Gene name - diaphanous
Cytological map position - 38E4-6
Symbol - dia
FlyBase ID: FBgn0011202
Genetic map position -
Classification - formin homology domains 1 and 2
Cellular location - cytoplasmic
|Recent literature||Rosa, A., Vlassaks, E., Pichaud, F. and Baum, B. (2015). Ect2/Pbl acts via Rho and polarity proteins to direct the assembly of an isotropic actomyosin cortex upon mitotic entry. Dev Cell 32: 604-616. PubMed ID: 25703349
Entry into mitosis is accompanied by profound changes in cortical actomyosin organization. This study delineate a pathway downstream of the RhoGEF Pbl/Ect2 that directs this process in Drosophila notum epithelial cells. The data suggest that the release of Pbl/Ect2 from the nucleus at mitotic entry drives Rho-dependent activation of Myosin-II and, in parallel, induces a switch from Arp2/3 (see Actin-related protein 2/3 complex, subunit 1) to Diaphanous-mediated cortical actin nucleation that depends on Cdc42, aPKC, and Par6. At the same time, the mitotic relocalization of these apical protein complexes to more lateral cell surfaces enables Cdc42/aPKC/Par6 to take on a mitosis-specific function-aiding the assembly of a relatively isotropic metaphase cortex. Together, these data reveal how the repolarization and remodeling of the actomyosin cortex are coordinated upon entry into mitosis to provide cells with the isotropic and rigid form they need to undergo faithful chromosome segregation and division in a crowded tissue environment.
|Matsubayashi, Y., Coulson-Gilmer, C. and Millard, T. H. (2015). Endocytosis-dependent coordination of multiple actin regulators is required for wound healing. J Cell Biol 210: 419-433. PubMed ID: 26216900
The ability to heal wounds efficiently is essential for life. After wounding of an epithelium, the cells bordering the wound form dynamic actin protrusions and/or a contractile actomyosin cable, and these actin structures drive wound closure. Despite their importance in wound healing, the molecular mechanisms that regulate the assembly of these actin structures at wound edges are not well understood. This paper, using Drosophila embryos, demonstrates that Diaphanous, SCAR, and WASp play distinct but overlapping roles in regulating actin assembly during wound healing. Moreover, that endocytosis was shown to be essential for wound edge actin assembly and wound closure. Adherens junctions (AJs) were identified as a key target of endocytosis during wound healing, and it is proposed that endocytic remodeling of AJs is required to form 'signaling centers' along the wound edge that control actin assembly. It is concluded that coordination of actin assembly, AJ remodeling, and membrane traffic is required for the construction of a motile leading edge during wound healing.
|Rousso, T., Schejter, E.D. and Shilo, B.Z. (2015). Orchestrated content release from Drosophila glue-protein vesicles by a contractile actomyosin network. Nat Cell Biol [Epub ahead of print]. PubMed ID: 26641716
Releasing content from large vesicles measuring several micrometres in diameter poses exceptional challenges to the secretory system. An actomyosin network commonly coats these vesicles, and is thought to provide the necessary force mediating efficient cargo release. This study describes the spatial and temporal dynamics of the formation of this actomyosin coat around large vesicles and the resulting vesicle collapse, in live Drosophila melanogaster salivary glands. The Formin family protein Diaphanous (Dia) was identified as the main actin nucleator involved in generating this structure, and Rho was uncovered as an integrator of actin assembly and contractile machinery activation comprising this actomyosin network. High-resolution imaging reveals a unique cage-like organization of myosin II on the actin coat. This myosin arrangement requires branched-actin polymerization, and is critical for exerting a non-isotropic force, mediating efficient vesicle contraction.
|Gao, Y., Shuai, Y., Zhang, X., Peng, Y., Wang, L., He, J., Zhong, Y. and Li, Q. (2019). Genetic dissection of active forgetting in labile and consolidated memories in Drosophila. Proc Natl Acad Sci U S A. PubMed ID: 31488722
Different memory components are forgotten through distinct molecular mechanisms. In Drosophila, the activation of 2 Rho GTPases (Rac1 and Cdc42), respectively, underlies the forgetting of an early labile memory (anesthesia-sensitive memory, ASM) and a form of consolidated memory (anesthesia-resistant memory, ARM). This study dissected the molecular mechanisms that tie Rac1 and Cdc42 to the different types of memory forgetting. Two WASP family proteins, SCAR/WAVE and WASp, act downstream of Rac1 and Cdc42 separately to regulate ASM and ARM forgetting in mushroom body neurons. Arp2/3 complex, which organizes branched actin polymerization, is a canonical downstream effector of WASP family proteins. However, this study found that Arp2/3 complex is required in Cdc42/WASp-mediated ARM forgetting but not in Rac1/SCAR-mediated ASM forgetting. Instead, Rac1/SCAR may function with formin Diaphanous (Dia), a nucleator that facilitates linear actin polymerization, in ASM forgetting. The present study, complementing the previously identified Rac1/cofilin pathway that regulates actin depolymerization, suggests that Rho GTPases regulate forgetting by recruiting both actin polymerization and depolymerization pathways. Moreover, Rac1 and Cdc42 may regulate different types of memory forgetting by tapping into different actin polymerization mechanisms.
|Thestrup, J., Tipold, M., Kindred, A., Stark, K., Curry, T. and Lewellyn, L. (2020). The Arp2/3 complex and the formin, Diaphanous, are both required to regulate the size of germline ring canals in the developing egg chamber. Dev Biol. PubMed ID: 31945342
Intercellular bridges are an essential structural feature found in both germline and somatic cells throughout the animal kingdom. Because of their large size, the germline intercellular bridges, or ring canals, in the developing fruit fly egg chamber are an excellent model to study the formation, stabilization, and growth of these structures. Within the egg chamber, the germline ring canals connect 15 supporting nurse cells to the developing oocyte, facilitating the transfer of materials required for successful oogenesis. The ring canals are derived from a stalled actomyosin contractile ring; once formed, additional actin and actin-binding proteins are recruited to the ring to support the 20-fold growth that accompanies oogenesis. These behaviors provide a unique model system to study the actin regulators that control incomplete cytokinesis, intercellular bridge formation, and growth. By temporally controlling their expression in the germline, this study has demonstrated that the Arp2/3 complex and the formin, Diaphanous (Dia), coordinately regulate ring canal size and growth throughout oogenesis. Dia is required for successful incomplete cytokinesis and the initial stabilization of the germline ring canals. Once ring canals have formed, the Arp2/3 complex and Dia cooperate to determine ring canal size and maintain stability. These data suggest that nurse cells must maintain a precise balance between the activity of these two nucleators during oogenesis.
|Kirkland, N. J., Yuen, A. C., Tozluoglu, M., Hui, N., Paluch, E. K. and Mao, Y. (2020). Tissue Mechanics Regulate Mitotic Nuclear Dynamics during Epithelial Development. Curr Biol. PubMed ID: 32413305
Cell divisions are essential for tissue growth. In pseudostratified epithelia, where nuclei are staggered across the tissue, each nucleus migrates apically before undergoing mitosis. Successful apical nuclear migration is critical for planar-orientated cell divisions in densely packed epithelia. Most previous investigations have focused on the local cellular mechanisms controlling nuclear migration. Inter-species and inter-organ comparisons of different pseudostratified epithelia suggest global tissue architecture may influence nuclear dynamics, but the underlying mechanisms remain elusive. This study used the developing Drosophila wing disc to systematically investigate, in a single epithelial type, how changes in tissue architecture during growth influence mitotic nuclear migration. Distinct nuclear dynamics were observed at discrete developmental stages, as epithelial morphology changes. Genetic and physical perturbations were used to show a direct effect of cell density on mitotic nuclear positioning. Rho kinase and Diaphanous, which facilitate mitotic cell rounding in confined cell conditions, are essential for efficient apical nuclear movement. Perturbation of Diaphanous causes increasing defects in apical nuclear migration as the tissue grows and cell density increases, and these defects can be reversed by acute physical reduction of cell density. These findings reveal how the mechanical environment imposed on cells within a tissue alters the molecular and cellular mechanisms adopted by single cells for mitosis.
|Lv, Z., Rosenbaum, J., Mohr, S., Zhang, X., Kong, D., Preiß, H., Kruss, S., Alim, K., Aspelmeier, T. and Großhans, J. (2020). The emergent yo-yo movement of nuclei driven by cytoskeletal remodeling in pseudo-synchronous mitotic cycles. Curr Biol. PubMed ID: 32470369
Many aspects in tissue morphogenesis are attributed to a collective behavior of the participating cells. Yet, the mechanism for emergence of dynamic tissue behavior is not well understood. This study reports that the "yo-yo"-like nuclear movement in the Drosophila syncytial embryo displays emergent features indicative of collective behavior. Following mitosis, the array of nuclei moves away from the wave front by several nuclear diameters only to return to its starting position about 5 min later. Based on experimental manipulations and numerical simulations, this study finds that the ensemble of elongating and isotropically oriented spindles, rather than individual spindles, is the main driving force for anisotropic nuclear movement. ELMO-dependent F-actin restricts the time for the forward movement and ELMO- and dia-dependent F-actin is essential for the return movement. This study provides insights into how the interactions among the cytoskeleton as individual elements lead to collective movement of the nuclear array on a macroscopic scale.
The diaphanous (dia) locus plays a critical role during cytokinesis: various combinations of dia mutations result in multinucleate spermatids, polyploid larval neuroblasts and adult follicle cells, and pupal lethality (Castrillon and Wasserman, 1994). Subcellular characterization of dia-deficient spermatids reveals that inactivation of Diaphanous results in defects in the interzonal microtubules, structures known to be crucial for the execution of cytokinesis, and also in the absence of an actomyosin contractile ring (Giansanti, 1998). Embryos deficient for dia were analyzed for three cell-cycle-regulated actin-mediated events during embryogenesis: formation of the metaphase furrow, cellularization and formation of the pole cells. In dia embryos, all three processes are defective. Diaphanous is a good candidate for a protein that functions at the cell cortex (the intracellular region just below the plasma membrane), through which microtubules signal actin organization (reviewed by Wasserman, 1998). Indeed, recent studies in budding yeast provide a clue to the potential position of Diaphanous in positioning and assembly of cytoskeletal structures. Diaphanous homologs, FH (formin homology domain) proteins Bni1p and Bnr1p, mediate cross-talk between cortical actin and the spindle structure; this cross-talk is necessary for correct positioning of the spindle in the bud neck (Lee, 1999; Miller, 1999). Since an interaction between the mitotic spindle and the cell cortex is also thought to determine the position of the animal cell contractile ring, Diaphanous could similarly act in a pathway linking microtubule and microfilament organization (Afshar, 2000).
FH proteins contain several conserved sequence elements distributed over the primary sequence (Wasserman, 1998). A centrally located, proline-rich FH1 domain in fission yeast has been demonstrated to serve as a binding site for the actin-binding protein profilin (Drosophila homolog: Chickadee) (Chang, 1997). Functional domain analysis of a fission yeast FH protein, Fus1, has identified an amino-terminal FH3 homology domain required for proper localization (Petersen, 1998). In addition, the amino-terminal domain of several FH proteins interacts with Rho-GTPase proteins (Evangelista, 1997; Imamura, 1997; Kohno, 1996; Watanabe, 1997). Much less is known about the function of the carboxyl-terminal halves of FH proteins, which include the highly conserved FH2 region and, typically, flanking coiled-coil domains (Afshar, 2000).
To investigate the role of Diaphanous in the actin-mediated processes of the syncytial blastoderm, the phenotype of dia-deficient embryos was examined during early development. To eliminate the maternal contribution of Diaphanous to the oocyte, homozygous dia mutant clones in the female germline were generated. Two alleles of dia were used for the generation of these clones. dia5 is a null allele that causes larval and pupal lethality. dia9 is weaker than dia5, with some homozygous mutant flies surviving to adulthood. Fertilized embryos produced by ovarian clones of dia mutant tissue exhibit severe developmental defects. Only about 3% of the embryos produced by dia5clones hatch, whereas about 20% of those from dia9clones survived through the larval and pupal stages. Although similar results have been obtained for both mutant alleles, the phenotype of the dia5embryos is more severe at all stages of embryogenesis. Therefore, all of the experimental results presented below are from embryos produced by dia5clones (hereafter referred to as dia mutant embryos) (Afshar, 2000).
Defects in dia mutant embryos first appear at nuclear cycle 11; earlier stages, as assessed by nuclear migration, division and organization, appear as wild-type. Abnormalities in nuclear and actin cytoskeletal organization affect almost two-thirds of all fertilized embryos at cycles 11-13 and a higher percentage at later stages. Among embryos of a similar stage, the surface area affected varies considerably, ranging from a set of small patches to the entire embryonic surface. This variability does not extend to all phenotypes, however, because 100% of the embryos fail to form any pole cells. Furthermore, greater than 95% of embryos are grossly defective at gastrulation, despite the fact that half receive a wild-type copy of dia paternally. Cuticle preparations of dia mutant embryos reveal a wide range of phenotypes, including failure in head involution, loss of head structures, reduction or absence of denticle bands and incomplete formation of the cuticle (Afshar, 2000).
To explore the nature of the defects seen in the absence of diaphanous function, wild-type and dia mutant embryos were stained at nuclear cycles 11-13 with the DNA dye DAPI and with an antibody directed against F-actin. In the wild type, nuclei are positioned at the embryo cortex at interphase of nuclear cycles 11-13; a structure referred to as the actin cap is situated between each nucleus and the plasma membrane. During the transition to prophase, filament reorganization results in a concentration of actin at the edge of the caps. At metaphase, the resulting rings of cortical actin, together with associated plasma membrane, invaginate to form metaphase furrows. As viewed from above, actin staining at these furrows appears as a hexagonal array over the embryonic surface. In the sagittal view, actin staining at the metaphase furrow appears as a line between the metaphase nuclei. In dia-deficient embryos, severe structural changes in the actin cytoskeleton are manifested after nuclear cycle 11. Formation of the hexagonal actin arrays is disrupted during prophase and metaphase and there is an absence of actin staining between the metaphase nuclei. Similar patterns of staining are obtained when dia embryos are stained with antibodies directed against anillin (Drosophila gene: Scraps) and Peanut, other components of the metaphase furrow. There is thus a failure in the formation of the metaphase furrow. Consistent with the known role of metaphase furrows in maintaining nuclear organization, the nuclei in dia mutant embryos frequently exhibit abnormal spacing and, in some cases, fuse in subsequent nuclear cycles. These irregularities are readily apparent in contrast to the uniform pattern observed in the wild type. In regions in which cortical actin staining is weak or absent, nuclei are frequently found displaced into the interior of the embryo, although the centrosomes remain at the surface (Afshar, 2000).
To investigate whether the absence of metaphase furrows results from a failure in membrane invagination, dia embryos were stained with antibodies directed against myosin (also known as Zipper). In wild-type embryos, myosin localizes to the embryonic cortex between the actin caps at each interphase, appears at the tip of the invaginating membrane at prophase and disappears at metaphase. In dia embryos, myosin staining, albeit very weak and irregular, is detected between the actin caps at the cortex during interphase. At prophase, myosin, where detectable, remains at the cortex, with no detectable membrane pinching or invagination. Therefore, despite the presence of myosin at the cortex between actin caps, the membrane invagination that precedes metaphase furrowing is absent in dia embryos (Afshar, 2000).
Immunolocalization was used to determine whether Diaphanous plays a role in the recruitment of anillin and Peanut, a Drosophila septin. In wild-type embryos both anillin and Peanut localize to the embryonic cortex, between the actin caps at interphase. During prophase and metaphase, they localize to the metaphase furrow and their pattern of staining is similar to that of actin. In dia embryos, the staining patterns of both anillin and Peanut are very weak during interphase. Similarly, in dia embryos the localization of both of these proteins is disrupted during prophase and metaphase, when the metaphase furrow is being formed in wild-type embryos. Diaphanous is thus required for recruitment and proper localization of anillin and Peanut as well as myosin to the regions of membrane invagination (Afshar, 2000).
Following the 13 syncytial nuclear division cycles, wild-type cellularization initiates. Actin-associated membrane invaginates between neighboring nuclei. This invagination is accompanied by the growth of microtubules, which extend from the pair of centrosomes above each nucleus, to form a basket-shaped structure that eventually surrounds each nucleus. The subsequent formation of individual cells involves further growth of the membrane and an actomyosin-mediated contractile event that pinches off the membrane at the bottom of each nucleus. In dia embryos, there is a variable defect in the organization of both actin- and microtubule-based structures during cellularization. In the least severe cases, the cellularization furrow is absent between some nuclei, without any noticeable defect in morphology or positioning of nuclei or microtubule structure. In more severely affected embryos, actin staining is absent at the furrow canals and irregular at some regions of the cortex. Surface regions that lack any organized actin display abnormalities in the positioning of both nuclei and microtubule baskets. Viewed from en face, such embryos display readily apparent irregularities in the hexagonal actin and microtubule arrays and nuclear positioning. These abnormalities are manifested as starburst arrays of nuclei and associated microtubules, with the nuclei tilted outward. In such regions, actin staining is faint or absent at the cortex, although some patchy staining is detected deeper in the embryo. In addition, gamma-tubulin staining reveals that centrosomal behavior is abnormal in these regions. This phenotype is similar to that observed in embryos treated with Cytochalasin D, in which the disruption of the cortical actin results in misorganization of the nuclei and mislocalization of microtubule baskets. In the most severely affected dia embryos the defective cortical actin and arrays of misoriented nuclei are observed over the entire embryonic surface. The localization of both anillin and Peanut in dia embryos is abnormal in regions of nuclear misorientation, but is wild-type elsewhere. For example, anillin, which can be detected in the nuclei of wild-type embryos only after cellularization, is present in the tilted nuclei in dia embryos during cellularization (Afshar, 2000).
Diaphanous is necessary for pole cell formation. During interphase of the wild-type cortical nuclear divisions, actin cap formation causes protrusion of the plasma membrane and cytoplasm around each nucleus, forming a cytoplasmic bud. At the posterior pole, in contrast to the rest of the embryo, these cytoplasmic buds grow extensively during nuclear cycle 10. Cytokinesis at the base of each bud results in the formation of a set of pole cells, progenitors of the adult germline. In dia mutant embryos, formation and growth of the cytoplasmic buds at the posterior pole is wild-type. Such buds, however, never cleave to produce pole cells. Rather, they regress in synchrony with the buds covering the rest of the embryonic cortex. Buds reform at the posterior pole at each nuclear cycle, but do not undergo cytokinesis. In some embryos, the number and size of the somatic buds are abnormal compared to wild-type embryos. Moreover, unlike the somatic nuclei, the posterior pole nuclei fail to initiate cellularization. To investigate the basis for the failure in pole cell formation, the presence was sought of an actomyosin contractile ring at the base of the posterior cytoplasmic buds. In contrast to wild-type embryos, dia embryos lack any concentration of actin or myosin at the base of the cytoplasmic buds, suggesting the contractile process does not start in the absence of Diaphanous (Afshar, 2000).
The phenotypic analysis of dia embryos indicates a requirement for Diaphanous for nuclear organization, metaphase furrow formation, cellularization and pole cell formation. To investigate the function of Diaphanous in these actin-mediated events, anti-Diaphanous sera was used to assay the spatial and temporal pattern of localization of Diaphanous during early embryonic development. During the first ten nuclear division cycles, no Diaphanous localization to specific structures was detected. Once the nuclei migrate to the cortex, however, Diaphanous staining appears as a hexagonal array at the surface of interphase and prophase embryos. At interphase, actin filaments remain in cap structures and Diaphanous localizes to the site of formation of the metaphase furrow prior to any significant redistribution of actin, which occurs at prophase. This is readily apparent in a sagittal view, revealing intense Diaphanous staining between the interphase actin caps. By prophase, Diaphanous is abundant at the tip of the metaphase furrow; this intense localization continues through metaphase. During cellularization, Diaphanous is enriched at the tip of the cellularization front and at the site of membrane invagination. As cellularization ends, Diaphanous localizes to the basal surface of each newly formed cell, where a contractile event pinches off the membrane at the base of the nuclei to produce individual cells (Afshar, 2000).
It is thought that specific signals, acting through Diaphanous, could assign actin cytoskeletal function for a specific cortical event. Different sets of Rho proteins and their regulators could recruit and/or activate Diaphanous in particular locations and at particular times. Diaphanous has a genetic interaction with Rho1 and the RhoGTPase exchange factor encoded by the Drosophila pebble gene. Pebble is strictly required for cytokinesis, but not for any membrane invagination events in the syncytial blastoderm. In addition, the immunolocalization of Diaphanous in dividing cells closely overlaps that of Pebble, being nuclear during telophase and interphase and located at the cleavage furrow at anaphase. Thus, one can imagine that Diaphanous function is controlled by Pebble at anaphase during cytokinesis, whereas other factors regulate Diaphanous function during early embryogenesis (Afshar, 2000).
Additional roles in cell regulation in the mouse have been revealed by studies of Diaphanous-related formin (DRF) Rho GTPase binding proteins mDia1 and mDia2. The DRF proteins couple Rho and Src during signaling and the regulation of actin dynamics. The DRFs are required for cytokinesis, stress fiber formation, and transcriptional activation of the serum response factor (SRF). 'Activated' mDia1 and mDia2 variants, lacking their GTPase binding domains, cooperate with Rho-kinase or ROCK to form stress fibers but independently activate SRF. Src tyrosine kinase associates and co-localizes with the DRFs in endosomes and in mid-bodies of dividing cells. Inhibition of Src also blocks cytokinesis, SRF induction by activated DRFs, and cooperative stress fiber formation with active ROCK (Tominaga, 2000).
The DRF proteins complex with multiple regulatory factors and bridge different signaling pathways. These multimeric complexes are likely to be highly dynamic, regulated by cycles of GTP hydrolysis and binding to signaling molecules such as Src. In yeast, deregulation of the yeast DRF Bni1 also triggers the formation of actin cables, suggesting that it is an active scaffold that recruits cellular machinery capable of polymerizing actin. In this regard, Bni1 or Bnr1 has been shown to bind multiple actin binding proteins, including elongation factor 1, profilin, and Bud6/Aip3p and appears to bridge two small GTPase regulated signaling pathways. Bni1 is found in high molecular weight complexes with docking components such as Spa2 that in turn bind constituent kinases from both the Cdc42/pheromone and Rho1/cell wall integrity pathways. These data suggest that the DRFs may bridge signals from the Rho GTPases and Src, which is known to contribute to multiple signaling pathways. Deregulation of the mouse DRFs results in activation of nuclear signaling and stress fiber formation, likely through obligate binding to Src. Recent results suggest that SRF activation can be triggered by depletion of G-actin pools. Is this mediated by the DRFs, profilin, or Src? Expression of an interfering mutant of profilin (H119E) that binds mDia1 and mDia2, but fails to bind actin, has no effect on mDia2 activation of SRF. This is a surprising result, although consistent with recently published data showing that this mutant profilin has no effect on activated RhoA or LPA induction of stress fibers. These data suggest that Src is the more important DRF-binding partner and begs the question: What is the role of profilin in Rho- or DRF-regulated actin dynamics? Profilin has been presumed to be the critical effector for the formin homology proteins in the regulation of actin polymerization. These findings indicate that this is not the case for the DRFs (Tominaga, 2000 and references therein).
Src and related family members have multiple roles in cell signaling and receptor trafficking. Src becomes activated in response to receptor-peptide growth factor binding and in response to ligands such as LPA that utilize G protein coupled receptors (GPCRs). Receptor clustering and the formation of clathrin-coated pits have been suggested to be essential for full activation of signaling by GPCRs. Sequestration of both EGF receptors and GPCRs appears to be dependent upon Src. Also, Rho has been shown to have a role in targeting Src. There is increasing evidence to suggest that actin dynamics and the Rho GTPases are involved in receptor trafficking and endocytosis. mDia1 has also been shown to associate with phagocytic cups. The roles of mDia1 and mDia2 in these processes are currently being evaluated. In complexes derived from brain extracts, profilin has been shown to be complexed with components of the endocytosis machinery clathrin and dynamin. The data clearly show an association of mDia1 and mDia2 with Src in endosomes. It will be important to explore the possibility that Src and the Rho GTPases form part of a complex that integrates components involved in both endocytosis and signaling. Do mDia1 and mDia2 bind other GTPases that regulate endocytosis or phagocytosis? RhoB, for example, has been shown to be targeted to endosomes or to have a role in vesicle dynamics. Other Rho-family members are being tested for their ability to utilize the DRFs as effectors (Tominaga, 2000 and references therein).
In addition to the DRFs, other Rho effectors localize to the cleavage furrow and have critical roles in the completion of cell division. The ROCK-related Citron kinase, for example, likely cooperates with mDia1 and mDia2 during cytokinesis in a manner analogous to the ROCK-DRF collaborative induction of stress fiber formation. But what is the importance of a DRF bridge between Src and Rho in the process of cytokinesis? Both Cdc42 and RhoA are required for cell division and RhoA has been shown to localize to the cleavage furrow. Does Rho localize Src via mDia1 or mDia2 in a manner similar to the observed localization of Src to the cell periphery? Consistent with the observation that Src co-localizes with the DRF proteins at the mid-body of dividing cells, microinjection of inhibitory anti-Src antibodies inhibits the completion of mitosis. Active Src localizes to whole spindle microtubules in addition to the mid-body. This indicates that Src is active prior to any association with the DRFs. During early stages of mitosis, Cdc2 kinase has been shown to phosphorylate Src on amino terminal residues, which may contribute to its activation. `Active Src then persists after metaphase when cyclin B is degraded and cdc2 kinase activity is low. Src then localizes with the DRF and is maintained in its active form as defined by antibody staining. It is hypothesized that the DRFs bind and stabilize Src and therefore protect it from inactivating kinases such as Csk. Src has known mitotic substrates such as Sam68, but their role(s) in cell division is not clear. Further studies will be needed to identify important Src substrates and mechanisms of Src activation, stabilization, or localization by DRFs during cytokinesis (Tominaga, 2000 and references therein).
The Diaphanous protein contains two domains shared by the formin proteins, encoded by the limb deformity gene in the mouse. The FH1 domain consists of an unusual proline-rich region near the middle of Diaphanous. This proline-rich domain contains six repeats of 5 to 8 consecutive prolines separated by short stretches rich in the amino acids glycine, methionine, alanine or arginine, with a few interspersed prolines. The FH2 domain, present in a region of about 130 amino acids, has been termed FH2. Both FH1 and FH2 domains are found in Bni1p, the product of a Saccharomyces cerevisiae gene required for normal cytokinesis in diploid yeast cells. It is striking that not only the sequence composition of the FH1 and FH2 domains, but also the spacing between these domains, is conserved among Diaphanous, Bni1p, and the formins (Castrillon, 1994).
Secondary structure analysis, using the algorithm devised by Stock and colleagues, has revealed two regions of the Diaphanous protein that are very likely to form coiled-coil domains. One of these, spanning amino acids 441 to 500, ends just at the beginning of the FH1 domain. The second, spanning amino acids 863 to 1053, begins 17 amino acids before the end of the FH2 domain, just after the region of highest sequence similarity. Thus it appears that coiled-coil domains flank the FH1 and FH2 domains of diaphanous. Moreover, sequence analysis also predicts the existence of coiled-coil domains at equivalent positions in mouse formin IV and Bni1p (Castrillon, 1994 and references therein).
date revised: 5 May 2000
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