Yeast formins

The RHO1 gene encodes a homolog of mammalian RhoA small GTP binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth sites, including the bud tip and the cytokinesis site, and is required for bud formation. Pkc1p, a yeast homolog of mammalian protein kinase C, and glucan synthase are targets of Rho1p. Using the two-hybrid screening system, a gene encoding a protein that interacts with the GTP-bound form of Rho1p has been cloned. This gene has been identified as BNI1, known to be implicated in cytokinesis and in the establishment of cell polarity in S.cerevisiae. Bni1p shares homologous domains (FH1 and FH2 domains) with proteins involved in cytokinesis and establishment of cell polarity, including formin in the mouse, Capu and Dia in Drosophila and FigA in Aspergillus. A temperature-sensitive mutation in which the RHO1 gene is replaced by the mammalian RhoA gene shows a synthetically lethal interaction with the bni1 mutation; the RhoA bni1 mutant accumulates cells with a deficiency in cytokinesis. Furthermore, this synthetic lethality is caused by the inability of RhoA to activate Pkc1p, but not glucan synthase. These results suggest that Rho1p regulates cytoskeletal reorganization at least through Bni1p and Pkc1p (Kohno, 1996).

As in many other eukaryotic cells, cell division in fission yeast depends on the assembly of an actin ring that circumscribes the middle of the cell. Schizosaccharomyces pombe cdc12 is an essential gene necessary for actin ring assembly and septum formation. Cdc12p is a member of a family of proteins including Drosophila Diaphanous, S. cerevisiae BNI1, and S. pombe fus1, all involved in cytokinesis or other actin-mediated processes. Using indirect immunofluorescence, cdc12p has been shown to be located in the cell division ring and not in other actin structures. When overexpressed, cdc12p is located at a medial spot in interphase that anticipates the future ring site. cdc12p localization is altered in actin ring mutants. cdc8 (tropomyosin homolog), cdc3 (profilin homolog), and cdc15 mutants exhibit no specific cdc12p staining during mitosis. cdc4 mutant cells exhibit a medial cortical cdc12p spot in place of a ring. Based on these patterns, a model is presented in which ring assembly originates from a single point on the cortex; a molecular pathway for the functions of cytokinesis proteins is suggested. cdc12 and cdc3 mutants show a synthetic-lethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin (Chang, 1997).

The RHO1 gene encodes a homolog of mammalian RhoA small G-protein in the yeast S. cerevisiae. Rho1p is required for bud formation and is localized at a bud tip or a cytokinesis site. Bni1p is a potential target of Rho1p. Bni1p shares the FH1 and FH2 domains with proteins involved in cytokinesis or establishment of cell polarity. In S. cerevisiae, there is an open reading frame (YIL159W) that encodes another protein, BNR1 (BNI1 Related), also having the FH1 and FH2 domains. Bnr1p interacts with another Rho family member, Rho4p, but not with Rho1p. Disruption of BNI1 or BNR1 does not show any deleterious effect on cell growth, but the bni1 bnr1 mutant shows a severe temperature-sensitive growth phenotype. Cells of the bni1 bnr1 mutant arrested at the restrictive temperature are deficient in bud emergence, exhibit a random distribution of cortical actin patches and often become multinucleate. These phenotypes are similar to those of the PFY1 mutant, which encodes profilin, an actin-binding protein. Yeast two-hybrid and biochemical studies demonstrate that Bni1p and Bnr1p interact directly with profilin at the FH1 domains. These results indicate that Bni1p and Bnr1p are potential targets of the Rho family members, interact with profilin and regulate the reorganization of actin cytoskeleton (Imamura, 1997).

The Saccharomyces cerevisiae BNI1 gene product (Bni1p) is a member of the formin family of proteins, which participate in cell polarization, cytokinesis, and vertebrate limb formation. During mating pheromone response, bni1 mutants show defects both in polarized morphogenesis and in reorganization of the underlying actin cytoskeleton. In two-hybrid experiments, Bni1p forms complexes with the activated form of the Rho-related guanosine triphosphatase Cdc42p, with actin, and with two actin-associated proteins, profilin and Bud6p (Aip3p). Both Bni1p and Bud6p (like Cdc42p and actin) localize to the tips of mating projections. Bni1p may function as a Cdc42p target that links the pheromone response pathway to the actin cytoskeleton (Evangelista, 1997).

The RHO1 gene encodes a homolog of mammalian RhoA small G protein in the yeast Saccharomyces cerevisiae. Bni1p is one of the downstream targets of Rho1p and regulates reorganization of the actin cytoskeleton through the interaction with profilin, an actin monomer-binding protein. A Bni1p-binding protein was affinity purified from the yeast cytosol fraction and was identified as Tef1p/Tef2p, a translation elongation factor 1alpha (EF1alpha). EF1alpha is an essential component of the protein synthetic machinery and also possesses the actin filament (F-actin)-binding and -bundling activities. EF1alpha binds to the 186 amino acids region of Bni1p, located between the FH1 domain, the proline-rich profilin-binding domain, and the FH2 domain, whose function is not known. The binding of Bni1p to EF1alpha thus inhibiting BN1p's F-actin-binding and -bundling activities. The BNI1 gene deleted in the EF1alpha-binding region does not suppress the bni1 bnr1 mutation, in which the actin organization is impaired. These results suggest that the Rho1p-Bni1p system regulates reorganization of the actin cytoskeleton through the interaction with both EF1alpha and profilin (Umikawa, 1998).

In Saccharomyces cerevisiae, the phosphatidylinositol kinase homolog Tor2 controls the cell-cycle-dependent organization of the actin cytoskeleton by activating the small GTPase Rho1 via the exchange factor Rom2. Four Rho1 effectors are known: protein kinase C 1 (Pkc1); the formin-family protein Bni1; the glucan synthase Fks, and the signaling protein Skn7. Rho1 has been suggested to signal to the actin cytoskeleton via Bni1 and Pkc1; rho1 mutants have never been shown to have defects in actin organization, however. The role of Rho1 in controlling actin organization has been further investigated and an analysis has been carried out on which of the Rho1 effectors mediates Tor2 signaling to the actin cytoskeleton. Some, but not all, rho1 temperature-sensitive (rho1ts) mutants arrest growth with a disorganised actin cytoskeleton. Both the growth defect and the actin organization defect of the rho1-2ts mutant are suppressed by upregulation of Pkc1 but not by upregulation of Bni1, Fks or Skn7. Overexpression of Pkc1, but not overexpression of Bni1, Fks or Skn7, also rescues a tor2ts mutant, and deletion of BNI1 or SKN7 does not prevent the suppression of the tor2ts mutation by overexpressed Rom2. Furthermore, overexpression of the Pkc1-controlled mitogen-activated protein (MAP) kinase Mpk1 suppresses the actin defect of tor2ts and rho1-2ts mutants. Thus, Tor2 signals to the actin cytoskeleton via Rho1, Pkc1 and the cell integrity MAP kinase cascade (Helliwell, 1998).

Proteins containing the formin homology (FH) domains FH1 and FH2 are involved in cytokinesis or establishment of cell polarity in a variety of organisms. The FH proteins Bni1p and Bnr1p are potential targets of the Rho family small GTP-binding proteins and bind to an actin-binding protein, profilin, at their proline-rich FH1 domains to regulate reorganization of the actin cytoskeleton in the yeast Saccharomyces cerevisiae. A novel Src homology 3 (SH3) domain-containing protein, encoded by YMR032w, interacts with Bnr1p in a GTP-Rho4p-dependent manner through the FH1 domain of Bnr1p and the SH3 domain of Ymr032wp. Ymr032wp weakly binds to Bni1p. Ymr032wp is homologous to cdc15p, which is involved in cytokinesis in Schizosaccharomyces pombe: this gene has been named HOF1 (homolog of cdc 15). Both Bnr1p and Hof1p are localized at the bud neck, and both the bnr1 and hof1 mutations show synthetic lethal interactions with the bni1 mutation. The hof1 mutant cells show phenotypes similar to those of the septin mutants, indicating that HOF1 is involved in cytokinesis. These results indicate that Bnr1p directly interacts with Hof1p as well as with profilin to regulate cytoskeletal functions in S. cerevisiae (Kamei, 1998).

Formins are involved in diverse aspects of morphogenesis, and share two regions of homology: FH1 and FH2. A new formin homology region, FH3, is described. FH3 is an amino-terminal domain that differs from the Rho binding site identified in Bni1p and p140mDia. The Schizosaccharomyces pombe formin Fus1 is required for conjugation, and is localized to the projection tip in cells of mating pairs. Genomic fus1+ was replaced with green fluorescent protein (GFP)- tagged versions that lacked either the FH1, FH2, or FH3 domain. Deletion of any FH domain essentially abolishes mating. FH3, but neither FH1 nor FH2, is required for Fus1 localization. An FH3 domain-GFP fusion protein localizes to the projection tips of mating pairs. Thus, the FH3 domain alone can direct protein localization. The FH3 domains of both Fus1 and the S. pombe cytokinesis formin Cdc12 are able to localize GFP to the spindle pole body in half of the late G2 cells in a vegetatively growing population. Expression of both FH3-GFP fusions also affect cytokinesis. Overexpression of the spindle pole body component Sad1 alters the distribution of both Sad1 and the FH3-GFP domain. Together these data suggest that proteins at multiple sites can interact with FH3 domains (Peterson, 1998).

Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. Bni1p is a potential target of Rho1p and Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using the yeast two-hybrid screening system, a gene was cloned encoding a protein that interacts with Bni1p. This protein, Spa2p, is localized at the bud tip and has been implicated in the establishment of cell polarity. The C-terminal 254 amino acid region of Spa2p, (amino acids 1213-1466), directly binds to a 162-amino acid region of Bni1p (amino acids 826-987). Genetic analyses reveal that both the bni1 and spa2 mutations show synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis shows that Bni1p is localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p is not localized at the bud tip and instead localizes diffusely in the cytoplasm. A mutant Bni1p, which lacked the Rho1p-binding region, also fails to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p (Fujiwara, 1998).

Alignment of the mitotic spindle with the axis of cell division is an essential process in Saccharomyces cerevisiae that is mediated by interactions between cytoplasmic microtubules and the cell cortex. A cortical protein, the yeast formin Bni1p, is required for spindle orientation. Two striking abnormalities are observed in bni1Delta cells. (1) The initial movement of the spindle pole body (SPB) toward the emerging bud is defective. This phenotype is similar to that previously observed in cells lacking the kinesin Kip3p and, in fact, BNI1 and KIP3 are found to be in the same genetic pathway. (2) Abnormal pulling interactions between microtubules and the cortex appear to cause preanaphase spindles in bni1Delta cells to transit back and forth between the mother and the bud. It is therefore proposed that Bni1p may localize or alter the function of cortical microtubule-binding sites in the bud. Additionally, evidence is presented that other bipolar bud site determinants together with cortical actin are also required for spindle orientation (Lee, 1999).

Proteins containing formin homology domains, FH1 and FH2, are involved in cytokinesis or establishment of cell polarity in a variety of organisms. Bni1p and Bnr1p are FH proteins and potential targets of the Rho family small GTP-binding proteins in S. cerevisiae. Bnr1p is localized at the bud neck to interact with Hof1p, a protein involved in cytokinesis. The overexpression of BNR1 causes a cytokinesis deficiency that is similar to the phenotypes of the septin mutants, including cdc3, cdc10, cdc11, and cdc12. The region required for the septin mutant phenotypes maps to Bnr1p (amino acids 35-500), which coincides with the region required for the bud-neck localization. To further isolate a gene interacting with BNI1 or BNR1, a multicopy suppressor of the bni1 bnr1 mutant was isolated. This gene encodes Smy1p, a kinesin-related protein. Bnr1p, but not Bni1p, directly interacts with the C-terminal region of Smy1p. The Smy1p-interacting region of Bnr1p was mapped to a region containing the FH2 domain. Bnr1p also directly interacts with Bud6p, a novel actin-binding protein. Bnr1p is thus a multifunctional protein that interacts with the septin system, a microtubule-dependent motor protein, and the actin system, to regulate cytoskeletal functions in S. cerevisiae (Kikyo, 1999).

In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle requires both the cytoplasmic microtubules and actin. Kar9p is a novel cortical protein that is required for the correct position of the mitotic spindle and the orientation of the cytoplasmic microtubules. Green fluorescent protein (GFP)- Kar9p localizes to a single spot at the tip of the growing bud and the mating projection. However, the cortical localization of Kar9p does not require microtubules, suggesting that Kar9p interacts with other proteins at the cortex. To investigate Kar9p's cortical interactions, cells were treated with the actin-depolymerizing drug, latrunculin-A. In both shmoos and mitotic cells, Kar9p's cortical localization is completely dependent on polymerized actin. Kar9p localization is also altered by mutations in four genes, spa2Delta, pea2Delta, bud6Delta, and bni1Delta, required for normal polarization and actin cytoskeleton functions and, of these, bni1Delta affects Kar9p localization most severely. Like kar9Delta, bni1Delta mutants exhibit nuclear positioning defects during mitosis and in shmoos. Furthermore, like kar9Delta, the bni1Delta mutant exhibits misoriented cytoplasmic microtubules in shmoos. Genetic analysis places BNI1 in the KAR9 pathway for nuclear migration. However, analysis of kar9Delta bni1Delta double mutants suggests that Kar9p retains some function in bni1Delta mitotic cells. Unlike the polarization mutants, kar9Delta shmoos have a normal morphology and diploids budded in the correct bipolar pattern. Furthermore, Bni1p localizes normally in kar9Delta. It is concluded that Kar9p's function is specific for cytoplasmic microtubule orientation and that Kar9p's role in nuclear positioning is to coordinate the interactions between the actin and microtubule networks (Westendorf, 1999).

The RHO1 gene encodes a yeast homolog of the mammalian RhoA protein. Rho1p is localized to the growth sites and is required for bud formation. Bni1p is one of the potential downstream target molecules of Rho1p. The BNI1 gene is implicated in cytokinesis and the establishment of cell polarity in Saccharomyces cerevisiae but is not essential for cell viability. In this study, a screen was performed for mutations that are synthetically lethal in combination with a bni1 mutation and two genes were isolated. They were the previously identified PAC1 and NIP100 genes, both of which are implicated in nuclear migration in S. cerevisiae. Pac1p is a homolog of human LIS1, which is required for brain development, whereas Nip100p is a homolog of rat p150 (Glued), a component of the dynein-activated dynactin complex. Disruption of BNI1 in either the pac1 or nip100 mutant results in an enhanced defect in nuclear migration, leading to the formation of binucleate mother cells. The arp1 bni1 mutant shows a synthetic lethal phenotype while the cin8 bni1 mutant does not, suggesting that Bni1p functions in a kinesin pathway but not in the dynein pathway. Cells of the pac1 bni1 and nip100 bni1 mutants exhibit a random distribution of cortical actin patches. Cells of the pac1 act1-4 mutant show temperature-sensitive growth and a nuclear migration defect. These results indicate that Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton. Bni1p lacking the Rho-binding region does not suppress the pac1 bni1 growth defect, suggesting a requirement for the Rho1p-Bni1p interaction in microtubule function (Fujiwara, 1999).

Cytokinesis in Saccharomyces cerevisiae occurs by the concerted action of the actomyosin system and septum formation. The roles of HOF1, BNI1, and BNR1 in cytokinesis have been examained, with a focus placed on Hof1p. Deletion of HOF1 causes a temperature-sensitive defect in septum formation. A Hof1p ring forms on the mother side of the bud neck in G2/M, followed by the formation of a daughter-side ring. Around telophase, Hof1p is phosphorylated and the double rings merge into a single ring that contracts slightly and may colocalize with the actomyosin structure. Upon septum formation, Hof1p splits into two rings, disappearing upon cell separation. Hof1p localization is dependent on septins. Synthetic lethality suggests that Bni1p and Myo1p belong to one functional pathway, whereas Hof1p and Bnr1p belong to another. These results suggest that Hof1p may function as an adapter, linking the primary septum synthesis machinery to the actomyosin system. The formation of the actomyosin ring is not affected by bni1Delta, hof1Delta, or bnr1Delta. However, Myo1p contraction is affected by bni1Delta but not by hof1Delta or bnr1Delta. In bni1Delta cells that lack the actomyosin contraction, septum formation is often slow and asymmetric, suggesting that actomyosin contraction may provide directionality for efficient septum formation (Vallen, 2000).

In eukaryotic cells, dynamic rearrangement of the actin cytoskeleton is critical for cell division. In the yeast Saccharomyces cerevisiae, three main structures constitute the actin cytoskeleton: cortical actin patches, cytoplasmic actin cables, and the actin-based cytokinetic ring. The conserved Arp2/3 complex and a WASP-family protein mediate actin patch formation, whereas the yeast formins (Bni1 and Bnr1) promote assembly of actin cables. However, the mechanism of actin ring formation is currently unclear. Actin filaments are shown to be required for cytokinesis in S. cerevisiae, and the actin ring is shown to be a highly dynamic structure that undergoes constant turnover. Assembly of the actin ring requires the formin-like proteins and profilin, but is not Arp2/3-mediated. Furthermore, the formin-dependent actin ring assembly pathway is regulated by the Rho-type GTPase Rho1 but not Cdc42. Finally, the formins are shown to not be required for localization of Cyk1/Iqg1, an IQGAP-like protein that is required for actin ring formation, suggesting that formin-like proteins and Cyk1 act synergistically but independently in assembly of the actin ring (Tolliday, 2002).

Formins, characterized by formin homology domains FH1 and FH2, are required to assemble certain F-actin structures including actin cables, stress fibers, and the contractile ring. FH1FH2 in a recombinant fragment from a yeast formin (Bni1p) nucleates actin filaments in vitro. It also binds to the filament barbed end where it appears to act as a 'leaky' capper, slowing both polymerization and depolymerization by 50%. FH1FH2 competes with tight capping proteins (including gelsolin and heterodimeric capping protein) for the barbed end. FH1FH2 forms a tetramer. The observation that this formin protects an end from capping but still allows elongation confirms that it is a leaky capper. This is significant because a nucleator that protects a new barbed end from tight cappers will increase the duration of elongation and thus the total amount of F-actin. The ability of FH1FH2 to dimerize probably allows the formin to walk processively with the barbed end as the filament elongates (Zigmond, 2003).

Microtubules regulate actin-based processes such as cell migration and cytokinesis, but the molecular mechanisms at work are not yet understood. In the fission yeast Schizosaccharomyces pombe, microtubule plus ends regulate cell polarity in part by transporting the kelch repeat protein tea1p to cell ends. This study identifies tea4p, a SH3 domain protein that binds directly to tea1p. Like tea1p, tea4p localizes to growing microtubule plus ends and to cortical sites at cell ends, and it is necessary for the establishment of bipolar growth. Tea4p binds directly to and recruits the formin for3p, which nucleates actin cable assembly. During 'new end take off' (NETO), formation of a protein complex that includes tea1p, tea4p, and for3p is necessary and sufficient for the establishment of cell polarity and localized actin assembly at new cell ends. These results suggest a molecular mechanism for how microtubule plus ends regulate the spatial distribution of actin assembly (Martin, 2005).

The possible homologs of tea1p and tea4p have generally not yet been well characterized. In S. cerevisiae, the nearest homologs of S. pombe tea1p, tea4p, and for3p are Kel1/Kel2p, Bud14p, and Bni1p, respectively. Although association between these factors has not been reported to date, mutant phenotypes and genetic interactions suggest that these proteins also function together to regulate cell polarity. In animal cells, the equivalents of tea1p and tea4p are less clear. A mammalian protein with some functional similarity (but low sequence homology) to tea4p is WISH/DIP. Like tea4p, this protein contains an N-terminal SH3 domain protein and interacts with mammalian formins, mDia1 and FHOD1, with an N-terminal region binding to FHOD1. Many kelch proteins regulate cytoskeletal processes in animal cells (Adams, 2000). Of note, the kelch repeat protein gigaxonin associates with MTs in neurons, Drosophila Kelch organizes ring canals, and Keap1 associates with the mid-body, adherens junctions, and focal adhesions and is required for cytokinesis. Further study of these proteins and their possible interactions will be a key part of understanding the molecular principles of cell morphogenesis (Martin, 2005).

Mammalian formins: Identification and effects of mutation

Rho small GTPase regulates cell morphology, adhesion and cytokinesis through the actin cytoskeleton. The protein p140mDia has been identified as a downstream effector of Rho. It is a mammalian homolog of Drosophila diaphanous, a protein required for cytokinesis, and belongs to a family of formin-related proteins containing repetitive polyproline stretches. p140mDia binds selectively to the GTP-bound form of Rho and also binds to profilin. p140mDia, profilin and RhoA are co-localized in the spreading lamellae of cultured fibroblasts. They are also co-localized in membrane ruffles of phorbol ester-stimulated sMDCK2 cells, which extend these structures in a Rho-dependent manner. The three proteins are recruited around phagocytic cups induced by fibronectin-coated beads. Their recruitment is not induced after Rho is inactivated by microinjection of botulinum C3 exoenzyme. Overexpression of p140mDia in COS-7 cells induces homogeneous actin filament formation. These results suggest that Rho regulates actin polymerization by targeting profilin beneath the specific plasma membranes via p140mDia (Watanabe, 1997).

The gene responsible for autosomal dominant, fully penetrant, nonsyndromic sensorineural progressive hearing loss in a large Costa Rican kindred localizes to chromosome 5q31 and has been named DFNA1. Deafness in the family is associated with a protein-truncating mutation in a human homolog of the Drosophila gene diaphanous. The truncation is caused by a single nucleotide substitution in a splice donor, leading to a four-base pair insertion in messenger RNA and a frameshift. The diaphanous protein is a profilin ligand and target of Rho that regulates polymerization of actin, the major component of the cytoskeleton of hair cells of the inner ear (Lynch, 1997).

Premature ovarian failure (POF) is a defect of ovarian development and is characterized by primary or secondary amenorrhea, with elevated levels of serum gonadotropins, or by early menopause. The disorder has been attributed to various causes, including rearrangements of a large 'critical region' in the long arm of the X chromosome. The identification is reported, in a family with POF, of a gene that is disrupted by a breakpoint. The gene is the human homolog of the Drosophila diaphanous gene; mutated alleles of this gene affect spermatogenesis or oogenesis and lead to sterility. The protein (DIA) is the first human member of the growing FH1/FH2 protein family. Members of this protein family affect cytokinesis and other actin-mediated morphogenetic processes that are required during early development. It is proposed that the human DIA gene is one of the genes responsible for POF and that it affects the cell divisions that lead to ovarian follicle formation (Bione, 1998).

A novel member of the Formin/Diaphanous family of proteins has been cloned and characterized. A 4kB mRNA is ubiquitously expressed but is found in abundance in the spleen. FHOS (formin homolog overexpressed in spleen) contains a 3414bp open reading frame and encodes an approximately 128kDa protein. FHOS has sequence homology to Diaphanous and Formin proteins within the formin homology (FH)1 and FH2 domains. FHOS also contains a coiled-coil, a collagen-like domain, two nuclear localization signals, and several potential PKC and PKA phosphorylation sites. FHOS-specific antiserum was generated and used to determine that FHOS is a predominantly cytoplasmic protein and is expressed in a variety of human cell lines. FHOS was mapped to chromosome 16q22 between framework markers WI-5594 and WI-9392 (Westendorf, 1999).

Formin-1 is the founding member of a family of genes of emerging biological and medical importance that share specific domains of homology, allowing them to be classified together as the formin homology proteins. Although deficiency mutations in formin-1 lead to profound developmental defects in limb and kidney formation, similar deficiency mutations in more distantly related members of this family (diaphanous and cappuccino in Drosophila and BNI1 in yeast) have ostensibly unrelated phenotypes. Murine and human formin-2 (Fmn2) are described; these genes bear a high degree of similarity to formin-1 and cappuccino and are more distantly related to daiphanous. The mouse gene, which encodes a putative 1567-amino-acid open reading frame and maps to mouse Chromosome 1, is expressed almost exclusively in the developing and mature central nervous system. Expression begins at embryonic day 9.5 in the developing spinal cord and brain structures and continues in neonatal and adult brain structures including the olfactory bulb, cortex, thalamus, hypothalamus, hippocampus and cerebellum. Human formin-2 has a similar expression pattern (Leader, 2000).

Until the discovery of formin-2, formin-1 was the only potential vertebrate ortholog of cappuccino. Assigning ortholog status between members of the cappuccino subfamily is difficult because, unlike members of the diaphanous subfamily, very little similarity is found among the cappuccino subfamily proteins in the N-terminal domain. For example, chicken formin-1 is considered to be the ortholog of mouse formin-1, isoform IV, because these two proteins share 39% identity in the N-terminus and have similar expression patterns. Even less identity would be expected of a Drosophila/murine ortholog relationship. Surprisingly, formin-2 contains a 100-amino-acid region in the N-terminus (revealed through BLASTP) which is 23%/42% identical/similar to cappuccino. However, based on sequence alone, this low level of similarity is not sufficient to conclude anything other than that formin-2 and formin-1 are both potential orthologs of cappuccino. It is clear, though, that mouse and human formin-2 are orthologs, given 79% N-terminal identity and 94% C-terminal identity, in addition to similar expression patterns (Leader, 2000).

Diaphanous structure

Mammalian and fungal Diaphanous-related formin homology (DRF) proteins contain several regions of conserved sequence homology. These include an amino-terminal GTPase binding domain (GBD) that interacts with activated Rho family members and formin homology domains that mediate targeting or interactions with signaling kinases and actin-binding proteins. DRFs also contain a conserved Dia-autoregulatory domain (DAD) in their carboxyl termini that binds the GBD. The GBD is a bifunctional autoinhibitory domain that is regulated by activated Rho. Expression of the isolated DAD in cells causes actin fiber formation and stimulates serum response factor-regulated gene expression. Inhibitor experiments show that the effects of exogenous DAD expression are dependent upon cellular Dia proteins. Alanine substitution of DAD consensus residues that disrupt GBD binding also eliminate DAD biological activity. Thus, DAD expression activates nuclear signaling and actin remodeling by mimicking activated Rho and unlatching the autoinhibited state of the cellular complement of Dia proteins (Alberts, 2001).

Formins constitute a family of eukaryotic proteins that are considered to function as a cytoskeleton organizer to regulate morphogenesis, cell polarity and cytokinesis. Fhos is a recently identified mammalian formin, which contains the conserved domains FH (formin homology) 1 and FH2 in the middle region and the Dia-autoregulatory domain (DAD) in the C-terminus. The role of Fhos in the regulation of cytoskeleton, however, has remained unknown. This study shows that Fhos, in an active form, induces the formation of actin stress fibers and localizes to the actin-based structure. Fhos appears to normally exist in a closed inactive form via an intramolecular interaction between the N-terminal region and the C-terminal DAD. Both FH1 and FH2 domains are required for the induction of the stress fiber formation. However, the N-terminal region of Fhos is required for the targeting of this protein to stress fibers, which is probably mediated via its F-actin-binding activity. Fhos occurs as a homotypic complex in cells. The self-association of Fhos seems to be mediated via the FH2 domain: the domains bind to each other in a direct manner. Thus, the mammalian formin Fhos, which directly binds to F-actin via the N-terminal region, forms a homotypic complex via the FH2 domain to organize actin cytoskeleton (Takeya, 2003).

Formins are involved in a variety of cellular processes that require the remodelling of the cytoskeleton. They contain formin homology domains FH1 and FH2, which initiate actin assembly. The Diaphanous-related formins form a subgroup that is characterized by an amino-terminal Rho GTPase-binding domain (GBD) and an FH3 domain, which bind somehow to the carboxy-terminal Diaphanous autoregulatory domain (DAD) to keep the protein in an inactive conformation. Upon binding of activated Rho proteins, the DAD is released and the ability of the formin to nucleate and elongate unbranched actin filaments is induced. This study presents the crystal structure of RhoC in complex with the regulatory N terminus of mammalian Diaphanous 1 (mDia1) containing the GBD/FH3 region, an all-helical structure with armadillo repeats. Rho uses its 'switch' regions for interacting with two subdomains of GBD/FH3. The FH3 domain of mDia1 forms a stable dimer, and the DAD-binding site has been identified. Although binding of Rho and DAD on the N-terminal fragment of mDia1 are mutually exclusive, their binding sites are only partially overlapping. On the basis of these results, a structural model for the regulation of mDia1 by Rho and DAD is reported (Rose, 2005).

Diaphanous-related formins (DRFs) regulate dynamics of unbranched actin filaments during cell contraction and cytokinesis. DRFs are autoinhibited through intramolecular binding of a Diaphanous autoinhibitory domain (DAD) to a conserved N-terminal regulatory element. Autoinhibition is relieved through binding of the GTPase RhoA to the N-terminal element. The crystal structure of the dimeric regulatory domain of the DRF, mDia1, is reported in this study. Dimerization is mediated by an intertwined six-helix bundle, from which extend two Diaphanous inhibitory domains (DIDs) composed of five armadillo repeats. NMR and biochemical mapping indicate the RhoA and DAD binding sites on the DID partially overlap, explaining activation of mDia1 by the GTPase. RhoA binding also requires an additional structurally independent segment adjacent to the DID. This regulatory construction, involving a GTPase binding site spanning a flexibly tethered arm and the inhibitory module, is observed in many autoinhibited effectors of Ras superfamily GTPases, suggesting evolutionary pressure for this design (Otomo. 2005a).

The conserved formin homology 2 (FH2) domain nucleates actin filaments and remains bound to the barbed end of the growing filament. This study reports the crystal structure of the yeast Bni1p FH2 domain in complex with tetramethylrhodamine-actin. Each of the two structural units in the FH2 dimer binds two actins in an orientation similar to that in an actin filament, suggesting that this structure could function as a filament nucleus. Biochemical properties of heterodimeric FH2 mutants suggest that the wild-type protein equilibrates between two bound states at the barbed end: one permitting monomer binding and the other permitting monomer dissociation. Interconversion between these states allows processive barbed-end polymerization and depolymerization in the presence of bound FH2 domain. Kinetic and/or thermodynamic differences in the conformational and binding equilibria can explain the variable activity of different FH2 domains as well as the effects of the actin-binding protein profilin on FH2 function (Otomo, 2005b).

Diaphanous related formins (DRFs) are cytoskeleton remodeling proteins that mediate specific upstream GTPase signals to regulate cellular processes such as cytokinesis, cell polarity, and organelle motility. Previous work on the Rho-interacting DRF mDia has established that the biological activity of DRFs is regulated by an autoinhibitory interaction of a C-terminal diaphanous autoregulatory domain (DAD) with the DRF N terminus. This autoinhibition is released upon competitive binding of an activated GTPase to the N terminus of the DRF. Analyzing autoregulation of the Rac1-interacting DRF FHOD1, in vitro binding studies were used to identify a 60-amino acid DAD at the protein C terminus that recognizes an N-terminal formin homology (FH) 3 domain. Importantly, the FH3 domain of FHOD1 does not overlap with the proposed Rac1-binding domain. The FHOD1 DAD was found to contain one functional hydrophobic autoregulatory motif, while a previously uncharacterized basic cluster that is conserved in all DRF family DADs also contributes to the FH3-DAD interaction. Simultaneous mutation of both motifs efficiently releases autoinhibition of FHOD1 in NIH3T3 cells resulting in the formation of actin stress fibers and increased serum response element transcription. A second putative hydrophobic autoregulatory motif N-terminal of the DAD belongs to a unique FHOD subdomain of yet undefined function. NMR structural analysis and size exclusion chromatography experiments revealed that the FHOD1 DAD is intrinsically unstructured with a tendency for a helical conformation in the hydrophobic autoregulation motif. Together, these data suggest that in FHOD1, DAD acts as signal sequence for binding to the well folded and monomeric FH3 domain and imply an activation mechanism that differs from competitive binding of Rac1 and DAD to one interaction site (Schonichen 2006).

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. This study found that yeast and mammalian formins bind actin monomers but that this activity requires their C-terminal DAD domains. Furthermore, the DAD works in concert with the FH2 to enhance nucleation without affecting the rate of filament elongation. This mechanism was dissected in mDia1, nucleation activity was mapped to conserved residues in the DAD, and DAD roles in nucleation and autoinhibition were demonstrated to be separable. Furthermore, DAD enhancement of nucleation was independent of contributions from the FH1 domain to nucleation. Together, these 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 (Gould, 2011).

Formins couple Rho and Src during signaling and the regulation of actin dynamics

The small GTPase Rho induces the formation of actin stress fibers and mediates the formation of diverse actin structures. What remains unclear is how Rho regulates its effectors to elicit such functions. GTP-bound Rho activates its effector mDia1 by disrupting the intramolecular interactions of mDia1. mDia1 protein is the first identified mammalian homolog of Drosophila Diaphanous, which is essential for cytokinesis, and belongs to a family of formin-homology (FH) proteins. FH proteins share structural features, including the tandemly aligned FH1 and FH2 domains in their carboxy-terminal halves. The FH1 domain contains repetitive polyproline sequences, which interact with an actin-monomer-binding protein, profilin. FH proteins regulate cellular morphogenic events, possibly in collaboration with profilin. Indeed, mDia1, profilin and RhoA have been found to co-localize in dynamic plasma-membrane structures such as phorbol-ester-induced membrane ruffles and phagocytic cups. Because the inactivation of Rho by microinjection of C3 exoenzyme inhibits such co-localization, endogenous Rho may regulate the subcellular assembly of filamentous actin (F-actin) through mDia1 signaling, although the type of actin structure formed under the control of Rho-mDia1 signaling has largely remained unknown. In addition, a genetic study of a Costa Rica family, members of which often develop deafness without other symptoms, has identified hDIA1 as a gene responsible for non-syndromic deafness. The protein encoded by this gene is the human counterpart of mDia1. In the affected hDIA1 protein, 21 aberrant amino acids are substituted for the C-terminal 52 amino acids. It remains unknown how this C-terminal truncation induces pathogenicity and why it is inherited as a dominant trait (Watanabe, 1999 and references therein).

Active mDia1 induces the formation of thin actin stress fibers, which are disorganized in the absence of activity of the Rho-associated kinase ROCK. Moreover, active mDia1 transforms ROCK-induced condensed actin fibers into structures reminiscent of Rho-induced stress fibers. Thus mDia1 and ROCK work concurrently during Rho-induced stress-fiber formation. Intriguingly, mDia1 and ROCK, depending on the balance of the two activities, induce actin fibers of various thicknesses and densities. Thus Rho may induce the formation of different actin structures affected by the balance between mDia1 and ROCK signaling (Watanabe, 1999).

Rho, a member of the Rho small G protein family, regulates the formation of stress fibers and focal adhesions in various types of cultured cells. The actions of ROCK and mDia, both of which have been identified as putative downstream target molecules of Rho, have been investigated in Madin-Darby canine kidney cells. The dominant active mutant of RhoA induces the formation of parallel stress fibers and focal adhesions, whereas the dominant active mutant of ROCK induces the formation of stellate stress fibers and focal adhesions, and the dominant active mutant of mDia induces the weak formation of parallel stress fibers without affecting the formation of focal adhesions. In the presence of C3 ADP-ribosyltransferase for Rho, the dominant active mutant of ROCK induces the formation of stellate stress fibers and focal adhesions, whereas the dominant active mutant of mDia induces only the diffuse localization of actin filaments. These results indicate that ROCK and mDia show distinct actions in reorganization of the actin cytoskeleton. The dominant negative mutant of either ROCK or mDia inhibits the formation of stress fibers and focal adhesions, indicating that both ROCK and mDia are necessary for the formation of stress fibers and focal adhesions. Moreover, inactivation and reactivation of both ROCK and mDia are necessary for the 12-O-tetradecanoylphorbol-13-acetate-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. The morphologies of stress fibers and focal adhesions in the cells expressing both the dominant active mutants of ROCK and mDia are not identical to those induced by the dominant active mutant of Rho. These results indicate that at least ROCK and mDia cooperatively act as downstream target molecules of Rho in the Rho-induced reorganization of the actin cytoskeleton (Nakano, 1999).

Mammalian Diaphanous-related formins (Drfs) act as Rho small GTPase effectors during growth factor-induced cytoskeletal remodeling and cell division. While both p140 mDia1 (herein called Drf1) and p134 mDia2 (Drf3) have been shown to bind in vitro to activated RhoA-C, and Drf3 has also been shown to bind to Cdc42, little is known about the cellular function of these GTPase effector pairs. Thus, the murine Drf genes have been targeted to address their various contributions to small GTPase signaling in cytoskeletal remodeling and development. Drf1 +/+, +/-, and -/- cell lines were derived from embryonic stem cells. While some Drf1 +/- lines have fewer actin stress fibers, several Drf1 +/- and -/- cells were more motile and had more abundant lamella and filopodia. Because the apparent 'gain-of-function' corresponds with elevated levels of Drf3 protein expression, it was hypothesized that the effects on the actin cytoskeleton are due to Cdc42 utilization of Drf3 as an effector. In this study, it was found that inactive Drf3 variants and microinjected Drf3 antibodies interfer with Cdc42-induced filopodia. In addition, Drf3 contains a previously unidentified CRIB-like motif within its GTPase binding domain (GBD). By fluorescent resonance energy transfer (FRET) analysis, it has been demonstrated that this motif is required for Cdc42 binding and Drf3 recruitment to the leading edge and, surprisingly, to the microtubule organizing center (MTOC) of migrating fibroblasts. These observations extend the role of the mammalian Drfs in cell signaling and demonstrate that Cdc42 not only activates Drf3, but guides the effector to sites at the cell cortex where it remodels the actin cytoskeleton (Peng, 2003).

The small GTPase RhoA controls activity of serum response factor (SRF) by inducing changes in actin dynamics. In PC12 cells, activation of SRF after serum stimulation is RhoA dependent, requiring both actin polymerization and the Rho kinase (ROCK)-LIM kinase (LIMK)-cofilin signaling pathway, previously shown to control F-actin turnover. Activation of SRF by overexpression of wild-type LIMK or ROCK-insensitive LIMK mutants also requires functional RhoA, indicating that a second RhoA-dependent signal is involved. This is provided by the RhoA effector mDia: dominant interfering mDia1 derivatives inhibit both serum- and LIMK-induced SRF activation and reduce the ability of LIMK to induce F-actin accumulation. These results demonstrate a role for LIMK in SRF activation, and functional cooperation between RhoA-controlled LIMK and mDia effector pathways (Geneste, 2002).

Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism

The transition of cell-matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II-driven contractility by a Rho target known as Rho-associated kinase (ROCK) has been shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, mechanical force was applied to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling leads to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein-tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppresses this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1, are sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurs in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. These experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force (Riveline, 2001).

mDia1 is a potent actin nucleation factor regulated by autoinhibition

Formin proteins are widely expressed in eukaryotes and play essential roles in assembling specific cellular actin-based structures. Formins are defined by a Formin Homology 2 (FH2) domain, as well as a proline-rich FH1 domain that binds the actin monomer binding protein, profilin, and other ligands. Constructs including FH2 of budding yeast Bni1 or fission yeast Cdc12 formins nucleate actin filaments in vitro. FH2-containing constructs of murine mDia1 (also called p140 mDia or Drf1) are much more potent actin nucleators than the yeast formins. FH1 is necessary for nucleation when actin monomers are profilin bound. mDia1 is a member of the Diaphanous formin subfamily (Dia), whose members contain an N-terminal Rho GTPase binding domain (GBD) and a C-terminal Diaphanous autoinhibitory domain (DAD). Based on cellular and in vitro binding studies, an autoinhibitory model for Dia formin regulation proposes that GBD binding to DAD inhibits Dia-induced actin remodeling, whereas Rho binding activates by releasing GBD from DAD. Supporting this model, these results show that an N-terminal mDia1 construct strongly inhibits actin nucleation by the C terminus. RhoA partially relieves inhibition but does so when bound to either GDP or GTP analogs. Both N- and C-terminal mDia1 constructs appear to be multimeric (Li, 2003).

The following evidence suggests that mDia1 is a potent nucleator that causes filament elongation in the barbed-end direction: (1) mDia1 increases filament concentration when incubated with actin monomers; (2) this filament increase occurs within seconds even in the presence of low monomer concentrations that do not spontaneously nucleate appreciably on this timescale; and mDia1-induced polymerization acceleration occurs below the pointed-end critical concentration (Li, 2003).

The ability to form barbed-end nuclei in the absence of profilin distinguishes mDia1 from fission yeast Cdc12, which tightly caps barbed ends but allows nucleation and barbed-end elongation when bound to profilin. mDia1 appears more similar to Bni1, which allows barbed-end elongation in the absence of profilin. However, mDia1 is at least 7-fold more potent than Bni1 at creating filaments. This difference may reflect a higher affinity of mDia1 for actin monomers or nucleation mechanism that is different from that of dimer stabilization proposed for Bni1. Because mDia1's ability to partially cap barbed ends might affect polymerization rates, it was not possible to draw conclusions on nucleus size from the present data (Li, 2003).

An mDia1 construct containing the FH1 domain can overcome profilin's inhibition of nucleation, suggesting that profilin-actin binding to FH1 provides a monomer able to interact with the nascent nucleus. mDia1 interacts with filament barbed ends, slowing but not stopping monomer release from this end. These results suggest that, as proposed for Bni1 as well as Cdc12 bound to profilin, mDia1 might nucleate and allow barbed-end elongation but remain bound at the barbed end (Li, 2003).

Gel filtration chromatography suggests that nucleation-competent mDia1 constructs are dynamic multimers, and the 5:1 ratio of mDia1:filaments produced during nucleation suggests that the functional mDia1 nucleation unit may be multimeric. More detailed multimerization studies, employing techniques that are not affected by molecular shape, are required to determine the multimerization parameters of these proteins. However, one possible scenario is that each mDia1 subunit in a multimer contributes one or more monomers to a nucleus. An alternative hypothesis is that the partial barbed-end capping by mDia1 slows elongation, which would cause an underestimation in the number of filaments assembled by mDia1 and thus an increase in the apparent mDia1:filament ratio (Li, 2003).

The N terminus of mDia1 strongly inhibits nucleation by the C terminus, supporting an autoinhibition model of regulation. This effect must be verified with full-length mDia1. Another issue to be resolved with full-length protein is the possibility that inhibition occurs in trans between subunits of a multimeric mDia1 complex. RhoA only partially relieves autoinhibition, suggesting that other activating molecules might be required for full relief of autoinhibition. The situation might be analogous to that for WASp and N-WASP, for which combinations of Cdc42, polyphosphoinositides, SH3 domain-containing proteins, and non-receptor tyrosine kinases can relieve autoinhibition cooperatively (Li, 2003).

There are at least nine distinct mammalian formin genes, some possessing several splice variants. This work raises many additional questions, including the following: What is the detailed mechanism of mDia1-mediated nucleation, and do other formins utilize the same mechanism? Are other formins regulated by autoinhibition? What factors in addition to RhoA are required for full relief of autoinhibition (Li, 2003)?

Cellular control of cortical actin nucleation

The contractile actin cortex is a thin layer of actin, myosin, and actin-binding proteins that subtends the membrane of animal cells. The cortex is the main determinant of cell shape and plays a fundamental role in cell division, migration, and tissue morphogenesis. For example, cortex contractility plays a crucial role in amoeboid migration of metastatic cells and during division, where its misregulation can lead to aneuploidy. Despite its importance, knowledge of the cortex is poor, and even the proteins nucleating it remain unknown, though a number of candidates have been proposed based on indirect evidence. This study used two independent approaches to identify cortical actin nucleators: a proteomic analysis using cortex-rich isolated blebs, and a localization/small hairpin RNA (shRNA) screen searching for phenotypes with a weakened cortex or altered contractility. This unbiased study revealed that two proteins generated the majority of cortical actin: the formin mDia1 and the Arp2/3 complex. Each nucleator contributes a similar amount of F-actin to the cortex but has very different accumulation kinetics. Electron microscopy examination revealed that each nucleator affected cortical network architecture differently. mDia1 depletion led to failure in division, but Arp2/3 depletion did not. Interestingly, despite not affecting division on its own, Arp2/3 inhibition potentiated the effect of mDia1 depletion. These findings indicate that the bulk of the actin cortex is nucleated by mDia1 and Arp2/3 and suggest a mechanism for rapid fine-tuning of cortex structure and mechanics by adjusting the relative contribution of each nucleator (Bovellan, 2014).

Dia and cell migration

Lysophosphatidic acid (LPA) stimulates Rho GTPase and its effector, the formin mDia, to capture and stabilize microtubules in fibroblasts. Whether mammalian EB1 and adenomatous polyposis coli (APC) function downstream of Rho-mDia in microtubule stabilization was investigated. A carboxy-terminal APC-binding fragment of EB1 (EB1-C) functions as a dominant-negative inhibitor of microtubule stabilization induced by LPA or active mDia. Knockdown of EB1 with small interfering RNAs also prevents microtubule stabilization. Expression of either full-length EB1 or APC, but not an APC-binding mutant of EB1, is sufficient to stabilize microtubules. Binding and localization studies showed that EB1, APC and mDia may form a complex at stable microtubule ends. Furthermore, EB1-C, but not an APC-binding mutant, inhibits fibroblast migration in an in vitro wounding assay. These results show an evolutionarily conserved pathway for microtubule capture, and suggest that mDia functions as a scaffold protein for EB1 and APC to stabilize microtubules and promote cell migration (Wen, 2004).

Cell migration requires spatial and temporal regulation of filamentous actin (F-actin) dynamics. This regulation is achieved by distinct actin-associated proteins, which mediate polymerization, depolymerization, severing, contraction, bundling or engagement to the membrane. Mammalian Diaphanous-related (mDia) formins, which nucleate, processively elongate, and in some cases bundle actin filaments, have been extensively studied in vitro, but their function in the cell has been less well characterized. The role of mDia2 activity in the dynamic organization of F-actin was studyed in migrating epithelial cells. mDia2 was found to localizes in the lamella of migrating epithelial cells, where it is involved in the formation of a stable pool of cortical actin and in maintenance of polymerization-competent free filament barbed ends at focal adhesions. Specific inhibition of mDia2 alters focal adhesion turnover and reduces migration velocity. It is suggested that the regulation of filament assembly dynamics at focal adhesions may be necessary for the formation of a stable pool of cortical lamella actin and the proper assembly and disassembly dynamics of focal adhesions, making mDia2 an important factor in epithelial cell migration (Gupton, 2007).

Dia-interacting protein modulates formin-mediated actin assembly at the cell cortex

Mammalian Diaphanous (mDia)-related formins and the N-WASP-activated Arp2/3 complex initiate the assembly of filamentous actin. Dia-interacting protein (DIP) binds via its amino-terminal SH3 domain to the proline-rich formin homology 1 (FH1) domain of mDia1 and mDia2 and to the N-WASp proline-rich region. This study investigated an interaction between a conserved leucine-rich region (LRR) in DIP and the mDia FH2 domain that nucleates, processively elongates, and bundles actin filaments. DIP binding to mDia2 was regulated by the same Rho-GTPase-controlled autoinhibitory mechanism modulating formin-mediated actin assembly. DIP was previously shown to interact with and stimulate N-WASp-dependent branched filament assembly via Arp2/3. Despite direct binding to both mDia1 and mDia2 FH2 domains, DIP LRR inhibited only mDia2-dependent filament assembly and bundling in vitro. DIP expression interfered with filopodia formation, consistent with a role for mDia2 in assembly of these structures. After filopodia retraction into the cell body, DIP expression induced excessive nonapoptotic membrane blebbing, a physiological process involved in both cytokinesis and amoeboid cell movement. DIP-induced blebbing was dependent on mDia2 but did not require the activities of either mDia1 or Arp2/3. These observations point to a pivotal role for DIP in the control of nonbranched and branched actin-filament assembly that is mediated by Diaphanous-related formins and activators of Arp2/3, respectively. The ability of DIP to trigger blebbing also suggests a role for mDia2 in the assembly of cortical actin necessary for maintaining plasma-membrane integrity (Eisenmann, 2007).

Mitf regulation of Dia1 controls melanoma proliferation and invasiveness

It is widely held that cells with metastatic properties such as invasiveness and expression of matrix metalloproteinases arise through the stepwise accumulation of genetic lesions arising from genetic instability and 'clonal evolution.' By contrast, this study shows that in melanomas invasiveness can be regulated epigenetically by the microphthalmia-associated transcription factor, Mitf, via regulation of the DIAPH1 gene encoding the diaphanous-related formin Dia1 that promotes actin polymerization and coordinates the actin cytoskeleton and microtubule networks at the cell periphery. Low Mitf levels lead to down-regulation of Dia1, reorganization of the actin cytoskeleton, and increased ROCK-dependent invasiveness, whereas increased Mitf expression leads to decreased invasiveness. Significantly the regulation of Dia1 by Mitf also controls p27Kip1-degradation such that reduced Mitf levels lead to a p27Kip1-dependent G1 arrest. Thus Mitf, via regulation of Dia1, can both inhibit invasiveness and promote proliferation. The results imply variations in the repertoire of environmental cues that determine Mitf activity will dictate the differentiation, proliferative, and invasive/migratory potential of melanoma cells through a dynamic epigenetic mechanism (Carreira, 2007).

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