REVIEWS Unleashing formins to remodel the actin and microtubule cytoskeletons Melissa A. Chesarone, Amy Grace DuPage and Bruce L. Goode Abstract | Formins are highly conserved proteins that have essential roles in remodelling the actin and microtubule cytoskeletons to influence eukaryotic cell shape and behaviour. Recent work has identified numerous cellular factors that locally recruit, activate or inactivate formins to bridle and unleash their potent effects on actin nucleation and elongation. The effects of formins on microtubules have also begun to be described, which places formins in a prime position to coordinate actin and microtubule dynamics. The emerging complexity in the mechanisms governing formins mirrors the wide range of essential functions that they perform in cell motility, cell division and cell and tissue morphogenesis. Virtually all eukaryotic cell types have morphologies that might be unique in their ability to directly regulate both are uniquely tailored to their physiological functions. The actin filaments and microtubules. Furthermore, only immense variation in cell shape depends crucially on an formins show a clearly established and robust ability to underlying network of dynamic, interconnected actin both nucleate actin polymers and dramatically accelerate and microtubule polymers. The dynamic assembly and polymer elongation. turnover of these filamentous networks is used to direct Formins are large (120–220 kDa), multidomain pro- cell polarity and to facilitate membrane and organelle teins that interact with many binding partners to perform traffic, cell adhesion, chromosome segregation, cell their functions (TABLE 1). Fungal species typically have 2 migration and cell division. or 3 formin genes, whereas mammals have 15 and some To construct these intricate fibrous arrays, cells make plant species have more than 20 (REFS 5,6,7). The potent use of a palette of proteins that bind to cyto skeletal activities of formins on actin and micro tubule dynam- polymers and work in concert to organize them into ics have been harnessed to the assembly of the diverse higher-order force-generating structures. The task of cytoskeletal structures that are required in a range of cell- assembling actin and microtubule polymers de novo ular functions, including cell morphogenesis, adhesion, requires active mechanisms, as there are abundant factors division and motility (FIG. 1). Formins are also implicated in cells that inhibit spontaneous polymer formation. in a growing number of diseases (BOX 1). These inhibitory factors include proteins that buffer In this review, we first summarize formin structure and free actin sub units (such as profilin and thymosins) and activities and then we describe regulatory control points tubulin subunits (such as stathmin), and factors that cap and mechanisms used to govern formin activities. polymer ends and sever or depolymerize polymers. To build new actin and microtubule polymers, cells deploy Deconstructing formins specialized proteins that catalyse polymer nucleation and Although no three-dimensional (3D) structure has yet elongation, protect growing polymer ends and attach been reported for any intact formin, a working model to the sides and/or ends of polymers to stabilize them for their general architecture can be assembled from against disassembly. the crystal structures of formin fragments (FIG. 2). In Rosenstiel Basic Medical Science Research Center, Recent work has shown that the formin family of pro- this model, formins are depicted as dimers, as recent Brandeis University, teins (which is conserved in plants, animals and fungi) biophysical data has confirmed that the purified full- Waltham, Massachusetts, has a central role in catalysing actin polymer assembly length formins mouse diaphanous 1 (mDia1; also known 02454, USA. and in stabilizing microtubules1,2. Various other proteins as DIAPH1) and mDia2 (also known as DIAPH3), Correspondence to B.G. that are capable of stimulating actin assembly are found and yeast Bni1 and Bni1-related protein 1 (Bnr1) are e-mail: [email protected] doi:10.1038/nrm2816 in cells, including the Arp2/3 complex, spire, cordon-bleu dimeric (B.G., unpublished observations). Below, we Published online (COBL), leiomodin (LMOD), and junction-mediating describe the general domain layout of animal, fungal 9 December 2009 and -regulatory protein (JMY).3,4. However, formins and plant formins. 62 | JANUARY 2010 | VOLUME 11 www.nature.com/reviews/molcellbio REVIEWS Table 1 | Binding proteins regulating formin localization and activity Protein* Formin target* Organism and/or cell type Function Refs Profilin All tested All cell types Recruits actin monomers to the FH1 domain to accelerate elongation 53 Bud6 Bni1 Saccharomyces cerevisiae Nucleation cofactor for Bni1, binds to the DAD and promotes the 41,149 assembly of actin cables Fus3 Bni1 S. cerevisiae Phosphorylates and localizes Bni1 to the tips of mating cells 95 Rho1 Bni1 S. cerevisiae Required for Bni1 localization to the bud neck and bud cortex 97,150 Spa2 Bni1 S. cerevisiae Helps localize Bni1 to the bud cortex 103,151 Bud14 Bnr1 S. cerevisiae Displaces the Bnr1 FH2 domain from growing barbed ends of 89 filaments and regulates actin cable architecture Spire CAPU Drosophila melanogaster Synergizes with CAPU to assemble actin meshworks in vivo and is 44,45 oocytes thought to inhibit CAPU in vitro Cdc15 Cdc12 Schizosaccharomyces pombe Binds directly to the amino terminus of Cdc12 and is required for the 152 assembly of the cytokinetic actin ring and for cell division Bud6 For3 S. pombe Binds to the DAD and helps localize For3 to cell tips and is required 42,153 for actin cable assembly Cdc42 For3 S. pombe Helps localize For3 to cell tips, is required for actin cable assembly 42 and interacts genetically with Bud6 Tea4 For3 S. pombe Helps localize For3 to cell tips, is required for actin cable assembly 104 and is the closest homologue of S. cerevisiae Bud14 by sequence ABI1 mDia1 Epithelial, melanoma and Helps localize mDia1 to lamellipodia, filopodia and cell adhesions 105,147 HeLa cells CLIP170 mDia1 (DIAPH1) Macrophages Binds the FH2 domain and helps localize mDia1 to sites of 17 (CLIP1) phagocytosis Gα12/13 mDia1 Fibroblasts Helps localize mDia1 to the leading edge of migrating cells 154 IQGAP1 mDia1 Fibroblasts and macrophages Binds to the DID and is required for mDia1 localization to the leading 93 edge and to phagocytic cups RHOA mDia1 Epithelial cells Required for mDia1 localization to adherens junctions and partially 26,100 activates mDia1 from autoinhibition RHOB mDia1 Melanoma cells Helps localize mDia1 to endosomes 155 RHOB mDia2 (DIAPH3) Fibroblasts Required for mDia2 localization to endosomes 25 RIF (RHOF) mDia2 Fibroblasts Helps localize mDia2 to filopodial tips 156 DIP (WISH, mDia2 HEK and HeLa cells Inhibits mDia2 FH2, suppresses filopodial protrusion and induces 88 NCKIPSD) membrane blebbing ROCK1 FHOD1 HeLa cells Binds the FH2 domain and phosphorylates and activates FHOD1 to 39,40 promote membrane remodelling α-catenin FMN1 Epithelial cells Helps localize FMN1 to adherens junctions and is required for 106 FMN1-dependent actin assembly at cell adhesion sites Cdc42 FMNL1 (FRL1) Macrophages Helps localize FMNL1 to the cell cortex 84 ABI1, Abelson interactor 1; Bnr1, Bni1-related protein 1; Bud, bud site selection protein; CAPU, Cappuccino; CLIP170, cytoplasmic linker protein 170; DAD, Dia autoregulatory domain; DID, Dia inhibitory domain; DIP, Dia-interacting protein; FH, formin homology; FHOD1, FH1/FH2 domain-containing protein 1; FMN1, formin 1; FMNL1, FMN-like protein 1; For3, formin 3; IQGAP1, IQ motif-containing GTPase activating protein 1; mDia, mouse diaphanous; RIF, Rho in filopodia; ROCK, Rho-activated kinase; Tea4, tip elongation aberrant protein 4. *Alternative protein names are provided in brackets. Formin polypeptides can be divided into two major structure, and it contains binding sites for profilin–actin Polyproline tract (FIG. 2a) A short protein motif, found in functional regions : the amino-terminal ‘regu- complexes. Profilin is a ubiquitous actin monomer- many actin regulatory scaffold latory’ region, which typically governs in vivo local- binding protein with separate binding sites for mono- proteins, that typically contains ization and can influence the activities of the carboxy meric actin (also called globular actin or G-actin) and five or more tandem proline terminus, and the ‘active’ region, which stimulates actin polyproline tracts8–10. Profilin is associated with most actin residues and binds profilin or 11 SH3 domains. assembly and, in some formins, interacts with micro- monomers in cells and, therefore, profilin–actin com- tubules. The C terminus of many formins includes a plexes are the predominant substrate for actin assem- Barbed end Dia autoregulatory domain (DAD), which can, in some bly in vivo. Interactions between profilin and the FH1 The rapidly growing end of cases, mediate auto inhibition through interactions with domain are crucial for the recruitment of actin mono- an actin filament, so-called the N terminus. mers to the active region12–15. The adjacent FH2 domain because of the arrowhead pattern created when myosin The C-terminal active region consists of the formin forms a head-to-tail doughnut-shaped dimer that binds. The slowly growing end homology 1 (FH1) and FH2 domains. The FH1 domain en circles the barbed end of the actin filament. In mDia1 is called the pointed end. is predicted to be rope-like, based on a lack of secondary or