple-color microscopy suggests that two factors interact to initiate actin formation and then separate as the filament grows. Interacting sets of actin assembly factors work together in cells, but the underlying mechanisms have remained obscure. We used triple-color single-molecule fluorescence microscopy to image the tumor suppressor adenomatous polyposis coli (APC) and the formin mDia1 during filament assembly. Complexes consisting of APC, mDia1, and actin monomers initiated actin filament formation, overcoming inhibition by capping protein and profilin. Upon filament polymerization, the complexes separated, with mDia1 moving processively on growing barbed ends while APC remained at the site of nucleation. Thus, the two assembly factors directly interact to initiate filament assembly and then separate but retain independent associations with either end of the growing filament.

The Formin DAD Domain Plays Dual Roles in Autoinhibition and Actin Nucleation

Formins are a large family of actin assembly-promoting proteins with many important biological roles. However, it has remained unclear how formins nucleate actin polymerization. All other nucleators are known to recruit actin monomers as a central part of their mechanisms. However, the actin-nucleating FH2 domain of formins lacks appreciable affinity for monomeric actin. Here, we found that yeast and mammalian formins bind actin monomers but that this activity requires their C-terminal DAD domains. Furthermore, we observed that the DAD works in concert with the FH2 to enhance nucleation without affecting the rate of filament elongation. We dissected this mechanism in mDia1, mapped nucleation activity to conserved residues in the DAD, and demonstrated that DAD roles in nucleation and autoinhibition are separable. Furthermore, DAD enhancement of nucleation was independent of contributions from the FH1 domain to nucleation. Together, our data show that (1) the DAD has dual functions in autoinhibition and nucleation; (2) the FH1, FH2, and DAD form a tripartite nucleation machine; and (3) formins nucleate by recruiting actin monomers and therefore are more similar to other nucleators than previously thought.

Displacement of Formins from Growing Barbed Ends by Bud14 Is Critical for Actin Cable Architecture and Function

Normal cellular development and function require tight spatiotemporal control of actin assembly. Formins are potent actin assembly factors that protect the growing ends of actin filaments from capping proteins. However, it is unresolved how the duration of formin-mediated actin assembly events is controlled, whether formins are actively displaced from growing ends, and how filament length is regulated in vivo. Here, we identify Bud14 as a high-affinity inhibitor of the yeast formin Bnr1 that rapidly displaces the Bnr1 FH2 domain from growing barbed ends. Consistent with these activities, bud14Delta cells display fewer actin cables, which are aberrantly long, bent, and latrunculinA resistant, leading to defects in secretory vesicle movement. Moreover, bud14Delta suppressed mutations that cause abnormally numerous and shortened cables, restoring wild-type actin architecture. From these results, we propose that formin displacement factors regulate filament length and are required in vivo to maintain proper actin network architecture and function.

Structure and activity of full‐length formin mDia1

Formins are a conserved family of actin assembly‐promoting factors with essential and diverse biological roles. Most of our biochemical understanding of formin effects on actin dynamics is derived from studies using formin fragments. In addition, all structural information on formins has been limited to fragments. This has left open key questions about the structure, activity and regulation of intact formin proteins. Here, we isolated full‐length mouse mDia1 (mDia1‐FL) and found that it forms tightly autoinhibited dimers that can only be partially activated by RhoA. We solved the structure of autoinhibited mDia1‐FL using electron microscopy and single particle analysis. Docking of crystal structures into the three dimensional reconstruction revealed that the fork‐shaped N‐terminal diaphanous inhibitory domain‐coiled coil domain region hangs over the ring‐shaped formin homology (FH)2 domain, suggesting that autoinhibition results from steric obstruction of actin binding. Deletion of the C‐terminal diaphanous autoregulatory domain extended mDia1 structure and activated it for actin assembly. Using total internal reflection fluorescence microscopy, we observed that RhoA‐activated mDia1‐FL persistently accelerated filament elongation in the presence of profilin similar to mDia1 FH1‐FH2 fragment. These observations validate the known activities of FH1‐FH2 fragments as reflecting those of the intact molecule. Our results further suggest that mDia1‐FL does not readily snap back into the autoinhibited conformation and dissociate from growing filament ends, and thus additional factors may be required to displace formins and restrict filament length.

Crystal Structure of a Coiled-Coil Domain from Human ROCK I

The small GTPase Rho and one of its targets, Rho-associated kinase (ROCK), participate in a variety of actin-based cellular processes including smooth muscle contraction, cell migration, and stress fiber formation. The ROCK protein consists of an N-terminal kinase domain, a central coiled-coil domain containing a Rho binding site, and a C-terminal pleckstrin homology domain. Here we present the crystal structure of a large section of the central coiled-coil domain of human ROCK I (amino acids 535–700). The structure forms a parallel α-helical coiled-coil dimer that is structurally similar to tropomyosin, an actin filament binding protein. There is an unusual discontinuity in the coiled-coil; three charged residues (E613, R617 and D620) are positioned at what is normally the hydrophobic core of coiled-coil packing. We speculate that this conserved irregularity could function as a hinge that allows ROCK to adopt its autoinhibited conformation.

Ligand-induced activation of a formin–NPF pair leads to collaborative actin nucleation

Bil1 binds to the regulatory sequence of Bud6, unmasking its ability to stimulate the activity of the formin Bni1 and promote actin nucleation.

Saccharomyces cerevisiae Kelch Proteins and Bud14 Protein Form a Stable 520-kDa Formin Regulatory Complex That Controls Actin Cable Assembly and Cell Morphogenesis

Background: Kelch proteins are required for cell morphogenesis, but their molecular functions have remained elusive. Results: S. cerevisiae Kel1 and Kel2 form a stable complex with the formin-binding protein Bud14 to regulate actin cable assembly. Conclusion: Kel1 and Kel2 directly regulate formins to control the actin cytoskeleton. Significance: These findings help resolve the roles of S. cerevisiae Kel1 and Kel2 in morphogenesis. Formins perform essential roles in actin assembly and organization in vivo, but they also require tight regulation of their activities to produce properly functioning actin structures. Saccharomyces cerevisiae Bud14 is one member of an emerging class of formin regulators that target the FH2 domain to inhibit actin polymerization, but little is known about how these regulators are themselves controlled in vivo. Kelch proteins are critical for cell polarity and morphogenesis in a wide range of organisms, but their mechanistic roles in these processes are still largely undefined. Here, we report that S. cerevisiae Kelch proteins, Kel1 and Kel2, associate with Bud14 in cell extracts to form a stable 520-kDa complex with an apparent stoichiometry of 2:2:1 Bud14/Kel1/Kel2. Using pairwise combinations of GFP- and red fluorescent protein-tagged proteins, we show that Kel1, Kel2, and Bud14 interdependently co-localize at polarity sites. By analyzing single, double, and triple mutants, we show that Kel1 and Kel2 function in the same pathway as Bud14 in regulating Bnr1-mediated actin cable formation. Loss of any component of the complex results in long, bent, and hyper-stable actin cables, accompanied by defects in secretory vesicle traffic during polarized growth and septum formation during cytokinesis. These observations directly link S. cerevisiae Kelch proteins to the control of formin activity, and together with previous observations made for S. pombe homologues tea1p and tea3p, they have broad implications for understanding Kelch function in other systems.