Drosophila Rho-kinase associates with the GTP-bound Drosophila Rho1 and can phosphorylate the vertebrate MRLC and MBS. Rho-kinase is involved in the establishment of planar polarity in adult structures such as the compound eye and wing. The Drosophila homolog of 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).
Myosins are a superfamily of actin-dependent molecular motor proteins, among which the bipolar filament forming myosin II has been the most studied. The activity of smooth muscle/non-muscle myosin II is regulated by phosphorylation of the regulatory light chains, which in turn are modulated by the antagonistic activity of myosin light chain kinase and myosin light chain phosphatase. The phosphatase activity is mainly regulated through phosphorylation of its myosin binding subunit Mypt [FlyBase term: Myosin binding subunit (Mbs)]. To identify the function of these phosphorylation events, the Drosophila homolog of MYPT has been molecularly characterized, and its mutant phenotypes have been analyzed. Drosophila MYPT is required for cell sheet movement during dorsal closure, morphogenesis of the eye, and oogenesis growth during oogenesis. These results indicate that the regulation of the phosphorylation of myosin regulatory light chains, or dynamic activation and inactivation of myosin II, is essential for its various functions during many developmental processes (Tan, 2003).
Myosins involved in a variety of essential processes that include muscular contraction, cytokinesis, vesicle transport, cell movement and cell shape change. Among the 17 subclasses of myosins, conventional myosins, known as myosin IIs, have been the most studied. Myosin IIs form bipolar filaments that drive contractile events by bringing together actin filaments of opposite polarity. Myosin II molecules are hexameric enzymes consisting of two heavy chains, two regulatory light chains (MRLCs - coded for by spaghetti squash\ in Drosophila), and two essential light chains. They can be subclassified into four groups based on their motor domain (or tail) sequences: (1) sarcomeric myosins, (2) vertebrate smooth muscle/non-muscle myosins, (3) Dictyostelium/Acanthamoeba type myosins and (4) yeast type myosins (Tan, 2003 and references therein).
The activity of smooth muscle/non-muscle myosin II is regulated by the phosphorylation of MRLC that is modulated by the antagonistic activity of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). MLCP is composed of three subunits: a catalytic subunit made up of protein phosphatase 1c ß (also called delta); a myosin binding or targeting subunit (MYPT), and a small subunit of unknown function. MYPT binds and confers the selectivity of PP1c for myosin (Hartshorne, 1998; Tan, 2003 and references therein).
The phosphatase activity of MLCP can be regulated in several ways (reviewed by Hartshorne, 1998; Somlyo, 2000). Rho-kinase (ROCK) phosphorylates an inhibitory phosphorylation site on MYPT and inhibits the phosphatase activity in smooth muscle. This phosphorylation may occur through ZIPK (leucine zipper interacting protein kinase)-like kinase or integrin-linked kinase. Myotonic dystrophy protein kinase phosphorylates the same inhibitory phosphorylation site, although it is not clear whether this phosphorylation event also goes through ZIPK. In addition, protein kinase C (PKC) can phosphorylate the ankyrin repeat region of MYPT, and thus attenuate the interaction of MYPT with PP1c and MRLC. Furthermore, CPI-17, a smooth muscle-specific inhibitor of MLCP, can also regulate the phosphatase activity of MLCP. Phosphorylation of CPI-17 by PKC, or ROCK, or protein kinase N, or p21-activated kinase (PAK) dramatically enhances the inhibition ability of CPI-17. Finally, MRLC can also be phosphorylated by ROCK and PAK, which itself is a substrate of Rac and Cdc42. Thus ROCK can regulate MRLC phosphorylation both through direct phosphorylation of MRLC and through inactivation of MLCP. Importantly, although the biochemistry of these phosphorylation events is well characterized, the physiological significance of these regulatory steps in vivo remains to be explored (Tan, 2003).
The in vivo function of non-muscle myosin II has been extensively analyzed in Drosophila melanogaster, Dictyostelium discoideum and Saccharomyces cerevisiae. Drosophila has a single non-muscle myosin II heavy chain encoded by zipper (zip), as well as a single non-muscle myosin II regulatory light chain encoded by spaghetti squash (sqh). Analysis of the phenotypes associated with mutations in zip and sqh have revealed that non-muscle myosin II regulates cell shape changes and cell movements in multiple processes such as cytokinesis, dorsal closure and oogenesis. In addition, mutations in both zip and sqh affect planar cell polarity during development (Tan, 2003).
The temporal requirement of zip has been studied in sqh2 mutant animals that carry a sqh transgene driven by a heat shock promoter. This analysis showed that sqh activity is needed for eye and leg imaginal discs morphogenesis. Also, during oogenesis, sqh is required for morphogenesis of interfollicular stalks, border cell migration, centripetal cell ingression, dorsal appendage cell migration, and rapid transport of the nurse cell cytoplasm into the oocyte. Inhibition of this transport was also observed in animals that carry homozygous sqh1 germline clones (GLCs) (Tan, 2003 and references therein).
The in vivo function of MRLC phosphorylation was determined by expression of sqh transgenes that contain mutated phosphorylation sites in a sqh null mutant background. Embryos carrying the null mutation sqhAX3 die, mostly during the first larval instar, and sqhAX3 GLCs develop extensive defects, including failure in cytokinesis, during oogenesis. SqhA20A21, in which both the primary and secondary phosphorylation sites have been changed to alanine, fails to rescue sqhAX3, indicating that phosphorylation of Sqh is important for myosin II function. In support of this, a change of serine 21 to glutamic acid (SqhE21), that presumably mimics constitutive phosphorylation of Sqh, substantially rescues the sqhAX3 oogenesis phenotype (Tan, 2003).
To gain further insight into the regulation of Zip and to define precisely the in vivo function of MLCP, the Drosophila homolog of the MYPT gene (DMYPT) has been cloned. DMYPT is essential for cell sheet movement during dorsal closure, morphogenesis during eye development, and ring canal growth during oogenesis. These results indicate that regulation of the phosphorylation state of MRLC, and dynamic activation and inactivation of myosin II, are essential for its various functions during many developmental processes (Tan, 2003).
A BLAST search of the Drosophila database with mammalian MYPT sequences reveals that the Drosophila genome has a single related gene, CG5891. CG5891 is predicted to encode a protein with limited homology to mammalian MYPT at the N terminus. However, sequence analysis of several cDNAs derived from CG5891 uncovered additional regions of homology between the mammalian and fly homologs, suggesting that the predicted CG5891 gene was incorrectly annotated. A representative cDNA, AT12677, encodes an ORF of 1101 amino acids (aa) that has been named Drosophila MYPT (DMYPT) to follow the nomenclature of the mammalian protein. A comparison of the compiled DMYPT cDNA and genome sequences shows that the DMYPT locus contains 18 exons and 17 introns. The start codon lies in the second exon and the stop codon in the last. Sequence alignment shows that DMYPT shares significant homology with human MYPTs in three regions: the N terminus containing several ankyrin repeats, the C terminus, and a short peptide in the middle that contains the highly conserved inhibitory phosphorylation site (Tan, 2003).
To characterize the consequences of loss of DMYPT function during development, mutations in the DMYPT gene were sought. Two P-element transposon insertions in the DMYPT locus have been defined molecularly by recovery of flanking genomic sequence. EP(3)3727, in the first intron, is homozygous viable and l(3)03802, in the tenth intron, is associated with zygotic lethality. Several deficiencies were identified that remove DMYPT sequences based on genetically defined breakpoints as well as their failure to complement l(3)03802. Df(3L)th102 deletes DMYPT entirely and thus serves as a complete loss-of-function allele for use in this study (Tan, 2003).
To determine whether the l(3)03802 P-element insertion within the DMYPT locus is responsible for the lethality, and to generate new deletion alleles, both DMYPT P-element insertions were excized using the Delta2-3 transposase. Mobilization of each element resulted in the recovery of both viable precise excisions and lethal imprecise excisions. Among the >200 excisions derived from l(3)03802, over half were viable, indicating that the lethality associated with the l(3)03802 chromosome is due to disruption of DMYPT and not another lethal hit. Thus l(3)03802 is renamed as DMYPT03802 and EP(3)3727 as DMYPT3727. Two of the strongest embryonic lethal excision lines, DMYPT2-188 and DMYPT2-199, like the original insert, DMYPT03802, fail to complement Df(3L)th102 and are described in detail below. Eleven of the 39 lethal excisions derived from DMYPT3727 failed to complement with DMYPT03802 and Df(3L)th102: this is consistent with the notion that they disrupt DMYPT activity (Tan, 2003).
To confirm that the DMYPT03802 insertion disrupts DMYPT function and that the cDNA derived from the DMYPT locus encodes all the functions associated with DMYPT activity, the original lethal P insertion was rescued with a transgene containing a heat shock promoter driving a DMYPT cDNA. Following 1-hour heat treatments daily from embryogenesis to eclosion, hs-DMYPT fully rescues DMYPT03802 homozygous animals to adulthood. Stopping heat treatment 1 to 2 days before eclosion led to incomplete rescue of DMYPT03802, with adults developing wing and leg defects similar to those noted for zip or sqh mutants partially rescued by a transgene. Stopping heat treatment 3 days prior to eclosion resulted in no rescue to adulthood. The complete rescue of the lethality associated with DMYPT03802 by the hs-DMYPT transgene demonstrates that loss of DMYPT activity is responsible for the lethal phenotype (Tan, 2003).
To assess the timing and cause of lethality associated with the DMYPT03802 insertion, embryos were collected and analyzed. Lethal phase analysis showed that 44% of homozygous DMYPT03802 animals died during embryogenesis, while the remaining 56% died during early first larval instar (485 total embryos counted). More than 80% of the dead mutant embryos displayed a failure of dorsal closure with a characteristic dorsal hole in their cuticles. The size of the hole in such flies is variable and is also influenced by the genetic background. Homozygous Df(3L)th102 embryos, as well as DMYPT03802/Df(3L)th102 embryos also showed dorsal closure defects. The embryonic cuticle phenotype of DMYPT03802/Df(3L)th102 is more severe (more embryos displayed large dorsal holes) than homozygous DMYPT03802, suggesting that DMYPT03802 is a hypomorphic allele. In addition, all of the embryonic lethal excision lines analyzed that were derived from DMYPT03802, and ten of the lethal excision lines from DMYPT3727, produced embryos with dorsal closure defects. Altogether, these results indicate that DMYPT is required for dorsal closure (Tan, 2003).
Dorsal closure involves a cell sheet movement where the dorsal-lateral ectoderm on both sides of the developing embryo moves toward the dorsal midline to cover a degenerative squamous epithelium, the amnioserosa. This epithelial cell sheet movement encloses the embryo in a continuous protective epidermis. Genetic loss-of-function studies have identified the Jun N-terminal kinase (JNK) signal transduction cascade as one of the key modulators of dorsal closure morphogenesis. Transcriptional targets of JNK signaling include decapentaplegic (dpp), a secreted morphogen related to the bone morphogenetic proteins (BMPs), and puckered (puc), a dual-specificity phosphatase that mediates a negative feedback loop of the JNK signal transduction pathway via dephosphorylation of JNK (Tan, 2003).
To determine whether the failure of dorsal closure in DMYPT mutants is due to an influence on JNK signaling, dpp expression was assayed in the leading cells of the ectoderm during closure. In situ hybridization revealed that the spatial and temporal expression pattern of dpp is normal in DMYPT mutant embryos, suggesting that DMYPT does not function through the JNK pathway during dorsal closure (Tan, 2003).
To further examine the cause of dorsal closure defects in the mutants, DMYPT mutant embryos were stained for markers that allowed analysis of cell polarity and shape in the dorsal ectoderm. Apically localized phosphotyrosine immunoreactivity similar to wild-type flies was observed. Moreover, there was normal basolateral fasciclin III immunostaining. Altogether, these results suggest that there are no gross defects in cell orientation or polarity. However, it was noticed that older mutant embryos begin to show abnormal cell shapes at the leading edge of the epidermis, which could account for the defects in dorsal closure observed in the DMYPT mutants (Tan, 2003).
Consistent with the late embryonic defects observed in DMYPT zygotic mutants, it was found that DMYPT is maternally contributed and ubiquitously expressed during embryogenesis. This maternal supply of DMYPT is likely the reason that the dorsal closure phenotype is variable among embryos and is influenced by genetic background. However, this question cannot be addressed directly since DMYPT is required during oogenesis (Tan, 2003).
During oogenesis, each cystoblast divides four times with incomplete cytokinesis and produces one oocyte and fifteen support nurse cells that are all connected through cleavage furrows. These cleavage furrows subsequently develop into ring canals. These open rings allow the nurse cells to transport cytoplasm into the oocyte, slowly from stage 6 to stage 10, then rapidly at stage 11. This fast phase of transport is referred to as 'dumping', and has been shown to require the activity of Sqh (MRLC). In sqh mutant germline egg chambers, dumping is blocked (Tan, 2003).
To analyze the role of DMYPT during oogenesis, homozygous mutant germline clones (GLCs) were generated of DMYPT03802 using the FLP-FRT/dominant female sterile technique. Females carrying DMYPT03802 homozygous GLCs lay few tiny eggs, about a quarter of the size of wild type eggs, that do not develop. Examination of the mutant egg chambers revealed that the dumping of nurse cell cytoplasm to the oocyte is blocked. This is similar to the dumpless phenotype observed with sqh homozygous mutant GLCs as well as for mutants in other actin binding proteins (Tan, 2003).
To investigate the basis of the dumpless phenotype associated with DMYPT03802 GLCs, actin filaments were stained using Texas Red phalloidin. The most obvious defect involves the ring canals. At stage 8, wild-type egg chambers have large bagel-shaped ring canals. In contrast, the ring canals of DMYPT03802 GLC egg chambers are very small (Tan, 2003).
To determine whether the ring canals of DMYPT03802 GLCs never enlarge, or whether they grow and then collapse, the ring canals were examined in different stage egg chambers. In wild-type egg chambers, ring canals grow from 1 µm at stage 2 to 10 µm at stage 11. In contrast, the ring canals of DMYPT03802 GLCs barely grow. Mutation of DMYPT in follicular cells have no effects on the ring canal growth, suggesting that DMYPT is required in the germline for ring canal growth. Presumably, these small ring canals cannot support the fast phase cytoplasmic transport and thus cause the dumpless phenotype resulting in tiny eggs (Tan, 2003).
In addition to actin, several other proteins, including Hu-li tai shao (Hts), Kelch, and phosphotyrosine (pY)-containing proteins, are recruited to ring canals as they form. Immunolocalization experiments have revealed that both Hts and Kelch are localized to the small DMYPT mutant ring canals. Interestingly, although pY staining is present in the mutant ring canals, an ectopic accumulation of pY staining was also observed in the nurse cells. The basis of this ectopic accumulation remains to be determined (Tan, 2003).
Next, the subcellular distribution of Zipper was examined. Mutation of Sqh causes Zip to form aggregates, thus an effect on Zip distribution in the absence of DMYPT was expected. Surprisingly, no major changes in Zip distribution were detectable between wild-type egg chambers and DMYPT GLCs. In both cases, Zip was uniformly distributed at low level with enhanced cell cortex localization. These observations are consistent with the result that DMYPT mutations have no effect on Zip localization during dorsal closure (Tan, 2003).
Previous studies have shown that the Rho family GTPases, Rac1, RhoA, and Cdc42, each play a role in dorsal closure, and may influence myosin activity through a RhoA mediated signal. Programmed overexpression of these genes by the eye-specific GMR promoter causes distinct rough eye phenotypes. To pinpoint the relationship of DMYPT with these GTPases, the effects of reducing DMYPT activity on the rough eye phenotypes was examined. Interestingly, reduction of DMYPT strongly enhances the eye phenotype caused by GMR-Rac7A. The eyes of GMR-Rac7A/DMYPT03802 flies are much smaller, with fewer bristles and hexagonal-shaped ommatidia, than those of GMR-Rac7A/OreR flies. Consistent with the idea that the P-insertion and the excisions are hypomorphic alleles, Df(3L)th102 enhances the GMR-Rac7A eye phenotype to an even greater extent than either DMYPT03802, DMYPT2-188 or DMYPT2-199. However, reduction of DMYPT has no effect on the size of the rough eye caused by either GMR-RhoA or GMR-Cdc42, although it does enhance the rough eye phenotype caused by GMR-RhoA since fewer bristles form. Together, these data suggest that DMYPT plays a role in eye development and functions downstream of, or in parallel with Rac and Rho (Tan, 2003).
RhoA functions downstream of Rac in determining ommatidia polarity in the eyes. Reducing the dosage of RhoA enhances the effect of sev-RacN17, a dominant negative form of Rac driven by the sevenless (sev) enhancer-promoter in the eye, and suppresses the activity of sev-RacV12, which encodes a constitutively active form of Rac. Consistently, overexpression of RhoA (sev-RhoA) rescues sev-RacN17, while reduction in the amount of Rac using a deficiency that uncovers Rac has no effect on the gain-of-function RhoA phenotype. Thus, similar to the Rho dependence on Rac function observed in mammalian fibroblasts, some developmental events in Drosophila also rely on a hierarchy of GTPase function (Tan, 2003).
Consistent with these observations, reducing the dosage of RhoA partially suppresses the rough eye phenotype caused by GMR-Rac. In fact, mutations of all the putative positive regulators of myosin activity (RhoA-Zip signaling pathway), including RhoA, Drok and zip itself, moderately suppress the rough eye phenotype of GMR-Rac, opposing the effect of DMYPT mutants. This suggests that the RhoA-Zip signaling pathway functions downstream of Rac, and that DMYPT is a negative regulator of the pathway (Tan, 2003).
Importantly, replacing the phosphorylation sites of Sqh with alanine remarkably suppresses the rough eye phenotype, while replacing them with glutamic acid to mimic phosphorylation slightly enhances the phenotype. This suggests that dephosphorylation of Sqh is important in eye morphogenesis and that DMYPT may be involved in regulating the dephosphorylation of myosin light chain in eye development (Tan, 2003).
To examine whether other myosins are also involved in this process, the effect of myosin VIIA, an unconventional myosin encoded by crinkled (ck), was included in the same assay. Myosin VIIA was chosen because ck and zip behave antagonistically in wing hair number determination in the Drosophila adult wing. Interestingly, ck behaves just the opposite of myosin II (Zip) during eye morphogenesis, since a reduction in ck activity enhances the GMR-Rac rough eye phenotype, nearly to the same extent as a reduction in DMYPT (Tan, 2003).
The regulation of MRLC phosphorylation is essential to modulate myosin II activity and can be controled by several distinct mechanisms. For instance, RhoA can activate its effector ROCK that in turn phosphorylates MYPT, either directly or indirectly. MYPT phosphorylation inhibits the phosphatase activity of MLCP and leads to elevation of MRLC phosphorylation. Phosphorylation of MRLC can also be increased by activation of MLCK, another downstream target of RhoA. Thus, the antagonistic activity of kinase and phosphatase is thought to engender a delicate balance of myosin II activity modulated through the phosphorylation state of its regulatory light chain (Tan, 2003).
To assess the relationship between DMYPT regulation of myosin II and signaling via the Rho GTPase family members, the Drosophila eye was examined since sensitive genetic interactions can be observed. RhoA function downstream of, or in parallel with, Rac has been implicated in regulation of orientation of ommatidia in the eye. Consistent with this, reducing the amount of RhoA, Drok and zip partially alleviates the eye defect associated with overexpression of Rac, while reducing the dosage of a putative negative regulator of myosin enhances the rough eye phenotype. Furthermore, expression of a non-phosphorylatable form of Sqh, which presumably reduces the activity of Zip, dramatically rescues the phenotype, while overexpression of a phospho-mimicking Sqh mutant, which should increase the activity of myosin, exacerbates the eye defects. Taken together, these data indicate that the regulation of myosin II activity via balancing the phosphorylation level of Sqh is critical for proper morphogenesis of the Drosophila eye. Based on these results, it is proposed that it is DMYPT that mediates myosin II downregulation in this system (Tan, 2003).
Interestingly, crinkled (myosin VIIA), an unconventional myosin, behaves antagonistically to Zip/myosin II in both eye morphogenesis and wing hair number restriction. This suggests that various myosins interact in different cell types to regulate reorganization of the actin cytoskeleton. It will be interesting to determine the specificity of functions of different myosins and their modes of regulation. Since there are many different myosins but only a single MYPT in Drosophila, it remains to be determined whether, and how, DMYPT interacts with other myosins (Tan, 2003).
In conclusion, the Drosophila homolog of mammalian MYPT, accordingly named DMYPT, has been identified. DMYPT plays multiple roles during Drosophila development. Loss of DMYPT function leads to blockage of rapid transport of nurse cell cytoplasm, inhibition of ring canal growth, failure of dorsal closure, defects of eye morphogenesis, and other unidentified processes during pupae development. Furthermore, the data indicate that dynamic regulation of myosin II activity via regulating phosphorylation level of myosin regulatory light chain by DMYPT is critical for the function of myosin II (Tan, 2003).
Animal cell cytokinesis is characterized by a sequence of dramatic cortical rearrangements. How these are coordinated and coupled with mitosis is largely unknown. To explore the initiation of cytokinesis, focus was placed on the earliest cell shape change, cell elongation, which occurs during anaphase B and prior to cytokinetic furrowing. Using RNAi and live video microscopy in Drosophila S2 cells, Rho-kinase (Rok) and myosin II were implicated in anaphase cell elongation. rok RNAi decreased equatorial myosin II recruitment, prevented cell elongation, and caused a remarkable spindle defect where the spindle poles collided with an unyielding cell cortex and the interpolar microtubules buckled outward as they continued to extend. Disruption of the actin cytoskeleton with Latrunculin A, which abolishes cortical rigidity, suppressed the spindle defect. rok RNAi also affected furrowing, which was delayed and slowed, sometimes distorted, and in severe cases blocked altogether. Codepletion of the Myosin binding subunit (Mbs) of myosin phosphatase, an antagonist of myosin II activation, only partially suppressed the cell-elongation defect and the furrowing delay, but prevented cytokinesis failures induced by prolonged rok RNAi. The marked sensitivity of cell elongation to Rok depletion was highlighted by RNAi to other genes in the Rho pathway, such as pebble, racGAP50C, and diaphanous, which had profound effects on furrowing but lesser effects on elongation. It is concluded that cortical changes underlying cell elongation are more sensitive to depletion of Rok and myosin II in comparison to other regulators of cytokinesis; this work suggests that a distinct regulatory pathway promotes cell elongation (Hickson, 2006 full text of article).
How the complex events of mitosis and cytokinesis are seamlessly coordinated remains largely a mystery. Cell elongation is a characteristic feature linking mitosis and cytokinesis in many cell types. However, it has not been apparent how much attention this event deserves; it could be construed as a secondary consequence of spindle extension or an early manifestation of the gradual recruitment of contractile elements that form the contractile ring. The current results suggest that, although it is inextricably linked with both mitosis and cytokinesis, there are distinctive genetic contributions to its success (Hickson, 2006).
One of the most striking findings was that depletion of rok function prevented anaphase cell elongation and caused a dramatic buckling of the spindle. Taking many observations into account, it is infered that the primary defect was one where the cortex failed to respond appropriately and, as a result, the spindle suffered a mechanical disruption as it encountered the unresponsive cortex. Thus, rok is required for remodeling the cell cortex during anaphase cell elongation, and perturbation of rok function disrupts the normal temporal coordination of cortex and spindle. In this regard, Rok might be required for the spindle to communicate with the cortex to stimulate elongation, or it might simply be required to execute elongation. In either case, the anaphase spindle extension alone is clearly insufficient to push the sides of the cell out and promote cell elongation. In addition, continued spindle elongation within the restricted confines of the rigid cortex demonstrates that there is no feedback signal from the cortex to the spindle. Thus, cell elongation and spindle extension are likely coupled only in a unidirectional manner: The cortex responds to the growing spindle, but the spindle does not sense an unyielding cortex (Hickson, 2006).
The data also clearly indicate that Rok is required for normal myosin II recruitment to the equatorial cortex. Myosin II is also required for cell elongation, suggesting that it is the relevant target of Rok action. In this regard, it is noted that a similar failure of cell elongation was observed in the neuroblasts of Drosophila larvae homozygous for sqh1, a hypomorphic spaghetti squash allele. These mutants show poorly elongated anaphase and telophase cells in which the segregated DNA masses were in tight apposition with the cell cortex. The similarity between those phenotypes and the ones described in this study in S2 cells strongly suggests that a similar Rok/myosin II pathway operates in vivo in the developing fly (Hickson, 2006).
Genetic-interaction studies have demonstrated that rok functions in the Pebble pathway to influence cytokinesis in the wing disc. However, rok mutant cells can divide at least several times to produce a substantial clone in the wing disc. Although this finding may lead one to question the importance of rok in cytokinesis, apparent dispensability in this context should not be taken as a lack of importance. Continued division of S2 cells with compromised rok function occurs in the face of major perturbations, and, after prolonged rok RNAi, frequent failures appear in cytokinesis. It is suggested that rok plays an integral part in promoting and coordinating cytokinesis and that successful cytokinesis with compromised rok function is testament to the robustness of the process (Hickson, 2006).
Given that cytokinesis is so robust, a consideration of how loss of rok function alters the normal progression of the process might provide more insight than a consideration of its overall success. In addition to the extreme defect in which furrowing is blocked, rok RNAi causes a pronounced delay in the onset of furrowing and reduction in the rate of ingression of furrows. Simultaneous depletion of Mbs prevented failures in cytokinesis but did not restore the normal timing of furrowing. Thus, Rok promotes whereas Mbs suppresses furrow ingression. Additionally, the normal timing requires Rok, indicating that its activity contributes to triggering the onset of furrowing. Studies in C. elegans (Piekny, 2002) have also found that Let-502 (the Rok ortholog) and MEL-11 (the Mbs ortholog) play antagonistic roles in furrow ingression, but in contrast to the observations in Drosophila S2 cells, in C. elegans, the activity of Let-502 appeared to control the speed of ingression without influencing the timing of onset of furrowing (Hickson, 2006).
Among the cytokinesis genes that were examined, only RNAi of rok and zipper (myosin II) gave a severe block to elongation, and, in the case of rok, this often gave a strong elongation effect without blocking furrowing. It was also found that RNAi of pebble and racGAP50c slowed elongation to half its normal rate while severely suppressing furrowing. RNAi of other cytokinesis genes, such as diaphanous, citron kinase, and anillin, did not interfere with elongation. Given these findings, it is suggested that elongation and furrowing, although they share some common functions, are differentially regulated and ought to be recognized as distinct subroutines in the overall process of cytokinesis. Given that Pebble is an upstream activator of Rho and Rok in cytokinesis, the finding that pebble RNAi gave a more mild elongation defect suggests that a different Rok activator promotes elongation: For example, Rok might be activated by other Rho-GEFs, as it is in interphase (Hickson, 2006).
Two processes likely contribute to cell elongation: equatorial contraction and polar relaxation. This duality may contribute to some of the apparent overlap in the regulation of elongation and furrowing. Rok mediated equatorial recruitment and activation of myosin II might contribute to equatorial contraction and provide one input into elongation. This input is likely to depend on Pebble and RacGAP50C which also localize to the equator and are known to influence Rho function. The partial defect in cell elongation induced by pebble or racGAP50c RNAi might be explained by disruption of this contraction. Other indications suggest that polar relaxation occurs in S2 cell cytokinesis and that rok RNAi interferes with the process. During elongation, the polar cortices bleb and appear to be actively remodeled as if signaled to do so by the approaching spindle poles, whereas following rok RNAi the segregating spindle poles push right up against the cortex. It is also noted that mitotic spindle poles have been shown in other systems to harbor active Rho (as evidenced through a GFP-Rho binding domain reporter, Rho-kinase and myosin II regulatory light chains phosphorylated on the Rho-kinase phosphorylation site. In addition, mammalian ROCKI/II are responsible for membrane blebbing during apoptosis and therefore clearly able to promote such an outcome. Thus, it is not inconceivable that a Rok/myosin II pathway could operate at the spindle poles during anaphase to promote polar relaxation. It is thought that Rok and myosin II function in two pathways, one governing equatorial contraction and the other polar relaxation, either of which can support cell elongation, whereas Pebble and RacGap50C interfere only with cortical contraction and hence result in a partial elongation defect (Hickson, 2006).
It seems likely that some divisions, particularly those that are asymmetric, might be particularly dependent on polar relaxation and hence be more sensitive to rok depletion than others. For example, asymmetric divisions might rely on differential actions at the spindle poles. Indeed, the sensitivity of the initial stages of polar-body formation in mouse eggs to an inhibitor of the mammalian Rok suggests that Rok may play a role in the formation of the cortical protrusion into which the spindle migrates in this highly asymmetric division (Hickson, 2006).
In summary, this study has uncovered pivotal roles for Rok in the earliest shape change of cytokinesis: anaphase cell elongation. A model of cell elongation and the onset of cytokinesis is depicted in a Model for Anaphase Cell Elongation and Initiation of Cytokinesis in Drosophila S2 Cells . As the spindle extends in anaphase B, Rok stimulates polar relaxation, allowing the spindle to push the sides of the cell out as it extends. In addition, Rok stimulates myosin II recruitment to the equatorial cortex, where it begins to contract in a broad zone. At the center of this broad zone, the contractile actin ring then forms and the cytokinetic furrow ingresses. Distinctions in gene requirements for anaphase cell elongation versus furrowing suggest distinctions between the two processes (Hickson, 2006).
It is intriguing that Rok and/or myosin II appears to be involved in the whole gamut of cell shape changes that occur during cell division: mitotic cell rounding, anaphase cell elongation, cytokinetic furrowing, and postmitotic spreading. This implies that the same fundamental machinery mediates each of these dramatic cytoskeletal rearrangements. Understanding how these events are regulated so as to ensure the appropriate response at the appropriate time is one of the challenges ahead (Hickson, 2006).
The pattern of the expression of MBS during development was analyzed by in situ hybridization using DMBS-L as a probe. A significant amount of MBS mRNA is uniformly detected in blastoderm stage embryos, and it is mostly of maternal origin. MBS is expressed ubiquitously throughout embryogenesis. In the imaginal discs from third instar larvae, the MBS transcript is uniformly detected. Tissue- and stage-specificity of the expression for each isoform remain to be analyzed (Mizuno, 2002).
MBS is located at the 72D region on the left arm of the third chromosome. The intron/exon structure of MBS was deduced from a comparison between the genomic and cDNA sequences. In this region, two P-element insertions, l(3)72Dd03802 and EP(3)3727, have been registered in FlyBase. Both of these and an EMS-induced mutation, l(3)72Dd3, fail to complement one another. l(3)72Dd03802 and l(3)72Dd3 are lethal during early larval stages, while EP(3)3727 results in development to adults with a mild wing defect. Excision of the P insertions reverses the lethal and wing phenotypes of l(3)72Dd03802 and EP(3)3727, respectively, indicating that the P insertions caused the mutations. Imprecise excision of the P insertion from EP(3)3727 produces a new mutation, P2r31, whose lethal phase spans from the third instar larval to early pupal stages (Mizuno, 2002).
A polyclonal antibody against a synthetic polypeptide corresponding to the carboxy-terminal region of MBS was developed, producing a major band of about 95 kDa and several minor bands on the immunoblot. Correspondence of these bands to DMBS-L and -S is not certain at this moment. The amounts of the MBS proteins were analyzed in the mutants. Mutants heterozygous between P2r31 and the strong alleles or Df(3L)th117, which deletes the region including MBS, survive to third instar larvae, and the amount of MBS is greatly reduced in the extracts prepared from these larvae. The result indicates that the mutations are correlated to the amount of MBS. MRLC is encoded by spaghetti-squash (sqh), and the levels of its phosphorylated form in the mutants were also examined. The levels of phospho-MRLC are significantly elevated in the mutants, indicating that the activity of myosin phosphatase is decreased in the mutants (Mizuno, 2002).
To further confirm that these mutations are at the MBS locus, a rescue experiment was performed. DMBS-L and DMBS-S cDNAs were driven under the heat shock promoter, and induction of either of them by heat shock significantly complements the lethality of both l(3)72Dd03802 and l(3)72Dd3. This may suggest that, despite the expression of multiple isoforms of MBS, they are functionally redundant. It is also possible that this functional redundancy is partial, and that overexpression of only one isoform would be sufficient for the viability of the fly (Mizuno, 2002).
From these results, it is concluded that they are the mutations in MBS, and l(3)72Dd03802, EP(3)3727, l(3)72Dd3 and P2r31 are referred to as DMBSP1, DMBSP2, DMBSE1 and DMBSP2r31, respectively. The strength of their phenotype can be ordered as DMBSE1=DMBSP1 > DMBSP2r31 > DMBSP2. Since DMBSE1 and DMBSP1 give identical results, only the results obtained with DMBSE1 are presented (Mizuno, 2002).
The animals homozygous for or transheterozygous between the strong MBS alleles are larval lethal and embryonic development seems to proceed normally. This would be because of the maternal contribution of MBS+ activity, a notion consistent with the observation that a significant amount of maternal mRNA is present in early-stage embryos. To analyze the function of MBS during embryogenesis, attempts were made to reduce the maternal contribution (Mizuno, 2002).
The mutants transheterozygous between the weak allele, DMBSP2, and the strong alleles or Df(3L)th117, survive to adults. Examination of such female flies by immunoblotting has revealed that the amount of MBS proteins is greatly reduced. When the females transheterozygous between DMBSP2 and Df(3L)th117 are mated with wild-type males, embryonic development proceeds normally in most of the embryos. However, about 25% of the embryos fail to hatch in matings involving males heterozygous for the strong alleles. Similar results were obtained with the females of the genotypes, DMBSE1/DMBSP2 or DMBSP1/DMBSP2. Furthermore, embryonic defects are suppressed by paternal expression of the MBS transgenes. These results indicate that embryos fail to develop when MBS is defective both maternally and zygotically, and that the maternal defect is rescued by the paternal expression of a wild-type gene (Mizuno, 2002).
About 80% of the dead embryos in the above experiments demonstrated the 'dorsal open' or 'dorsal hole' phenotype, which can be typically seen in embryos defective in the dorsal closure. In the remaining lethal embryos, the pattern of dorsal hairs was disturbed along the dorsal midline. These phenotypic variations would be due to the residual activity of maternal MBS derived from the weak allele, DMBSP2. The results indicate that MBS is required in the process of dorsal closure (Mizuno, 2002).
To examine whether defects in the dorsal closure in the embryos lacking MBS or overexpressing wild-type Rho kinase are due to an aberrant activation of nonmuscle myosin II, the genetic interactions with zipper (zip), which encodes the heavy chain of nonmuscle myosin II, were analyzed. About 25% of the progeny from crossing the females transheterozygous with DMBSP2 and Df(3L)th117 to the males heterozygous for DMBSE1 are embryonically lethal. It was expected that a reduction in the gene dosage of zip+ would suppress the defects in the MBS mutant or Rho-kinase-expressing embryos. When DMBSP2/Df(3L)th117 females are mated with males heterozygous for both DMBSE1 and zipEbr, half of the embryos defective for both maternal and zygotic MBS should be heterozygous for zipEbr. As expected, the embryonic lethality was reduced to nearly half that of the corresponding cross. Similarly, the heterozygosity for zipEbr considerably suppresses lethality due to ectopic wild-type Rho kinase expression. These results strongly suggest that either loss of MBS+ or overexpression of wild-type Rho-kinase causes hyperactivation of nonmuscle myosin II through increasing the levels of phosphorylation of MRLC (Mizuno, 2002).
zipEbr is a point mutation reported to be highly sensitive to genetic backgrounds. About 70% of the flies transheterozygous between zipEbr and zip02957 have malformed wings with varying degrees of severity. Although zipEbr is recessive, a considerable percentage of the flies heterozygous for both zipEbr and the mutations in the components of the Rho signaling pathway such as DRho1 and DRhoGEF2 produced similar defects. A half reduction of Drok, which encodes Rho-kinase, also dominantly enhances zipEbr. This indicates the involvement of the Rho signaling pathway and its effector, Rho-kinase, in the myosin function of adult wing morphogenesis. When the flies are also heterozygous for DMBSE1, wing malformation is significantly suppressed, suggesting that MBS functions antagonistically to the Rho signaling pathway (Mizuno, 2002).
Neuronal cells must extend a motile growth cone while maintaining the cell body in its original position. In migrating cells, myosin contraction provides the driving force that pulls the rear of the cell toward the leading edge. The function of myosin light chain phosphatase, which down-regulates myosin activity, has been characterized in Drosophila photoreceptor neurons. Mutations in the gene encoding the myosin binding subunit of this enzyme cause photoreceptors to drop out of the eye disc epithelium and move toward and through the optic stalk. This phenotype is due to excessive phosphorylation of the myosin regulatory light chain Spaghetti squash rather than another potential substrate, Moesin, and the phenotype requires the nonmuscle myosin II heavy chain Zipper. Myosin binding subunit mutant cells continue to express apical epithelial markers and do not undergo ectopic apical constriction. In addition, mutant cells in the wing disc remain within the epithelium and differentiate abnormal wing hairs. It is suggested that excessive myosin activity in photoreceptor neurons may pull the cell bodies toward the growth cones in a process resembling normal cell migration (Lee, 2004).
Nonmuscle myosin II consists of a hexamer of two myosin heavy chains (MHC), two myosin light chains (MLC), and two myosin regulatory light chains (MRLC). Phosphorylation of key serine and threonine residues on MRLC stimulates the ATPase activity of MHC and promotes its assembly into filaments, leading to stress fiber contraction. Mutations in the Drosophila orthologs of these myosin subunits have provided insight into the developmental functions of myosin II. Mutations in zipper (zip), which encodes MHC, cause defects in cytokinesis, closure of the dorsal embryonic epidermis over the amnioserosa, axon patterning, and myofibril formation. spaghetti squash (sqh), encoding MRLC, is required for cytokinesis, oogenesis, and imaginal disc eversion (Lee, 2004 and references therein).
Actin-binding proteins of the ezrin, radixin, and moesin (ERM) family are thought to link transmembrane proteins to the actin cytoskeleton. ERM proteins are activated by phosphorylation of a conserved threonine residue, which inhibits association between the N-terminal FERM domain and C-terminal actin-binding domain of the protein, freeing them to bind to other substrates. Moesin-like (Moe) is the only representative of this family in Drosophila. Moe mutants have abnormal oocyte polarity because defects in the anchorage of actin filaments to the oocyte cortex disrupt the localization of maternal determinants. In addition, Moe mutant cells in the wing disc undergo an epithelial-to-mesenchymal transition and adopt invasive migratory behavior (Lee, 2004 and references therein).
Interestingly, genetic and biochemical studies implicate the same kinase and phosphatase in the regulation of both nonmuscle myosin II and Moesin. Rho-associated kinase (ROCK/Rok) has been shown to phosphorylate MRLC in both mammalian and Drosophila systems. Myosin light chain kinase (MLCK) also can phosphorylate and activate MRLC; MLCK seems to act at the periphery of the cell, whereas ROCK is active in more central regions. Although ERM proteins are positively regulated by Rho GTPases, it is not clear whether they are directly phosphorylated by ROCK or by phosphoinositide-regulated kinases. However, in Drosophila wing disc development Moe seems to act antagonistically to Rho1 and rok (Lee, 2004 and references therein).
A major antagonist of the Rok/myosin signaling pathway is myosin light chain phosphatase (MLCP). This serine/ threonine protein phosphatase is a heterotrimer consisting of a catalytic subunit (PP1cdelta), a 20-kDa protein of unknown function, and the myosin binding subunit (MBS) that targets MLCP to its substrates, which include both MRLC and Moesin. Phosphorylation by Rok of a specific threonine within a conserved motif in MBS has been shown to inhibit MLCP activity; this suggests that Rok can positively activate MRLC and Moesin both by direct phosphorylation of these two substrates and also by inhibition of MBS. Like zip mutants, Drosophila Myosin binding subunit (Mbs) mutants fail to complete dorsal closure, suggesting that this process requires spatially regulated myosin activation. Mbs is also required for the growth of ring canals during oogenesis, and genetic interactions suggest that it opposes the functions in imaginal disc development of zip, Rho1, and rok. Likewise, Caenorhabditis elegans mel-11, which encodes MBS, and let-502, which encodes Rok, have opposite functions in embryonic elongation (Lee, 2004 and references therein).
Photoreceptor differentiation progresses across the Drosophila eye disc from posterior to anterior and is preceded by an epithelial indentation known as the morphogenetic furrow (MF). Cells in the MF undergo a transient contraction along the apical-basal axis and constrict their apical surfaces. After emerging from the MF, some of these cells assemble into ommatidial clusters, differentiate into photoreceptors, and extend axons through the optic stalk into the brain. Mbs mutations have been identified in a screen for genes required for normal photoreceptor differentiation. Findings on the role of Mbs in photoreceptor development suggest that photoreceptor neurons require Mbs to reduce myosin activity and thus prevent their cell bodies from migrating toward their axon terminals (Lee, 2004).
Mbs exerts its effects on eye development by regulating the phosphorylation state of the Sqh MRLC subunit of nonmuscle myosin II. The level of phosphorylated Sqh is greatly increased in Mbs mutant clones in both the eye and wing discs, and nonphosphorylatable or phosphomimetic forms of Sqh strongly modulate the severity of the Mbs phenotype. In addition, the effect of zip dosage on the Mbs phenotype indicates that p-Sqh acts through Zip to control photoreceptor localization. In vivo data show that in the eye disc Mbs is not required to dephosphorylate Moe. If dephosphorylation of Moe by Mbs occurs in vivo, it may be limited to specific tissues or developmental stages (Lee, 2004).
The identity of the kinase antagonized by Mbs in the eye is less clear. Although it has been reported that Rok can phosphorylate Sqh in vitro and that p-Sqh levels are reduced in rok mutant larvae, normal levels of p-Sqh were detected in rok2 eye disc clones. In addition, overexpression of Rok-CAT in the eye disc has no visible effect on photoreceptor differentiation or localization, and does not seem to enhance the Mbs phenotype. Rok may have a more significant effect on Sqh phosphorylation in other tissues; the lethality caused by overexpression of constitutively active Mbs is partially suppressed by coexpression of the catalytic domain of Rok. Myosin seems to be a downstream effector of Rho and Rok in wing and leg development, and the MEL-11 myosin phosphatase antagonizes the LET-502 Rho kinase in C. elegans development, supporting a role for Rok in phosphorylating Sqh in some cell types (Lee, 2004 and references therein).
Another kinase that might phosphorylate Sqh in the eye disc is MLCK. It has been reported that MLCK phosphorylates MRLC at the periphery of fibroblast cells, whereas ROCK acts in the central domain of these cells. Drosophila Stretchin-MLCK is a very large compound gene that produces multiple alternatively spliced transcripts, and no mutations in this gene have been identified, preventing the analysis of its interactions with Mbs. Another possible kinase is p21-activated kinase (PAK), which has been shown to increase the level of phosphorylated MRLC in cultured cells and to phosphorylate MRLC in vitro. Interestingly, overexpression of a myristylated form of PAK in Drosophila photoreceptors causes their cell bodies to detach from the eye disc epithelium and enter the brain, strongly resembling the Mbs mutant phenotype. Pak mutant photoreceptors develop normally except for axon guidance defects, suggesting that Pak is not essential for myosin activation in these cells. However, a second Pak gene, mushroom bodies tiny, is required for late photoreceptor morphogenesis and adherens junction integrity, and a third Pak gene is present in the genome, raising the possibility that these enzymes have redundant functions and complicating any analysis of their interactions with Mbs (Lee, 2004 and references therein).
The excessive myosin activity present in Mbs mutant photoreceptors causes them to adopt a more basal location in the eye disc and sometimes to enter the optic stalk. Several possible mechanisms for this phenotype have been addressed. Myosin can affect the shape of cultured cells by promoting the assembly of stress fibers and focal adhesions, and a transient accumulation of p-Sqh accompanies the apical constriction and apical-basal contraction of cells in the morphogenetic furrow. It was therefore interesting to enquire whether loss of Mbs might induce these cell shape changes in ectopic regions of the eye disc, resulting in mutant cells that formed a constitutive furrow. However, visualization of the apical surface of mutant clones by p-Tyr or phalloidin staining did not reveal any ectopic apical constriction of cells surrounding the photoreceptor clusters, suggesting that myosin phosphorylation is not sufficient to induce the cell shape changes that occur in the morphogenetic furrow. In addition, the integrity of the epithelial surface surrounding the photoreceptor clusters indicates that loss of Mbs specifically affects the localization of photoreceptor cells (Lee, 2004).
Another possibility is that Mbs mutant cells might undergo an epithelial to mesenchymal transition and become migratory. This phenotype has been reported for wing disc cells mutant for Moe, which encodes a potential substrate of Mbs. However, Mbs mutant cells in the wing disc remain within the epithelium and show no change in their apical-basal localization, although p-Sqh is up-regulated to a similar extent in both the wing and eye discs. In addition, Mbs mutant photoreceptors seem to retain some aspects of their epithelial character; they continue to express the epithelial apical junction proteins Patj, Crumbs, and E-cadherin. These proteins are present apical to mislocalized nuclei, suggesting that the entire cell is affected rather than the position of the nucleus within the cell. In contrast, the nuclei of klarsicht or Glued mutant cells are basally located within the cell due to defective dynein function (Lee, 2004).
The model that is favored is that unregulated myosin generates a traction force that pulls photoreceptor cell bodies toward their axon terminals. This would explain why the Mbs phenotype is specific to photoreceptors rather than wing disc cells or undifferentiated cells in the eye disc. It also would explain why the movement of mutant cells is directed toward the optic stalk or, in a disco background, toward the axon terminals within the eye disc. This abnormal force also might be accompanied by changes in adhesion to other cells or the substrate. Loss of Mbs could reduce the adhesion of epithelial cells to their neighbors, preventing them from withstanding the normal forces involved in axon extension. However, Mbs clones do not show the smooth borders characteristic of changes in adhesive properties (Lee, 2004).
It is not known whether the force generated by excessive myosin activity is located at the growth cone or in the cell body, although the latter model is favored because the highest levels of p-Sqh are found in apical regions of both wild-type and Mbs mutant cells. In vertebrate growth cones, two isoforms of the heavy chain of nonmuscle myosin II seem to have different locations and functions. MHCIIB is more peripheral and is required for axon outgrowth, whereas MHCIIA is central and is required for cell adhesion. Drosophila has only a single zip gene, which may perform both functions. The importance of MHCIIB in generating the traction force that allows growth cone extension suggests that this force might be increased in the absence of MLCP activity. There is a precedent for the idea that axon outgrowth can exert a pulling force on the cell body, because it has been shown that chick motor neurons will migrate out of the spinal cord along their axons if their movement is not blocked by boundary cap cells (Lee, 2004 and references therein).
The other possibility is that the actomyosin contraction takes place within the cell body, detaching it from surrounding cells and pulling it toward the growth cone. This would resemble the normal function of myosin in retracting the rear of migrating cells. Cell detachment and shrinkage has been reported for fibroblasts treated with an inhibitor of MLCP activity. Myosin light chain phosphatase activity may be specifically required in neuronal cells to allow axon extension to occur without triggering a migratory response in the cell body (Lee, 2004).
Ring canals, also known as stable intercellular bridges, are derived from the contractile rings of incomplete cytokinesis (IC) in most organisms. Formation of ring canals is necessary to generate functional eggs and sperm in multiple organisms including insects, birds, mammals and various plants. How the constriction of a contractile ring is arrested and how an arrested contractile ring is transformed into a ring canal is unknown. This paper describes the function of the Drosophila Myosin binding subunit of myosin phosphatase (DMYPT, Myosin binding subunit or Mbs) in both processes. DMYPT is highly enriched in the cytoplasm of cells undergoing IC during oogenesis. DMYPT mutations in germ cells, but not in somatic follicle cells, result in over-constriction of contractile rings and ring canals. This leads to formation of small ring canals and mis-regulation of centriole migration during female germline cyst formation (see Germline cyst formation during Drosophila oogenesis). The results suggest that there may be two parallel mechanisms to prevent the contractile rings from being completely closed, physical resistance and inhibition of myosin II activity via DMYPT (Ong, 2010b).
The engine of cytokinesis is non-muscle myosin II (henceforth referred to as myosin II) whose activity is negatively regulated by myosin light chain phosphatase (MLCP). MLCP is composed of three subunits: a catalytic subunit type I serine/threonine protein phosphatase 1ß (PP1cß), myosin phosphatase target subunit (MYPT, also known as myosin binding subunit MBS), and a small subunit M20 of unknown function. As its name implies, MYPT binds myosin II and confers the selectivity of PP1cß for myosin II (Ito, 2004). MYPT is the main target of regulation for the phosphatase activity of MLCP. There are five members in the human MYPT family: MYPT1, MYPT2, MYPT3, MBS85, and TIMAP. MYPT1, MYPT2, and MBS85 contain a leucine zipper motif at the C-terminus and highly conserved, inhibitory, Rho kinase phosphorylation sites in the center region. MYPT3 and TIMAP do not have these regulatory phosphorylation sites; instead, they have SH3 sites and a C-terminal prenylation motif, CAAX (Ong, 2010b).
Drosophila has two MYPT homologues: the MYPT1-like DMYPT, which is a pleiotropic essential gene, and the MYPT3-like MYPT-75D with unknown function. Animals homozygous for DMYPT03802, a hypomorphic mutation caused by a P-element insertion, die as embryo. DMYPT is also required for the viability of larvae and pupae. The lethality caused by DMYPT03802 can be rescued with a heat-shock driven DMYPT (hsDMYPT) transgene. Vitellarium egg chambers of homozygous DMYPT03802 germline clones (GLCs) have small ring canals, resulting in failure of nurse cell dumping (the fast phase of nurse cell cytoplasm transport) and sterility. Mature ring canal markers HtsRC, filamentous actin, and phospho-tyrosine epitopes were detected in the small ring canals of homozygous DMYPT03802 GLCs. However, it was not known how a lack of DMYPT function leads to formation of small vitellarium ring canals and whether DMYPT plays a role during early oogenesis, specifically in germline cyst formation (Ong, 2010b).
This study shows that DMYPT is required in germ cells to cell autonomously control germ cell ring canal size, prior to ring canal growth, probably by regulating cytokinesis arrest during germ cell IC. DMYPT protein is highly enriched in cells undergoing IC during fly oogenesis. DMYPT loss-of-function (LOF) mutations in germ cells, but not in the somatic follicle cells, cause over-constriction of contractile rings and ring canals, during germ cell IC. DMYPT is the first molecule identified that arrests contractile ring constriction and/or maintains the initial size of ring canals, directly or indirectly (Ong, 2010b).
The data show that DMYPT is highly enriched in germ cells undergoing IC. Moreover, loss of DMYPT function causes over constriction of contractile rings and dramatically increases constriction of ring canals just prior to ring canal growth. A schematic view of contractile ring constriction and ring canal formation is provided. As was described in Ong (2010a), in wild type flies, during each germline cystocyte mitotic division a contractile ring constricts and is then arrested when it reaches its maximal constriction point. A fusome plug forms in the arrested contractile ring and facilitates conversion of the contractile ring into a ring canal. The fusome plug then fuses with the fusome from earlier mitotic divisions and grows to form a mature fusome. The ring canal does not change in size during the subsequent mitotic divisions. When all four mitotic divisions are finished, the fusome starts to degrade, and eventually disappears. Ring canals start to grow at stage nona 2b1, after a slight constriction. Similar events occur in DMYPT heterozygotes. In the homozygous DMYPT mutants, contractile rings constrict to a greater degree than those in heterozygotes resulting in smaller nascent ring canals. The ring canals remain at that size until the fusome starts to degrade. Although ring canals constrict only slightly after the final mitotic division in the presence of DMYPT, they constrict dramatically in its absence. These observations have several possible explanations (Ong, 2010b).
It is possible that DMYPT only functions to arrest contractile ring constriction. Without DMYPT, contractile rings constrict marginally more than normal. These over-constricted contractile rings may be structurally compromised so that events immediately preceding ring canal growth result in further constriction or even collapse of the nascent ring canals (Ong, 2010b).
Alternatively, MYPT could function both during contractile ring constriction arrest and following the 4th mitotic division to prevent ring canal constriction prior to ring canal growth. This latter hypothesis posits that myosin II is active just before ring canal growth and that DMYPT is required to prevent additional constriction. Yet a third explanation is that MYPT functions to arrest contractile ring constriction and also plays a role in stabilizing ring canals in a manner independent of myosin II inactivation (Ong, 2010b).
Another factor that may be complicating this analysis is that a DMYPT-like protein functionally overlaps with DMYPT. Although the possibility of MYPT-75D's involvement in IC has not been ruled out, DMYPT and MYPT-75D do not function redundantly, at least not totally, in this process because LOF mutations in DMYPT alone are sufficient to cause the IC defect. It could however be the case that MYPT-75D is active early in the process to assist MYPT in constriction arrest, but is inactivated or plays a minor role at later times. Thus the observed mutant phenotype is most prevalent at times when DMYPT is the sole mediator of constriction arrest. Whether MYPT-75D LOF mutants affect IC is currently being examined (Ong, 2010b).
Finally, there may be two parallel mechanisms to prevent the contractile rings from closing completely, physical resistance and inhibition of myosin II activity via myosin phosphatase. Contractile rings in germ cells lacking DMYPT do eventually arrest, and the diameter of ring canals in these cells do not noticeably decrease until after all four mitotic divisions are finished and the fusome starts to degenerate. Thus it is possible that during mitotic divisions there is a physical limitation on contractile ring constriction. Following the final division this physical limitation is decreased as the fusome disintegrates. Consequently lack of DMYPT function alone only has a mild effect on contractile ring constriction during mitosis and the phenotype becomes more severe as the physical resistance decreases. The molecular nature of the physical resistance is currently being examined (Ong, 2010b).
DMYPT is, thus far, the only known protein that, when mutated, results in over constriction of both contractile rings and ring canals. The result suggests that DMYPT may be required for contractile ring constriction arrest and/or to maintain the initial size of ring canals. DMYPT will be a molecular tool to identify additional genes functioning in IC and to elucidate the molecular mechanism that mediates IC and germ line cyst formation (Ong, 2010b).
Alessi, D., MacDougall, L. K., Sola, M. M., Ikebe, M. and Cohen, P. (1992). The control of protein phosphatase-1 by targeting subunits. The major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1. Eur. J. Biochem. 210: 1023-1035. 1336455
Arquier, N., Perrin, L., Manfruell, P. and Sémériva, M. (2001). The Drosophila tumor suppressor gene lethal(2)giant larvae is required for the emission of the Decapentaplegic signal. Development 128: 2209-2220. 11493541
Begum, N., et al. (2000). Regulation of myosin-bound protein phosphatase by insulin in vascular smooth muscle cells: evaluation of the role of Rho kinase and phosphatidylinositol-3-kinase-dependent signaling pathways. Mol. Endocrinol. 14(9): 1365-76. 10976915
Broustas, C. G., et al. (2002). Phosphorylation of the myosin-binding subunit of myosin phosphatase by Raf-1 and inhibition of phosphatase activity. J. Biol. Chem. 277(4): 3053-9. 11719507
Hickson, G. R., Echard, A. and O'Farrell, P. H. (2006). Rho-kinase controls cell shape changes during cytokinesis. Curr. Biol. 16(4): 359-70. Medline abstract: 16488869
Fujioka, M., Takahashi, N., Odai, H., Araki, S., Ichikawa, K., Feng, J., Nakamura, M., Kaibuchi, K., Hartshorne, D. J., Nakano, T. and Ito, M. (1998). A new isoform of human myosin phosphatase targeting/regulatory subunit (MYPT2): cDNA cloning, tissue expression, and chromosomal mapping. Genomics 49: 59-68. 9570949
Hartshorne, D. J., Ito, M. and Erdodi, F. (1998). Myosin light chain phosphatase: subunit composition, interactions and regulation. J. Muscle Res. Cell Motil. 19: 325-341. 9635276
Ito, M., Nakano, T., Erdodi, F. and Hartshorne, D. J. (2004). Myosin phosphatase: structure, regulation and function. Mol. Cell. Biochem. 259: 197-209. PubMed Citation: 15124925
Kaibuchi, K., Kuroda, S. and Amano, M. (1999). Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells. Ann. Rev. Biochem. 68: 459-486. 10872457
Kawano, Y., Fukata, Y., Oshiro, N., Amano, M., Nakamura, T., Ito, M., Matsumura, F., Inagaki, M. and Kaibuchi, K. (1999). Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo. J. Cell Biol. 147: 1023-1038. 10579722
Kimura, K., et al. (1996). Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273: 245-248. 8662509
Lee, A. and Treisman, J. E. (2004). Excessive myosin activity in Mbs mutants causes photoreceptor movement out of the Drosophila eye disc epithelium. Mol. Biol. Cell. 15: 3285-3295. 15075368
Mitonaka, T., Muramatsu, Y., Sugiyama, S., Mizuno, T. and Nishida, Y. (2007). Essential roles of myosin phosphatase in the maintenance of epithelial cell integrity of Drosophila imaginal disc cells. Dev. Biol. 309 (1): 78-86. Medline abstract: 17662709
Mizuno, T., Tsutsui, K. and Nishida, Y. (2002). Drosophila myosin phosphatase and its role in dorsal closure. Development 129: 1215-1223. 11874917
Ong, S. and Tan, C. (2010a). Germline cyst formation and incomplete cytokinesis during Drosophila melanogaster oogenesis. Dev. Biol. 337: 84-98. PubMed Citation: 19850028
Ong, S., Foote, C. and Tan, C. (2010b). Mutations of DMYPT cause over constriction of contractile rings and ring canals during Drosophila germline cyst formation. Dev. Biol. 346(2): 161-9. PubMed Citation: 20542024
Piekny, A. J., Wissmann, A. and Mains, P. E. (2000). Embryonic morphogenesis in Caenorhabditis elegans integrates the activity of LET-502 Rho-binding kinase, MEL-11 myosin phosphatase, DAF-2 insulin receptor and FEM-2 PP2c phosphatase. Genetics 156(4): 1671-89. 11102366
Piekny, A. J. and Mains, P. E. (2002). Rho-binding kinase (LET502) and myosin phosphatase (MEL-11) regulate cytokinesis in the early Caenorhabditis elegans embryo. J. Cell Sci. 115: 2271-2282. Medline abstract: 12006612
Takizawa, N., Niiro, N. and Ikebe, M. (2002). Dephosphorylation of the two regulatory components of myosin phosphatase, MBS and CPI17. FEBS Lett. 515(1-3): 127-32. 11943207
Tan, C., Stronach, B. and Perrimon, N. (2003). Roles of myosin phosphatase during Drosophila development. Development 130: 671-681. Medline abstract: 12505998
Wissmann, A., Ingles, J., McGhee, J. D. and Mains, P. E. (1997). Caenorhabditis elegans LET-502 is related to Rho-binding kinases and human myotonic dystrophy kinase and interacts genetically with a homolog of the regulatory subunit of smooth muscle myosin phosphatase to affect cell shape. Genes Dev. 11: 409-422. 9042856
Wissmann, A., Ingles, J. and Mains, P. E. (1999). The Caenorhabditis elegans mel-11 myosin phosphatase regulatory subunit affects tissue contraction in the somatic gonad and the embryonic epidermis and genetically interacts with the Rac signaling pathway. Dev. Biol. 209(1): 111-27. 1020874
date revised: 28 December 2011
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.