Formins are key regulators of actin nucleation and elongation. Diaphanous-related formins, the best-known subclass, are activated by Rho and play essential roles in cytokinesis. In cultured cells, Diaphanous-related formins also regulate cell adhesion, polarity and microtubules, suggesting that they may be key regulators of cell shape change and migration during development. However, their essential roles in cytokinesis hamper testing of this hypothesis. Loss- and gain-of-function approaches were used to examine the role of Diaphanous in Drosophila morphogenesis. Diaphanous has a dynamic expression pattern consistent with a role in regulating cell shape change. Constitutively active Diaphanous was used to examine its roles in morphogenesis and its mechanisms of action. This revealed an unexpected role in regulating myosin levels and activity at adherens junctions during cell shape change, suggesting that Diaphanous helps coordinate adhesion and contractility of the underlying actomyosin ring. This hypothesis was tested by reducing Diaphanous function, revealing striking roles in stabilizing adherens junctions and inhibiting cell protrusiveness. These effects also are mediated through coordinated effects on myosin activity and adhesion, suggesting a common mechanism for Diaphanous action during morphogenesis (Homem, 2008).
Previous analysis of Diaphanous-related formins (DRF) function in morphogenesis was hampered by mammalian DRF redundancy and the key role of DRFs in cytokinesis. The genetic tools available in Drosophila were used to partially circumvent these difficulties. Dia is known to regulate cellularization. The analysis revealed new roles during morphogenesis. In embryos with severely reduced Dia function, gastrulation is disrupted. Apical constriction of central furrow cells is delayed or blocked in a subset of cells, suggesting a possible role for Dia in regulated apical constriction. Previous work suggested that an unknown Rho effector regulates actin during this process; perhaps this is Dia. Adjacent cells, which are stretched during invagination, become massively multinucleate in dia5M mutants. This may reflect the fact that the cells of the blastoderm undergo an incomplete form of cytokinesis, remaining connected to the underlying yolk by actin-lined yolk canals. These canals normally close at the onset of gastrulation in ventral furrow cells; this may prevent cell membrane rupture initiated at the yolk canal under stress. Dia may regulate yolk canal closure; it localizes there at the end of cellularization and similar defects are seen in mutants lacking the septin Peanut, a yolk canal component. Alternately, Dia may stabilize cortical actomyosin or its connections to AJs, with its absence weakening cortical integrity under stress (Homem, 2008).
Dia also plays a key role during germband retraction. The most striking effect of reduced Dia/Rho1 function is altered amnioserosal cell protrusiveness. They normally make stable AJs and form a tight tissue boundary with the epidermis; this is destabilized in dia/Rho1 and dia5M/Z mutants. Altered protrusive activity was seen in other contexts: dia5M/Z ectoderm exhibited cortical blebbing, and dia/Rho1 and dia5M/Z amnioserosal cells had altered protrusiveness during dorsal closure. Reduced Dia function destabilized AJs, and the striking increase in cell protrusiveness in dia/Rho1 mutants was substantially rescued by increasing Myosin phosphorylation. These data support the hypothesis that Dia normally helps restrict actomyosin contractility to AJs, and in its absence, AJs are destabilized and myosin activity outside AJs stimulates protrusiveness. Interestingly, in cultured cells, fully assembled AJs inhibit Rac1 and lamellipodial activity while increasing Rho and contractility, and myosin is required for mouse Dia1-induced AJ strengthening (Homem, 2008).
Given these morphogenetic roles, the mechanisms of action of Dia was explored, initially hypothesizing that it would primarily affect actin. DiaCA expression did elevate F-actin levels (reducing Dia did not dramatically decrease F-actin, but residual Dia may suffice). However, surprisingly, Dia activation also increased Myosin accumulation and cell contractility, and some effects of reduced Dia function were rescued by increasing Myosin activation. This suggests a surprising role for Dia in regulating Myosin levels/activity. The data also suggest that Dia stabilizes AJs, supporting earlier work in cultured cells. There are intimate connections between AJs and cortical actomyosin: AJs help organize the apicolateral actin ring while cortical actin stabilizes AJs. Interestingly, in cultured cells Myosin-mediated contraction of actin filaments is essential for cell-cell contact expansion, and mouse Dia1 AJ stabilization requires myosin activity, providing further evidence that contractility, AJ stability and Dia activity are mechanistically linked. The actomyosin network may also stabilize AJs by preventing endocytosis. Perhaps Dia, like formin 1, directly binds AJs. Linear actin filaments promoted by Dia at AJs might recruit α-catenin, which then might inhibit Arp2/3-induced actin branching, facilitating formation of a belt of unbranched actin. Thus, Dia is poised to act at the interface between AJs, cortical actin and contractility (Homem, 2008).
The Yin-Yang relationship between adhesion and protrusiveness is a very interesting one, underlying epithelial-mesenchymal transitions. Stable AJs probably actively inhibit protrusiveness, and Dia may assist this, acting in a reinforcing loop that concentrates actomyosin activity at AJs. There are interesting parallels with the role of Dia in cytokinesis. Dia stabilizes Myosin at the midzone. In its absence Myosin is delocalized around the cortex, leading to abnormal protrusiveness. Future work will reveal how Dia fits into the regulatory network coordinating adhesion and cytoskeletal regulation (Homem, 2008).
From these data, a mechanistic model was developed for the regulation of actin, Myosin and AJs by Dia during cell shape change, in which Dia activation stabilizes actin and active Myosin at AJs. In radially symmetric amnioserosal cells, Dia promotes organization/activation of an apical actomyosin network linked to AJs, inducing precocious apical cell constriction. Activation of endogenous Dia may regulate normal amnioserosal constriction: this now needs to be tested. Dia activation in cells where AJs are planar polarized, such as epidermal cells, promotes actomyosin organization preferentially at cell borders where AJs are enriched. This leads to cell widening or helps cells resist elongation, generating groove-cell-like morphology. Once again, Dia may be normally activated specifically in groove cells to modulate their shape (Homem, 2008).
How does Dia activation activate Myosin? It does not occur primarily through SRF, which triggers Myosin accumulation but not cell shape changes. Myosin activation via MLCK partially mimicked DiaCA, suggesting that Myosin activation is an important part of the process. However, MLCK did not precisely mimic DiaCA, suggesting that Dia acts preferentially at specific sites such as AJs rather than globally activating Myosin. The dual effects on actin and Myosin suggests the speculative possibility that feedback mechanisms exist to coordinate the Rho-regulated actin and Myosin pathways. Recent work revealed that active Dia1 can activate RhoA by binding the Rho-GEF LARG, and strong genetic interactions were seen between dia and the LARG relative RhoGEF2, making this idea more plausible. Future work is needed to test this hypothesis (Homem, 2008).
The Drosophila gene diaphanous is required for cytokinesis. Males homozygous for the dia1 mutation are sterile due to a defect in cytokinesis in the germline. Females trans-heterozygous for dia1 and a deficiency are sterile and lay eggs with defective eggshells; failure of cytokinesis is observed in the follicle cell layer. Null alleles are lethal. Death occurs at the onset of pupation due to the absence of imaginal discs. Mitotic figures in larval neuroblasts have been found to be polyploid, apparently due to a defect in cytokinesis (Castrillon, 1994).
The male-sterile dia1 allele has been identified in a P element screen for mutations affecting spermatogenesis. Although both spermatocytes and spermatids are initially present in dia1 testes, these cells degenerate and are not replenished. By 5 days after eclosion, most mutant testes are devoid of germinal contents. Female fertility is not significantly affected, and the viability of both males and females is normal (Castrillon, 1994).
During spermatogenesis, a spermatogonium (the product of a stem cell division) undergoes four rounds of mitotic division to give rise to a cyst of 16 spermatocytes; meiosis then produces a cyst of 64 haploid spermatids. Wild-type chromosomal segregation and cytokinesis result in spermatids that each contain two major cytological structures of identical size and shape: a pale round nucleus, and an adjacent dark nebenkern. The nebenkern results from the fusion of all the mitochondria in a spermatid; its size thus serves as a marker for the amount of cytoplasm inherited by a spermatid. The contents of testes from 50 newly eclosed dia1 males were examined. dia1 testes contain far fewer spermatids than normal, due to the reduced germinal content. The majority of spermatids were large and multinucleate. These abnormal spermatids contained either 2, 4 or 8 nuclei, with the size of the nebenkerne proportional to the number of nuclei. Of 159 unelongated spermatids identified, 51 (32%) contained one nucleus (phenotypically normal); 26 (16%) contained two nuclei; 81 (51%) contained four nuclei, and 1 (1%) contained 8 nuclei. The presence of spermatids containing 2 or 4 nuclei within a common cytoplasm can be explained by a failure in cytokinesis in one or both of the meiotic divisions. Likewise, the rare spermatid containing 8 nuclei can be explained by a failure of cytokinesis in three consecutive cell divisions, the first being the mitotic division preceding meiosis. The nuclei in defective spermatids are almost always of wild-type size, indicating that chromosome segregation is normal in spite of the failure of cytokinesis. In contrast, in mutants that cause nondisjunction during meiosis, nuclei are of variable size. The finding that the initial failure of cytokinesis can occur at distinct points along the spermatogenesis pathway suggests why the germline in dia1 testes is eventually depleted. Since the 5-9 stem cells present in each testis continually divide to give rise to spermatogonia, failure of cytokinesis during stem cell divisions should result in the permanent inactivation of these cells (Castrillon, 1994).
In trans to a chromosomal deficiency or a null allele such as dia2, the dia1 allele exhibits an oogenesis phenotype. Although the viability of such trans-heterozygous adults is normal and the male germline phenotype is similar to that of dia1 males, female fertility is dramatically decreased. The ovaries of dia1/dia2 females are smaller than wild type. Egg chambers of all stages are present, the great majority of which contain 15 nurse cells and one oocyte. However, the eggs laid by dia1/dia2 females are shorter than wild-type and have short, fused, or extra dorsal appendages. Only 10% of the eggs hatch. This eggshell phenotype suggested a defect in the somatically derived follicle cells, which surround each developing egg chamber and secrete the eggshell. Indeed, follicle cells in mutant ovaries have an abnormal appearance when viewed by differential interference contrast microscopy. Their nuclei vary considerably in size relative to wild-type controls. Some of the cells appear to contain two nuclei. To visualize the follicle cell layer better, ovaries were double labelled with a fluorescent Concanavalin A derivative (to stain plasma membranes) and with propidium iodide (to stain nuclei) and examined by confocal microscopy. Individual follicle cells containing two nuclei are frequently observed in egg chambers from mutant mothers; such cells are not found in wild-type egg chambers. Therefore, it appears that cytokinesis fails to occur in some follicle cells. The abnormally large nuclei seen are likely due to nuclear fusion following the failure of cytokinesis (Castrillon, 1994).
Lethal dia mutations, including null mutations, are generated by imprecise excision of the P element in the dia1 allele. Null alleles such as dia2 result in early pupal lethality and anabsence of imaginal discs. This phenotype is consistent with dia being an essential mitotic gene. Due to the presence of maternal gene products, an embryo with a null mutation in such a gene can develop into a larva. However, since imaginal disc cells divide during the larval stages, by which time zygotic gene expression is required, such larvae will have defective or absent discs and will die at the onset of pupation. Homozygotes for less severe alleles such as dia3 also die as early pupae, but third instar larvae contain imaginal discs, albeit somewhat smaller than normal. Homozygotes for the weakest lethal allele, dia9, have imaginal discs of normal appearance but die as late pupae or pharate adults. Very few (less than 1%) of dia9 flies eclose. These flies are sickly and have a weak ërough eyeí phenotype, consistent with a mitotic defect that affects a small fraction of cells. Testes from these surviving adults are almost completely devoid of germinal content (Castrillon, 1994).
The larval central nervous system (brain and ventral ganglion) is a rich source of dividing cells (neuroblasts) and is the most suitable tissue for examining mitosis in larvae. Chromosome morphology and segregation can be examined in aceto-orcein-stained preparations of the larval CNS. In wild type, a mitotic figure consists of 3 pairs of major chromosomes. In larvae homozygous for a weak lethal allele, dia9, only a small fraction of neuroblasts are polyploid and the number of mitotic figures is not affected. Homozygotes for a stronger allele, dia3, exhibit ploidy ranging from 2n, 4n and 8n to extreme hyperploidy. In addition, significantly fewer mitotic figures are present in dia3 homozygotes than in wild type. However, the fraction of mitotic figures in anaphase is the same as in wild type, whereas mutations that disrupt spindle function result in a decrease in the number of anaphases. In addition, chromosome morphology is generally normal, although a small number of mitotic figures contain highly condensed chromosomes. Homozygotes for a null allele, dia2 , have very few mitotic figures, and all are enormously hyperploid. The morphology of anaphase figures provides direct evidence that cytokinesis is defective in dia3 cells. Cleavage furrows are sometimes evident in wild-type anaphase figures, especially in well-isolated cells. However, cleavage furrows have not been observed in any dia3 anaphases and the cells appear completely round even when the chromosomes have finished migrating to opposite poles. Despite the cytokinesis defect, chromosome segregation appears to be relatively normal in polyploid cells. In bipolar dia3 anaphases, the spindles are well organized and the chromosomes are equally segregated, with no lagging chromosomes or other abnormalities. This is true even in anaphases that are clearly hyperploid. In more extremely hyperploid cells, anaphases are typically multipolar. Such multipolar spindles have also been observed in other mutants that produce hyperploid cells. Taken together, the male-sterile, female-sterile and lethal phenotypes associated with dia mutations demonstrate that dia is required for cytokinesis in both the soma and germline and in mitosis as well as meiosis (Castrillon, 1994).
In animal cells, cytokinesis is accomplished by the contractile ring, a transient structure containing actin and myosin II (see Zipper) filaments, that is anchored to the equatorial cortex. Interactions between these filaments lead to the constriction of a ring that pinches the dividing cell in the middle like an ever tightening purse string until cleavage is completed. Male meiosis was examined in mutants of the chickadee (chic) locus, a Drosophila gene that encodes profilin, a low molecular weight actin-binding protein that modulates F-actin polymerization. These mutants are severely defective in meiotic cytokinesis. Difficulties in meiotic cytokinesis are immediately obvious because of the characteristic appearance of spermatids directly after their formation at the so-called onion stage. Wild-type onion stage spermatids contain a single phase-light nucleus and a similarly sized phase-dark Nebenkern (a mitochondrial derivative). Failures in cytokinesis result in abnormally large Nebenkern associated with multiple normal-sized nuclei. The resulting phenotypes fall into multiple groups: in testes of males homozygous for chic a large fraction of onion-stage spermatids contain a single Nebenkern of larger than normal size, associated with two or more normal-sized nuclei. A substantial proportion have two nuclei with an intermediate-size Nebekern, but most frequently, these aberrant spermatids contain four nuclei and a very large Nebenkern. These phenotypes reflect failures of cytokinesis at either one or the other or both meiotic divisions, respectively, that would prevent proper subdivision of mitochondria and nuclei into daughter spermatids (Giansanti, 1998).
To investigate the relationships between the central spindle and the contractile ring, meiosis was examined in the cytokinesis-defective mutants KLP3A and diaphanous. The KLP3A gene encodes a kinesin-like protein that accumulates in the central spindle midzone during anaphase and telophase of both meiotic divisions. Accordingly, mutations in this gene disrupt central spindle formation and cause frequent failures in meiotic cytokinesis. To check whether the defect in central spindle integrity observed in KLP3A mutants also affects actin ring assembly, KLP3A mutant testes were stained with rhodamine-labeled phalloidin. The results of this experiment clearly show that most mutant ana-telophases (90%) are completely devoid of actin rings. The rare ana-telophases that exhibit thin and incomplete actin rings also contain more densely packed central spindles than those of cells completely lacking contractile rings. Despite the absence of the contractile ring, KLP3A mutants do not exhibit aberrant actin accumulations or problems in aster migration like those described above for chic and tsr mutants (Giansanti, 1998).
The diaphanous gene encodes a protein that interacts with profilin through its proline-rich domain. All the ana-telophases present in testes homozygous for dia mutants are completely devoid of actin rings. There are severe defects in the central spindle, similar to those observed in chic and KLP3A. The effects on the actomyosin contractile ring and the central spindle observed in chic and dia mutants could be specific consequences of lesions in the corresponding gene products. Alternatively, these effects could result from a more general disruption of the actin cytoskeleton. To discriminate between these possibilities, wild-type testes were treated with cytochalasin B prior to fixation and staining. Cytochalasin B binds the barbed ends of actin filaments and promotes the conversion of ATP-actin monomers to ADP-actin, preventing proper assembly of the contractile ring in most cell types. Remarkably, incubation with this drug produces an almost exact phenocopy of strong chic alleles. No F-actin staining is observed in any contractile ring-like structures at the equator of ana-telophase cells (Giansanti, 1998).
In all cases examined, the central spindle and the contractile ring in meiotic ana-telophases were simultaneously absent. Together, these results suggest a cooperative interaction between elements of the actin-based contractile ring and the central spindle microtubules: when one of these structures is disrupted, the proper assembly of the other is also affected. In addition to effects on the central spindle and the cytokinetic apparatus, another consequence of chic mutations was observed: a large fraction of chic spermatocytes exhibit abnormal positioning and delayed migration of asters to the cell poles. A similar phenotype is seen in testes treated with cytochalasin B and has been noted previously in mutants at the twinstar locus. These observations all indicate that proper actin assembly is necessary for centrosome separation and migration, and that the central spindle and the contractile ring are interdependent structures (Giansanti, 1998).
The best candidate at present for mediating interactions between the central spindle and cortical actin, at least during male meiosis, is the KLP3A kinesin-like protein. This protein could interact directly with both the central spindle microtubules and components of the contractile ring. Alternatively, KLP3A could transport to the spindle midzone molecules that mediate F actin-microtubule interactions. At the moment, it is not possible to discriminate between these possibilities, nor is there any information on the proteins that bind to or might be transported by KLP3A. It is believed, however, that the isolation and characterization of additional mutations causing cytological phenotypes similar to those of KLP3A, chic, and dia, will eventually provide substantial insight into the mechanisms underlying microtubule-actin interaction during cytokinesis (Giansanti, 1998).
The Drosophila formin homology (FH) protein Diaphanous has an essential role during cytokinesis. To gain insight into the function of Diaphanous during cytokinesis and explore its role in other processes, embryos deficient for Diaphanous were generated and three cell-cycle-regulated actin-mediated events during embryogenesis were analyzed: formation of the metaphase furrow, cellularization, and formation of the pole cells. In dia embryos, all three processes are defective. Actin filaments do not organize properly to the metaphase and cellularization furrows and the actin ring is absent from the base of the presumptive pole cells. Furthermore, plasma membrane invaginations that initiate formation of the metaphase furrow and pole cells are missing. Immunolocalization studies of wild-type embryos reveal that Diaphanous localizes to the site where the metaphase furrow is anticipated to form, to the growing tip of cellularization furrows, and to contractile rings. In addition, the dia mutant phenotype reveals a role for Diaphanous in recruitment of myosin II, anillin and Peanut to the cortical region between actin caps. There findings thus indicate that Diaphanous has a role in actin cytoskeleton organization and is essential for many, if not all, actin-mediated events involving membrane invagination. Based on known biochemical functions of FH proteins, it is proposed that Diaphanous serves as a mediator between signaling molecules and actin organizers at specific phases of the cell cycle (Afshar, 2000).
The proto-oncogenic kinase Abelson (Abl) regulates actin in response to cell signaling. Drosophila Abl is required in the nervous system, and also in epithelial cells, where it regulates adherens junction stability and actin organization. Abl acts at least in part via the actin regulator Enabled (Ena), but the mechanism by which Abl regulates Ena is unknown. A novel role is described for Abl in early Drosophila development, where it regulates the site and type of actin structures produced. In Abl's absence, excess actin is polymerized in apical microvilli, whereas too little actin is assembled into pseudocleavage and cellularization furrows. These effects involve Ena misregulation. In abl mutants, Ena accumulates ectopically at the apical cortex where excess actin is observed, suggesting that Abl regulates Ena's subcellular localization. Other actin regulators were also examined. Loss of Abl leads to changes in the localization of the Arp2/3 complex and the formin Diaphanous, and mutations in diaphanous or capping protein beta enhance abl phenotypes (Grevengoed, 2003).
Different actin regulators play fundamentally different biochemical roles. Models often picture all of these regulators modulating actin assembly and disassembly at a single site, but of course individual cells target different actin regulators to distinct sites, creating actin structures with diverse functions. Syncytial embryos provide an excellent example. During interphase, they assemble actin-based microvillar caps above each nucleus. As they enter prophase, caps are disassembled and actin polymerization is redirected to the pseudocleavage furrows. This is likely to require new machinery: both Arp2/3 and the formin Dia are required for pseudocleavage furrow formation, but not for actin caps. Cellularization also requires distinct machinery to polymerize/disassemble apical microvilli and to recruit and modulate actin at the cellularization front. For transitions to occur smoothly, two fundamental changes have to occur: the location at which actin polymerization occurs must change, and a different constellation of actin regulators must be deployed to produce the distinct actin structures observed (Grevengoed, 2003).
The data support a hypothesis in which the balance of activity of different actin regulators at distinct sites is tightly regulated, influencing the nature of the actin structures produced. One regulator is Abl. In its absence, Ena localizes ectopically to the cortical region, upsetting the temporal and spatial balance of actin regulators. This leads to a change in both the location and nature of actin polymerization during mitosis. Excess actin is polymerized into microvillar projections that extend from the apical region of the furrows, whereas insufficient actin is directed to the pseudocleavage furrows. Similarly, during cellularization in ablM mutants, actin polymerization continues to be directed to apical microvilli, whereas in a wild-type embryo this ceases early in cellularization (Grevengoed, 2003).
The data also suggest that there is cross-talk between different modulators of actin polymerization, and that the balance of their activities determines the outcome. Although many actin modulators are unaffected in ablM mutants, both the Arp2/3 complex and Dia are recruited to sites of ectopic actin polymerization. However, genetic analysis suggests that although Ena mislocalization plays a critical role in the actin alterations seen in ablM mutants, Dia and Arp2/3 mislocalization may not. In fact, reduction of the dose of Dia enhanced the ablM phenotype. Dia normally promotes actin polymerization lining the furrows. In ablM mutants, the balance of actin polymerization is already shifted to the apical microvilli because of ectopic Ena localization. Reduction in the dose of Dia might further reduce actin polymerization in pseudocleavage furrows, resulting in the observed enhancement of the ablM phenotype. The abnormal recruitment of Dia to the apical regions in ablM mutants may also reduce pseudocleavage furrow formation (Grevengoed, 2003).
It will now be important to investigate how the cell regulates the distinct types of actin polymerization required for distinct cellular and developmental processes. One mechanism of cross-talk may involve direct or indirect recruitment of one type of actin modulator by another. Abl's ability to interact with both Ena and the Arp2/3 regulator WAVE1 is interesting in this regard. However, the recruitment of Arp3 and Dia to ectopic actin structures observed in ablM mutants may have a more simple explanation. Both are thought to have a higher affinity for newly polymerized, ATP-bound actin, which is likely to be increased where ectopic actin polymerization appears to occur (Grevengoed, 2003).
Drosophila Abl also functions in other contexts. It has a role in embryonic morphogenesis, where it also acts, at least in part, via Ena. However, in this context Abl also affects AJ stability. Since Ena is normally highly enriched in AJs, it is hypothesized that Abl helps restrict Ena localization to AJs, and thus helps initiate the proper organization of actin underlying AJs. In Abl's absence, Ena may localize to sites other than AJs, leading to ectopic actin polymerization at those sites and reduction in actin polymerization at AJs (analogous to the divergent effects on apical actin and pseudocleavage/cellularization furrows). Since the cortical actin belt underlying the AJ plays an important role in its stability, this could explain the phenotype of abl mutants. A similar model may help explain the roles of Abl and Ena in axon outgrowth. The network of actin filaments in the growth cone is complex, with different types of actin in filopodia and in the body of the growth cone. By regulating Ena localization, Abl may influence the balance of the different types of actin, thus influencing growth cone motility. Likewise, in fibroblasts, where Ena/VASP proteins regulate motility, the Arp2/3 regulators N-WASP and WAVE localize to sites at the leading edge distinct from those where Mena is found. Whether Abl or Arg regulate the localization of Ena/VASP family proteins in mammals remains to be determined. Likewise, it is possible that deregulation of Ena/VASP proteins underlie some of the alterations in cell behavior in Bcr-Abl–transformed lymphocytes. Experiments to test whether Ena/VASP activity is important for either mammalian Abl's normal function or for the pathogenic effects of Bcr-Abl will help answer these questions (Grevengoed, 2003).
The steroid hormone 20-hydroxyecdysone (ecdysone) is the key regulator of postembryonic developmental transitions in insects and controls metamorphosis by triggering the morphogenesis of adult tissues from larvae. The Rho GTPase, which mediates cell shape change and migration, is also an essential regulator of tissue morphogenesis during development. Rho activity can modulate gene expression, in part, by activating LIM kinase (LIMK) and consequently affecting actin-induced SRF transcriptional activity. A link has been established between Rho-LIMK-SRF signaling and the ecdysone-induced transcriptional response during Drosophila development. Specifically, Rho GTPase, via LIMK, regulates the expression of several ecdysone-responsive genes, including those encoding the ecdysone receptor itself, a downstream transcription factor (Br-C), and Stubble, a transmembrane protease required for proper leg formation. Stubble and Br-C mutants exhibit strong genetic interactions with several Rho pathway components in the formation of adult structures, but not with Rac or Cdc42. In cultured SL2 cells, inhibition of Rho, F-actin assembly, or SRF blocks the transcriptional response to ecdysone. Together, these findings indicate a link between Rho-LIMK signaling and steroid hormone-induced gene expression in the context of metamorphosis and thereby establish a novel role for the Rho GTPase in development (Chen, 2004).
Metamorphosis in Drosophila is stringently controlled by pulses of the steroid hormone ecdysone at discrete developmental stages. During larval-pupal transition, ecdysone triggers coordinated changes in tissue morphology that involve histolysis of larval tissues and the initiation of adult structures. Rho GTPase-mediated signaling pathways have been implicated in several aspects of morphogenesis during Drosophila embryo formation. However, a role for Rho signaling in metamorphosis has not yet been reported. Among the downstream mediators of Rho signaling are the LIM kinases, and a closely related Drosophila ortholog of mammalian LIM kinases (designated Dlimk) is specifically expressed at relatively high levels in late larval and pupal stages, suggesting a potential role in Rho-LIMK signaling during this transition. In adult flies, Dlimk is expressed at substantially higher levels in males than in females, consistent with a potential evolutionarily conserved role in spermatogenesis, a process in which mammalian LIMK2 has been implicated. Dlimk mRNA is uniformly expressed throughout eye, wing, and leg imaginal discs (Chen, 2004).
The malformed legs in DlimkD522A flies closely resemble leg defects in flies in which Rho signaling is perturbed through genetic disruption of Rho1, DrhoGEF2 (a guanine nucleotide exchange factor for Rho1), sqh (myosin light chain), and zipper (nonmuscle myosin heavy chain). Sqh and zipper are downstream targets of Drok and regulate actomyosin contractility. Loss-of-function mutants of Rho1 or DrhoGEF2 strongly suppress the severity of wing defects associated with Dlimk expression. Reducing Rho activity by overexpressing the potent Rho inhibitor, p190 RhoGAP, also efficiently suppresses Dlimk-induced wing defects. Moreover, reducing levels of Diaphanous or Drok, two Rho targets that promote actin assembly, also substantially reduces the severity of Dlimk-induced wing defects. A loss-of-function allele of blistered, the Drosophila SRF ortholog, also suppresses the Dlimk-induced wing defects, suggesting that regulation of SRF-dependent transcription by Rho-LIMK signaling plays a role in wing morphogenesis. Significantly, in mammalian cells, LIMK and Diaphanous cooperate to regulate SRF activity (Geneste, 2002). Reducing levels of the Rho-related GTPases, Rac1, Rac2, and Cdc42, or the Rac activator, Myoblast city (Mbc), or the Rac/Cdc42 effector target, PAK, has very little effect on the Dlimk-induced wing phenotype. Thus, it appears that in the developing leg and wing, Dlimk specifically mediates a Rho-actin signaling pathway required for imaginal-disc morphogenesis (Chen, 2004).
Cells migrating through a tissue exert force via their cytoskeleton and are themselves subject to tension, but the effects of physical forces on cell behavior in vivo are poorly understood. Border cell migration during Drosophila oogenesis is a useful model for invasive cell movement. This migration requires the activity of the transcriptional factor Serum response factor (SRF) and its cofactor MAL-D and evidence is presented that nuclear accumulation of MAL-D is induced by cell stretching. Border cells that cannot migrate lack nuclear MAL-D but can accumulate it if they are pulled by other migrating cells. Like mammalian MAL, MAL-D also responds to activated Diaphanous, which affects actin dynamics. MAL-D/SRF activity is required to build a robust actin cytoskeleton in the migrating cells; mutant cells break apart when initiating migration. Thus, tension-induced MAL-D activity may provide a feedback mechanism for enhancing cytoskeletal strength during invasive migration (Somogyi, 2004).
The conserved protein structure, in particular the conserved RPEL motifs (MHD), as well as the functional interactions with SRF suggested that mammalian and fly MAL proteins might be regulated in similar ways. In a series of interesting experiments, activation of mammalian SRF and nuclear accumulation of MAL have shown to respond to changes in actin dynamics in NIH-3T3 cells. The N-terminal RPEL motifs of MAL were required for this regulation, which has also been called the Rho-actin pathway. One of the strongest activators of MAL/SRF was an activated form of Diaphanous, which acts downstream of Rho. To determine whether MAL-D could be subject to the same regulation, a corresponding activated form of Drosophila Diaphanous (HA-diaCA) was made and overexpressed in border cells. Border cell migration was blocked by HA-diaCA; however, nuclear accumulation of MAL-D was nevertheless stimulated. This effect was observed on endogenous MAL-D but was most obvious when looking at border cell clusters cooverexpressing MAL-D and HA-diaCA. In border cells, as in follicle cells, overexpressed MAL-D was predominantly cytoplasmic. In contrast, when HA-diaCA was present, MAL-D was predominantly nuclear. When both proteins were expressed at high levels, the nuclear pool of MAL-D was still detectable, but MAL-D was mainly cytoplasmic, suggesting that nuclear translocation was saturable. Thus, the ability of the Rho pathway to activate MAL proteins appears to be conserved in Drosophila (Somogyi, 2004).
What is the molecular mechanism for MAL-D regulation by tension? Given that the actin cytoskeleton and tension or cell shape changes are interdependent, it is likely that this regulation is related to the regulation of MAL/SRF by actin dynamics (the Rho pathway). Two models were proposed to explain the effect of actin on MAL and SRF. The simplest model is that free G-actin sequesters MAL in the cytoplasm, and depletion of this G-actin pool by actin polymerization results in MAL translocation/activation. Observations in border cells do not fit very well with this simple model. In normal cells, even very highly overexpressed MAL-D is almost exclusively cytoplasmic, indicating practically unlimited capacity in the cytoplasm. Expression of constitutively active Diaphanous, which should 'release' MAL-D by causing actin polymerization, did cause accumulation of MAL-D in the nucleus. But further overexpression of MAL-D led to more protein in the cytoplasm, not in the nucleus as would be expected if G-actin depletion in the cytoplasm (induced by active Diaphanous) were the trigger for nuclear translocation. Finally, even though endogenous MAL-D is expressed at low levels, overexpression of a nonpolymerizable form of actin in border cells did not appear to sequester MAL-D in the cytoplasm. These data seem more consistent with the alternative 'active' model of MAL activation, wherein a subpopulation of actin or an actin protein complex accumulates when actin polymerization is favored, leading to MAL nuclear translocation and activity (Somogyi, 2004).
There are two general ways in which regulation of MAL by actin and by tension might be related. Changes in actin dynamics, as induced by activated Diaphanous, may induce changes in tension, which could then affect MAL. For example, RhoA activation can induce formation of stress fibers, which are contractile structures. Conversely, changes in cell tension could affect RhoA, Diaphanous, and thereby actin dynamics, which then in turn directly regulate MAL. In fact, RhoA and Diaphanous, two of the most potent activators of SRF/MAL, have been shown to be important mediators of mechanosensitive changes at focal adhesions. The physical interaction observed between the conserved N-terminal domain of MAL and unpolymerized forms of actin suggests that regulation of MAL by actin is quite direct and thus supports this type of relationship. Tension applied to cell-matrix attachments or cell-cell interactions may also locally increase actin polymerization by other means and thereby activate MAL. A more speculative link to MAL regulation is offered by actin itself. A specific conformation of actin, or a specific protein complex containing actin, may be induced by tension and serve as the signal that is perceived by MAL. This would be consistent with the idea that a particular subpopulation of actin is responsible for the active regulation of MAL. It would be an elegant way for hard-working migratory cells to regulate strength as needed by the actin cytoskeleton. It is usually thought that actin-myosin supplies force and tension; the MAL/SRF system suggests a role for the complex actin cytoskeleton in force perception as well (Somogyi, 2004).
The physical interaction of the plasma membrane with the associated cortical cytoskeleton is important in many morphogenetic processes during development. At the end of the syncytial blastoderm of Drosophila the plasma membrane begins to fold in and forms the furrow canals in a regular hexagonal pattern. Every furrow canal leads the invagination of membrane between adjacent nuclei. Concomitant with furrow canal formation, actin filaments are assembled at the furrow canal. It is not known how the regular pattern of membrane invagination and the morphology of the furrow canal is determined and whether actin filaments are important for furrow canal formation. Both the guanyl-nucleotide exchange factor RhoGEF2 and the formin Diaphanous (Dia) are required for furrow canal formation. In embryos from RhoGEF2 or dia germline clones, furrow canals do not form at all or are considerably enlarged and contain cytoplasmic blebs. Both Dia and RhoGEF2 proteins are localised at the invagination site prior to formation of the furrow canal. Whereas they localise independently of F-actin, Dia localisation requires RhoGEF2. The amount of F-actin at the furrow canal is reduced in dia and RhoGEF2 mutants, suggesting that RhoGEF2 and Dia are necessary for the correct assembly of actin filaments at the forming furrow canal. Biochemical analysis shows that Rho1 interacts with both RhoGEF2 and Dia, and that Dia nucleates actin filaments. These results support a model in which RhoGEF2 and dia control position, shape and stability of the forming furrow canal by spatially restricted assembly of actin filaments required for the proper infolding of the plasma membrane (Grosshans, 2005).
This morphological analysis of the mutant phenotypes reveals a new function of RhoGEF2 and dia in the formation of the furrow canal. This function is consistent with the co-localisation of both proteins with F-actin at the furrow canal and the reduced amounts of F-actin in RhoGEF2 and dia mutants. Biochemical analysis demonstrates actin polymerisation by Dia and thus supports the model that RhoGEF2 and Dia organise actin filaments to control the formation of the furrow canals. Furthermore, evidence is provided that the previously characterised genes nullo and sry- alpha act in a genetic pathway in parallel to RhoGEF2 and dia, suggesting that they control two distinct aspects of furrow canal formation. This conclusion is based on the assumption that amorphic situtations were used in these experiment. The possibility cannot be excluded that RhoGEF2 and dia stabilise the furrow canal rather than control its initial formation. A function in the formation is supported by the observation that the proportion of nuclei in multinuclear cells does not increase in the course of cellularisation (Grosshans, 2005).
The following arguments support the hypothesis that RhoGEF2 and dia act in the same genetic pathway that controls spatially restricted assembly of actin filaments. In both dia and RhoGEF2 mutants the morphology of the furrow canal is disrupted. The furrow canals are much larger than normal and filled with cytoplasmic blebs. Both proteins are localised at the furrow canal and both precede the appearance of the cellularisation front. The localisation of both proteins does not depend on F-actin. However, they are directly or indirectly involved in the assembly of F-actin since the amount of F-actin is reduced at the furrow canal of the mutant embryos. The strongest argument for a functional connection is that Dia localisation at the furrow canal depends on RhoGEF2 during the early phase of cellularisation. Rho1 may mediate this functional link by direct interactions with RhoGEF2 and Dia. However, the findings do not show that RhoGEF2 exclusively functions via dia. Other targets of Rho1-GTP, like citron kinase, protein kinase N or Rho kinase may be activated in parallel to Dia. Although a reduction of MyoII at the furrow canal was observed during the first half of cellularisation in embryos from RhoGEF2 germline clones, correspondingly lower MyoII levels are also observed in embryos from dia germline clones, which indicates that the reduction of MyoII may be a consequence of reduced F-actin levels. Consistent with the reduction of F-actin at the furrow canal, levels of MyoII were also reduced in the mutant embryos. In contrast to the reduction at the furrow canal, cortical F-actin appeared to be increased in some embryos from dia germline clones. This increase was variable and not observed in all of the experiments, however (Grosshans, 2005).
The difference in the RhoGEF2 and dia mutant phenotypes clearly shows that dia has additional functions and may be controlled by other not yet identified factors besides RhoGEF2. Whereas RhoGEF2 mutants pass through the cleavage cycles without obvious defects, dia is involved in formation of pole cells and pseudo cleavage furrows. As a possible consequence of these additional functions, dia mutants in contrast to RhoGEF2 mutants often have a more disrupted F-actin array, larger furrow canals and a more disturbed cellularisation than RhoGEF2 mutants. Furthermore in the early phase of cellularisation Dia localisation depends on RhoGEF2, whereas later, after the furrow has formed, Dia becomes enriched to a certain degree at the cellularisation front independently of RhoGEF2. One gene that may act in parallel to RhoGEF2 to control Dia localisation is Abl. Embryos from Abl germline clones have reduced amounts of Dia at the furrow canal and show a disrupted F-actin array similar to that observed in dia and RhoGEF2 mutants. However, the molecular link between Abl and Dia is elusive and no abnormalities in the morphology of the furrow canal in Abl mutants have been described. Thus Dia may be controlled and activated by multiple pathways including RhoGEF2 among others (Grosshans, 2005).
It is not known how the position of the invaginating plasma membrane is determined. RhoGEF2 and Dia are not likely to be part of a pattern formation process, but their localisation reflects an early readout of this pattern, since the nuclei and centrosomes are properly arranged in RhoGEF2 and dia mutants. RhoGEF2 and Dia proteins are early markers for these sites and precede furrow canal formation because specific staining was detected for both Dia and RhoGEF2 when the nuclei were still spherical and when the cellularisation front was not yet visible. Other factors beside RhoGEF2 and Dia are also involved in furrow canal formation, because many furrow canals still form in RhoGEF2 and dia mutants, which indicates that there is genetic redundancy (Grosshans, 2005).
At present it can only be speculated about which factors and mechanisms are responsible for RhoGEF2 localisation. Candidates may be among the group of genes involved in furrow canal formation. However, for all of these mutations no ultrastructural analysis has been reported that would allow the morphological defect to be defined and their function for furrow canal formation to be compared with the function of RhoGEF2 and dia. Among this group are Rab11 and nuf, which encode a GTPase of the recycling endosome and its putative effector. Considering the assumed biochemical activities, it is conceivable that vesicle targeting is important for transporting factors to the site of membrane invagination. This raises the possibility that RhoGEF2 is transported by such vesicles to the sites of membrane infolding. Analysis of RhoGEF2 protein distribution in nuf and Rab11 mutants and the phenotype of double mutants may address this hypothesis. Alternatively, RhoGEF2 may be transported to the site of the future furrow canal along microtubules that form open baskets around the nuclei, or other recruiting factors may precede at the site of membrane invagination (Grosshans, 2005).
Furthermore, slow as molasses (slam) is required for timed formation of the furrow and invagination of the membrane in the first half of cellularisation. Like Dia and RhoGEF2 Slam protein localises to the furrow canal and localisation precedes furrow canal formation. Slam may act by recruiting MyoII to the furrow canal, but the biochemical activities of Slam have not been defined. Although the membrane does not invaginate initially in slam mutants, a complete F-actin array is visible. Thus despite the overlapping localisation of Slam, RhoGEF2 and Dia, their functions are clearly distinguishable (Grosshans, 2005).
How do RhoGEF2 and Dia act in furrow canal formation? If the biochemical activity of Dia is considered to nucleate actin filaments and the enlarged and labile furrow canals in the dia mutants, it is conceivable that Dia organises and assembles a coat of F-actin at the site of membrane invagination and furrow canal formation. The coat of F-actin may be important for the compactness and stability of the furrow canal to prevent infoldings of the cytoplasm. Such a function may be related to the function of F-actin in endocytic events. The subset of actin filaments controlled by RhoGEF2 would not significantly contribute to pulling in the plasma membrane, since membrane invagination proceeds with normal speed in RhoGEF2 mutants. Alternatively, RhoGEF2 and Dia may perform their function independently of actin polymerisation. Although the amount of F-actin is reduced in the mutants, the possibility that the polymerisation activity of Dia is not required for all or part of its function cannot be excluded. Dia may also influence the organisation of microtubules, as interactions of mDia1 with microtubules and EB1, a microtubule-associated protein, have been described (Grosshans, 2005).
The differences in protein localisation and mutant phenotypes of RhoGEF2 and nullo suggest that they have distinct activities. In contrast to the frequently missing furrow canals in single mutants, their complete absence in embryos lacking both gene functions clearly implies, however, that their functions are redundant from a genetic point of view. These results show that RhoGEF2 and dia are required for the formation of a compact and stable furrow canal. If one of the two pathways is disturbed, the furrow canal can still form, albeit with a lower and variable efficiency that depends on the conditions. For example the nullo phenotype is strongly temperature sensitive. However, if both pathways are affected, furrow canals do not form at all. Future studies will resolve how the actin filaments are involved in bending the plasma membrane that leads to the furrow canal and will further demonstrate how RhoGEF2 protein is expressed in the hexagonal array to serve as a template for local actin polymerisation (Grosshans, 2005).
The correct localization of myosin II to the equatorial cortex is crucial for proper cell division. A collection of genes was examined that causes defects in cytokinesis and revealed (with live cell imaging) two distinct phases of myosin II localization. Three genes in the rho1 signaling pathway, pebble (a Rho guanidine nucleotide exchange factor), rho1, and rho kinase, are required for the initial recruitment of myosin II to the equatorial cortex. This initial localization mechanism does not require F-actin or the two components of the centralspindlin complex, the mitotic kinesin pavarotti/MKLP1 and racGAP50c/CYK-4. However, F-actin, the centralspindlin complex, formin (diaphanous), and profilin (chickadee) are required to stably maintain myosin II at the furrow. In the absence of these latter genes, myosin II delocalizes from the equatorial cortex and undergoes highly dynamic appearances and disappearances around the entire cell cortex, sometimes associated with abnormal contractions or blebbing. These findings support a model in which a rho kinase-dependent event, possibly myosin II regulatory light chain phosphorylation, is required for the initial recruitment to the furrow, whereas the assembly of parallel, unbranched actin filaments, generated by formin-mediated actin nucleation, is required for maintaining myosin II exclusively at the equatorial cortex (Dean, 2005).
This study has discovered three steps in the myosin II localization/activation process that involve distinct groups of genes: (1) an initial recruitment of myosin II to the equatorial cortex that is independent of F-actin and centralspindlin but requires rho1 signaling; (2) a secondary stabilization of myosin II at the midzone that requires F-actin and a second set of genes that are likely involved in building a specific type of actin network, and (3) the activation of furrowing once myosin II is localized that depends on centralspindlin (Dean, 2005).
Rho1, its activating guanidine nucleotide exchange factor pebble, and rho kinase are each required for the initial recruitment of myosin II to the equatorial cortex. Rho1 has been implicated in two pathways that are important for cytokinesis. In the first pathway, rho1 signals to F-actin through the formin diaphanous. However, proteins on this F-actin pathway, including F-actin itself, are not essential for the initial myosin II recruitment to the equatorial cortex. However, rho kinase, another downstream target of rho1, is essential. Because rho kinase phosphorylates the myosin II RLC, it is possible that phosphorylation of the RLC is essential for myosin II recruitment to the furrow. This hypothesis could not be directly tested, because the myosin II heavy chain forms large aggregates when the RLC is depleted by RNAi (Dean, 2005).
Phosphorylation of the RLC both activates the motor domain and, in some myosins, increases bipolar thick filament formation. Because F-actin is not required for myosin II recruitment, activation of the motor is unlikely to be the mechanism by which phosphorylation of the RLC would cause recruitment of myosin II to the equatorial cortex. It is quite possible, however, that the rho kinase-mediated myosin II phosphorylation leads to thick filament assembly and that this assembly is important for localization of myosin to the equatorial cortex. Indeed, in Dictyostelium, it is clear that bipolar thick filament formation is sufficient for myosin II localization to the midzone of a mitotic cell. The nonactin-based mechanism of recruitment of myosin II filaments remains unknown (Dean, 2005).
In contrast to the lack of F-actin involvement in the early recruitment of myosin II to the equatorial cortex at anaphase, F-actin disruption by Latrunculin A results in a failure to maintain myosin II in the equatorial region. Interestingly, the downstream rho1 effectors diaphanous/formin and chickadee/profilin are also necessary for myosin II maintenance at the equatorial midzone. Although the loss of these genes could deplete F-actin, phalloidin staining has shown that F-actin is still present in all of the RNAi-treated cells. In addition, these RNAi-treated cells still contract, unlike when F-actin is completely disrupted with LatA. Thus, myosin II appears to be interacting with F-actin in the cortex as it disperses in dynamic patches throughout the cortex of these diaphanous- or chickadee-depleted cells (Dean, 2005).
It is suggested that the role of diaphanous/formin and chickadee/profilin in maintaining the myosin II contractile ring is through the creation of specific F-actin structures. In particular, formin- and profilin-mediated nucleation results in unbranched actin filaments because profilin promotes the barbed-end growth of formin-capped actin filaments. Indeed, electron microscopy has shown that F-actin in the cleavage furrow mainly consists of unbranched, bundled filaments. These parallel filaments contrast with Arp2/3-mediated nucleation, which creates a highly branched actin filament network. Indeed, Arp2/3, although essential for lamellipodia formation, is not required for cytokinesis in Drosophila cells. The hypothesis here is that once myosin II is recruited to the equatorial cortex of the cell by a rho kinase-dependent mechanism, possibly localized activation of RLC phosphorylation, it is retained there because of its higher affinity for parallel, unbranched actin filaments than to branched actin networks. Consistent with this hypothesis, myosin II is depleted from the lamellipodia in migrating cells where Arp2/3 is localized and branched F-actin networks are formed but is enriched in the lamella where F-actin filaments are more likely to be aligned in parallel bundles. Thus, it is proposed that high rho1 signaling to Diaphanous at the cleavage furrow maintains a higher concentration of parallel actin filaments in this region compared with the rest of the cortex, and these parallel filaments serve to selectively retain myosin II at the equator to form a stable contractile ring. In the absence of these parallel actin filaments, myosin II can bind branched F-actin throughout the cortex, perhaps occasionally organizing them into parallel bundles that cause increased myosin recruitment corresponding to the flashes of cortical myosin accumulation, but these interactions are unstable (Dean, 2005).
Live-cell imaging shows that when pavarotti or racGAP50c are depleted, the cells do not display significant contractions despite recruiting myosin II to the equatorial cortex. Although there is some modest membrane contractile activity in these cells, it is clear that significant contraction or furrowing requires both components of the centralspindlin complex. It is surprising that only these proteins were found to be necessary for cortical contraction at sites of myosin II localization. Data from fixed cells, as well as earlier studies, indicated that Drosophila cells do not undergo equatorial contractions during mitosis when Diaphanous or Chickadee is depleted. However, live-cell imaging shows that when either of these two genes is depleted in S2 cells, not only is myosin II transiently localized to the equatorial cortex before dispersing, but cells do indeed display transient equatorial contraction. It is difficult to recognize these events in fixed cells because of their transient nature and the somewhat irregular shapes of cells depleted of these proteins. This work highlights the importance of live-cell imaging in the study of dynamic processes such as cytokinesis (Dean, 2005).
In addition to the suppression of furrowing, depletion of centralspindlin also leads to an inability to retain F-actin exclusively at the equatorial cortex during cytokinesis. This similar phenotype of the centralspindlin complex and the F-actin affecting proteins suggests that centralspindlin may be an upstream regulator of F-actin filament formation. Indeed kinase-dead mutants of Pavarotti have been shown to accumulate at the spindle poles and are associated with an abnormal accumulation of F-actin near the centrosomes. Centralspindlin may be acting indirectly by helping to localize an important actin-affecting protein at the central spindle, or it may act more directly on the cortex. Because RacGAP50c has been shown to bind Pebble in vitro, it has been hypothesized that centralspindlin affects the F-actin cortex through rho1 signaling by the localization and/or activation of Pebble. However, RacGAP50c depletion does not lead to a lack of myosin II recruitment as does Pebble or Rho1 depletion, and, thus, centralspindlin must act in a rho1-independent manner. For instance, the racGAP activity of centralspindlin may itself be important for signaling to the F-actin cortex. Finally, centralspindlin cannot be the major actomyosin ring positioning signal because myosin II is properly recruited in its absence (Dean, 2005).
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date revised: 25 August 2008
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