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).
Mammalian Rho-kinase/ROK alpha, one of the targets of Rho, has been shown to bind to Rho in GTP-bound form and to phosphorylate the myosin light chain (MLC) and the myosin binding subunit (MBS: see Drosophila Myosin binding subunit) of myosin phosphatase, resulting in the activation of myosin. Thus, Rho-kinase/ROK alpha has been suggested to play essential roles in the formation of stress fibers and focal adhesions. A two-hybrid analysis demonstrates that Rok interacts with the GTP-bound form of the Drosophila Rho1 at the conserved Rho-binding site. Rok can phosphorylate MLC and MBS, preferable substrates for bovine Rho-kinase, in vitro. These results suggest that Rok is an effector of Rho1 (Mizuno, 1999).
To test the physical interaction between Rok and various Rho-GTPases, a pull-down assay using GST-GTPase fusion proteins and in vitro translated Rok was used. Rok binds to the constitutively active form of Drosophila RhoA, but not to constitutively active Rac1 or Cdc42. Mutating a key amino acid within the effector-binding domain (T37A) abolishes the interaction with Rok. These results suggest that Rok is an effector specific for Drosophila RhoA (Winter, 2001).
Studies in mammalian cells have identified several downstream substrates for Rho-kinase/ROCK (Amano, 2000). In particular, Rho-kinase regulates the phosphorylation of the nonmuscle myosin regulatory light chain (MRLC) primarily at Ser-19 and secondarily at the adjacent Thr-18 (Amano, 1996; Kimura, 1996). Phosphorylation of MRLC at these sites results in a conformational change that allows myosin II to form filaments and increases its actin-dependent ATPase activity (Winter, 2001).
The amino acid sequence around the phosphorylation site of MRLC is highly conserved between mammalian MRLC and the Drosophila homolog, encoded by spaghetti squash (sqh). Therefore the phosphorylation of the Drosophila MRLC was assessed using an antibody that recognizes mammalian MRLC only when Ser-19 is phosphorylated. Immunoblot analysis shows that this antibody specifically recognizes phosphorylated Sqh in larval extracts -- the single ~20 kDa band in wild-type extract is absent both in extracts of a sqh null, and when wild-type extract is treated with phosphatase. While phosphorylated Sqh is detectable in Drok2 mutant extracts, its level is greatly reduced, whereas the Sqh protein level is not affected in Drok2 mutants. Previous work with bovine Rho-kinase has established that expression of the N-terminal catalytic domain gives rise to a constitutively active kinase (Amano, 1997). Raising the level of Rok activity in vivo by transient expression of the catalytic domain of Rok (Drok-CAT) results in elevated phosphorylation of Sqh as compared to controls in which a kinase-dead form (Drok-CAT-KG) was used. Taken together, these experiments indicate that Rok is required for maintaining the proper level of MRLC phosphorylation in vivo, and that such regulation depends on its kinase activity (Winter, 2001).
The effect of loss of Rok function on MRLC phosphorylation was examined at the cellular level. In wild-type wing cells, phospho-MRLC is enriched at the cortex of the pupal wing cells, whereas in Drok2 mutant cells this perimembrane staining is reduced or absent. Thus, rok is cell autonomously required for maintaining the level of cortical phospho-MRLC in the pupal wing (Winter, 2001).
The next question considered was whether MRLC/Sqh is an effector for Rok in regulating hair number in response to Fz/Dsh signaling. Use was made of a series of mutant sqh transgenes with point mutations in the primary (Ser-21) and secondary (Thr-20) phosphorylation sites, changing them either to glutamic acid (phosphomimetic), or to nonphosphorylatable alanine. These sqh transgenes are under control of the endogenous promoter and are expressed at levels similar to the native protein. Remarkably, whereas 100% of Drok2 hemizygous animals die before the wandering third instar stage, introducing one copy of a sqh transgene carrying the E20E21 double mutation (mimicking phosphorylation on both sites) results in 4% hemizygous Drok2 survival to adulthood. Likewise, one copy of an analogous transgene expressing SqhE21 also results in Drok2 hemizygotes surviving to adulthood (albeit a lower percentage), with a large fraction surviving to late-stage pupae. No rescue was observed when transgenes expressing the alanine substituted forms (SqhA20A21 or SqhA21) were introduced into the Drok2 background. These observations support the notion that MRLC is a key target (either directly or indirectly) for Rok kinase in vivo, since mimicking its phosphorylation, even in an unregulated fashion, partially rescues Drok2 organismal lethality (Winter, 2001).
Moreover, the multiple hair defect resulting from rok loss of function is almost completely suppressed by the presence of the sqhE20E21 transgene in the rescued adults. Taken together with the modulation of MRLC phosphorylation by Rok, these results demonstrate that the regulation of MRLC phosphorylation is a principal function of Rok in regulating F-actin prehair number (Winter, 2001).
Mechanisms that regulate axon branch stability are largely unknown. Genome-wide analyses of Rho GTPase activating protein (RhoGAP) function in Drosophila using RNA interference has identified p190 RhoGAP as essential for axon stability in mushroom body neurons, the olfactory learning and memory center. RhoGAP inactivation leads to axon branch retraction, a phenotype mimicked by activation of GTPase RhoA and its effector kinase Drok and modulated by the level and phosphorylation of myosin regulatory light chain. Thus, there exists a retraction pathway from RhoA to myosin in maturing neurons, which is normally repressed by RhoGAP. Local regulation of RhoGAP could control the structural plasticity of neurons. Indeed, genetic evidence supports negative regulation of RhoGAP by integrin and Src, both implicated in neural plasticity (Billuart, 2001).
Drosophila Rho-kinase (Drok), an effector of RhoA, is essential for transducing signals to the actin cytoskeleton in wing cells (Winter, 2001). Since the effector domain mutant analysis of RhoA suggests that a cytoskeletal pathway is important for axon retraction, tests were performed to see if the Drok pathway is involved. Carboxy-terminal truncation of mammalian Rho-kinase/ROCK results in its constitutive activation. Expression of an analogous activated form (Drok-CAT; Winter, 2001) in MB neurons led to truncated dorsal lobes similar to the phenotypes of p190 RNAi and weak RhoA activation. A presumptive kinase-dead point mutation (Drok-CAT.KG; Winter, 2001) has no effect, indicating that Drok signaling is dependent on its kinase activity. Developmental studies indicate that the Drok-CAT phenotypes also results from axon retraction, as does the p190 RNAi phenotype (Billuart, 2001).
Neuroblast clones homozygous for Drok2 (Winter, 2001) do not show apparent defects in cell proliferation, because the adult clones contain dorsal axon lobes contributed by later born neurons. Close examination of Drok2 neuroblast clones reveals that 10 of 17 contain at least one axon that extends significantly further than the heterozygous neurons within the same MB. Although this phenotype is subtle, it is not seen in 19 control clones, the parental chromosome for the Drok2 mutant, nor in many other genotypes studied. Thus, it is concluded that Drok is required to limit dorsal axon extension (Billuart, 2001).
Biochemical and genetic evidence indicates that a key output for Drok signaling in vivo is the regulation of phosphorylation of myosin regulatory light chain (MRLC) encoded by spaghetti squash (sqh) (Winter, 2001). To test if endogenous MRLC is part of the axon retraction pathway regulated by p190, genetic interaction experiments were performed by reducing the dose of endogenous sqh in the context of the p190 dsRNA expression. Marked suppression of the phenotype was observed in flies heterozygous for a null mutation of sqh (sqhAX3). In contrast, expression of a phosphomimetic mutant, Sqh-E20E21, markedly enhanced the p190 phenotype, whereas analogous expression of a nonphosphorylable form (Sqh-A21) had no effect. Further, truncation of the medial lobe was frequently observed when Sqh-E20E21 was expressed with the intermediate p190 RNAi line. This is evident from the FasII staining, showing that the medial ß axons (strongly FasII positive) only extend a fraction of the length of the medial lobe. This phenotype was only observed in the strongest p190 RNAi lines, never in the intermediate line alone. Taken together, these results strongly suggest that Drok and phosphorylation of Drosophila MRLC participate in mediating axon retraction as a result of p190 inactivation (Billuart, 2001).
Drosophila Rho-kinase associates with the GTP-bound Drosophila Rho1 and can phosphorylate the vertebrate MRLC and myosin binding subunit (MBS). Rho-kinase is involved in the establishment of planar polarity in adult structures such as the compound eye and wing. Drosophila Myosin-binding subunit (MBS) has been characterized to elucidate the functions of myosin phosphatase in morphogenesis, revealing that MBS functions in dorsal closure and that it acts antagonistically to the Rho signaling cascade and its effector Rho-kinase (Mizuno, 2002).
Drosophila Rho-kinase physically interacts with Rho1 in the GTP form and phosphorylates the vertebrate MBS in vitro. The sequence at the putative phosphorylation site of vertebrate MBS is well conserved in Drosophila MBS, and tests were performed to see whether DRho-kinase phosphorylates MBS in vitro. The GST-fused DMBS-L was expressed and purified from Escherichia coli, and was found to be phosphorylated by wild-type Drosophila Rho-kinase but not by kinase-dead DRho-kinaseK116A (Mizuno, 2002).
Thr594 may correspond to the major phosphorylation site in vertebrate MBS. The threonine residue was replaced with an alanine, and this recombinant Drosophila MBS was used as a substrate. The level of phosphorylation was significantly reduced, indicating that Thr594 is the major site phosphorylated by Drosophila Rho-kinase. It has been reported that mammalian MBS is phosphorylated at several sites by Rho-kinase (Kawano, 1999), and there presumably are other phosphorylation sites in Drosophila MBS as well (Mizuno, 2002).
Drosophila Rho-kinase is thought to be responsible for the inactivation of myosin phosphatase through phosphorylation of MBS. If this inactivation turns out to have a considerable effect on the levels of phosphorylated MRLC, it can be expected that the phenotypes in the MBS mutant embryos and in the embryos overexpressing DRho-kinase would be similar. When wild-type Rho-kinase was expressed with the arm-GAL4 driver, about 80% of the embryos failed to hatch. A similar result was obtained with the 69B-GAL4 driver that induces the target gene in the ectoderm. Most of the lethal embryos show a dorsal open or dorsal hole phenotype, and the pattern of dorsal hairs is disturbed along the dorsal midline in the remaining embryos as observed in the MBS mutant embryos. Examination of the cell shape and the F-actin distribution reveals the same aberrations as those in the MBS mutant embryos (Mizuno, 2002).
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