1 Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
2 CREST, Japan Science and Technology Corporation, Honmachi, Kawaguchi, Saitama 332-0012, Japan
*Author for correspondence (e-mail: nishida{at}bio.nagoya-u.ac.jp)
Accepted 12 December 2001
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SUMMARY |
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Key words: Rho-kinase, MBS, zipper, spaghetti-squash, Rho, RhoGEF, Morphogenesis, Drosophila melanogaster
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INTRODUCTION |
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Dorsal closure is a process well suited to studies on the molecular and cellular basis of morphogenesis, and a number of loci involved in this process have been identified from their "dorsal open" or "dorsal hole" phenotypes, which are characterized by large holes in their dorsal cuticle (Noselli, 1998). They can be grouped into at least four classes; genes involved in the Jun amino-terminal kinase (JNK) signaling cascade, genes encoding the components of the Decapentaplegic (Dpp)-mediated signal transduction pathway, genes involved in the Rho GTPase-mediated signaling pathway, and genes encoding cytoskeletal proteins and membrane-associated molecules for cell adhesion. Activation of the JNK signaling cascade is required in the dorsal-most cells of the lateral epidermis, the leading edge cells, to induce the expression of Dpp (Noselli, 1998
). One of the target genes of Dpp signaling is zipper (zip), which encodes the heavy chain of nonmuscle myosin II, and Dpp signaling in the leading edge cells activates the transcription of zip (Ariquier et al., 2001
).
At the leading edge of the lateral epidermis, filamentous actin (F-actin) and nonmuscle myosin II are prominently accumulated. The supracellular purse-string composed of the actomyosin contractile apparatus provides one of the major forces for promoting dorsal closure (Young et al., 1993). An analysis of the zip mutations demonstrated an embryonic lethality due to defects in dorsal closure, indicating that nonmuscle myosin II is required for the morphogenetic processes in dorsal closure (Young et al., 1993
). Genetic interactions between zip and mutations in the components of the Rho signaling pathway suggest that nonmuscle myosin II is regulated by the Rho signals (Halsell et al., 2000
).
Nonmuscle myosin II is a hexamer composed of two of each of three subunits; the heavy chain, the regulatory light chain (MRLC) and the essential light chain. The force-generating activity of actomyosin is mainly regulated by phosphorylation and dephosphorylation of MRLC. Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and Rho-kinase/Rok, one of the effectors of the Rho GTPase, are responsible for the phosphorylation of MRLC (Tan et al., 1992
; Amano et al., 1996
). However, myosin phosphatase dephosphorylates MRLC, leading to the inactivation of nonmuscle myosin II. Myosin phosphatase is a heterotrimer composed of a catalytic subunit belonging to protein phosphatase 1c (PP1c), the myosin-binding subunit (MBS) and M20 (Alessi et al., 1992
; Hartshorne et al., 1998
). MBS plays the regulatory roles of myosin phosphatase as a target of the upstream signals and as a determinant of substrate specificity. Myosin phosphatase is negatively regulated through phosphorylation of MBS by Rho-kinase/Rok
(Kimura et al., 1996
; Kawano et al., 1999
). Thus, Rho-kinase/Rok
doubly activates nonmuscle myosin II through direct phosphorylation of MRLC and inactivation of myosin phosphatase by phosphorylating MBS (Kaibuchi et al., 1999
).
We have previously identified the Drosophila homolog of Rho-kinase that is encoded by DRhk, and have demonstrated that DRho-kinase associates with the GTP-bound DRho1 and phosphorylates the vertebrate MRLC and MBS (Mizuno et al., 1999). Recently, the same gene has been characterized genetically as Drok, and has been demonstrated to be involved in the establishment of planar polarity in adult structures such as the compound eye and wing (Winter et al., 2001
). We have identified the Drosophila homolog of MBS 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.
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MATERIALS AND METHODS |
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Cloning of DMBS cDNA
PCR primers were designed on the basis of the DNA sequences of two EST clones (GH27673 and GM02173) sequenced by the Berkeley Drosophila Genome Project. The primer sequences are 5'-AGCAATAGCGATACAACAGCGCTAC-3' and 5'-GGTACCGTCTGCTTATCTTAACGTACTC-3'. Two embryonic cDNA libraries (Brown and Kafatos, 1988) were used as templates. Pyrobest DNA polymerase (Takara Shuzo Co. Ltd., Japan) was used for PCR, which was performed according to the following protocol: 30 cycles of 94°C for 15 seconds, 55°C for 30 seconds, and 70°C for 5 minutes. The PCR products were cloned into pBluescript and sequenced.
Generation of an antibody to DMBS and immunoblotting
A rabbit polyclonal anti-DMBS-C1 antibody was raised against a synthetic polypeptide, KEKESGSERTSRS, corresponding to the carboxyl-terminal portion of DMBS shared by DMBS-L and DMBS-S, and was affinity purified. The polyclonal antibody and the mouse monoclonal anti-Pnut antibody (Neufeld and Rubin, 1994) obtained from the Developmental Studies Hybridoma Bank were used for immunoblotting after dilution to 1/700 and 1/20, respectively. Sqh, the Drosophila homolog of MRLC, and its phosphorylated form were detected using the anti-Sqh antibody (Jordan and Karess, 1997
) and rabbit polyclonal anti-phospho-MRLC antibody (Matsumura et al., 1998
), respectively, as described (Winter et al., 2001
). The latter antibody has been raised against a synthetic peptide based on the mouse sequence, and was provided by F. Matsumura. It reacts with the Drosophila protein.
Generation of transgenic lines and rescue of DMBS mutants
The DMBS-L and -S cDNAs were cloned into pP[CaSpeR-hs] and hs-DMBS-L and hs-DMBS-S transgenic lines were generated by the standard procedure. For rescue experiments, each two of the independent transgenes of hs-DMBS-L or hs-DMBS-S on the third chromosome were used, and the transgenes were combined with either DMBSE1 or DMBSP1 by recombination. Matings were performed between w; DMBS/TM3, Sb and w; DMBS hs-DMBS-L/TM3, Sb or w; DMBS hs-DMBS-S/TM3, Sb, and the cultures were either kept at 25°C or treated at 37°C for 1 hour twice a day throughout development. The full-length DRhk+ cDNA was cloned into pUAST (Brand and Perrimon, 1993) to generate UAS-DRhk+ transgenic lines.
Immunohistochemistry and confocal microscopy
Phalloidin staining
Embryos were fixed for 20 minutes in 7.2% formaldehyde/PBS: heptane, 1:1, and were devitellinized by hand. Embryos were incubated in rhodamine-conjugated phalloidin (Molecular Probes, Inc.) for 20 minutes, and were then washed with 0.2% Triton X-100/PBS three times. The DMBS mutant embryos were distinguished from the paternally rescued embryos by using the blue balancer, TM3, P[ry+t7.2=HZ2.7]DB2.
Immunostaining
Fixation and staining were carried out by the standard procedures. The primary antibodies and dilutions used are as follows. A mouse monoclonal anti-phosphotyrosine antibody (UBI) at a dilution of 1/1000; the rabbit polyclonal anti-myosin heavy chain antibody (Jordan and Karess, 1997), a gift from R. Karess, at a dilution of 1/1000; and the anti-phospho-MRLC antibody (Matsumura et al., 1998
) at a dilution of 1/20. The secondary antibodies used were a rhodamine-conjugated anti-mouse IgG antibody (TAGO) or a FITC-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories). The DMBS mutant embryos were distinguished as described above.
For the observation of MRLC distribution, the GFP-Sqh transgenic line (Sisson et al., 2000) provided by R. Karess was used. Embryos were fixed for 20 minutes in 4% paraformaldehyde/PBS: heptane, 1:1, and were devitellinized by vigorous shaking for 10 seconds after substitution of the aqueous layer with methanol. Then, embryos were rehydrated twice with 0.2% saponin/PBS for 10 minutes before mounting.
All images were collected on a Zeiss 512 laser scanning confocal microscope and processed with Adobe Photoshop software.
Expression and purification of DMBS recombinant proteins
For the production of GST-fused DMBS-L, the coding sequence of DMBS-L was amplified by PCR using the primer set, 5'-TCTGAATTCATGTCCTCGCTGGACGCACGCAAC-3' and 5'-GGTACCTCATTTACTTAATTTGCTAATTAC-3', to create the EcoRI restriction sites immediately before the first codon. The amplified DNA fragment was cloned into pGEX-3T-1 (Pharmacia) to fuse with GST at the N terminus. The construct was verified by DNA sequencing. Mutagenesis at the putative phosphorylation site in DMBS was done by PCR using the following primers: 5'-AGGGAGACTCGACGGTCTGCCCAAGGTGTC-3' and 5'-CCAGGGTGACACCTTGGGCAGACCGTCGAG-3'. Purification of the GST-fusion proteins was carried out essentially as described previously (Frangioni and Neel, 1993).
Immunoprecipitation, immunoblotting and in vitro kinase assay
Immunoprecipitation and immunoblotting of the HA-tagged DRho-kinase were performed as described previously (Mizuno et al., 1999). The immunoprecipitated DRho-kinase was incubated with 0.6 µg of GST-fused DMBS in the kinase buffer and was assayed as described previously (Mizuno et al., 1999
).
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RESULTS |
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They contained the three conserved motifs, and the amino acid identities to their corresponding regions of human MYPT2 (Fujioka et al., 1998) are 57.8%, 55.6% and 77.8%, respectively, from the amino terminus (Fig. 1A). The similarity to vertebrate MBSs is restricted to these regions. Vertebrate MBSs and the homolog of Caenorhabditis elegans, MEL-11, contain seven ankyrin repeats (Fujioka et al., 1998
; Wissmann et al., 1997
), but the sequences in the fourth and seventh repeats diverged in Drosophila. In the Drosophila Genome Database, no other sequence similar to MBS was found, and we consider them as Drosophila homologs of MBS. We refer to the longer and shorter forms as Drosophila MBS-long (DMBS-L) and Drosophila MBS-short (DMBS-S), respectively (Fig. 1A).
The two cDNAs should derive from a single gene by alternative splicing (Fig. 3A). The DMBS-L-specific, 7th exon encodes a sequence of 129 amino acid residues. Furthermore, two in-frame consecutive splicing acceptor sites are present 5' of the 4th exon (data not shown), and splicing variations at this site added one more amino acid residue in DMBS-L as compared to DMBS-S. We also obtained several partial cDNA fragments different from DMBS-L and DMBS-S (data not shown). Thus, the DMBS gene encodes multiple forms of DMBS through differential splicing. Splicing variants have been reported also for the vertebrate MBSs and MEL-11 of C. elegans (Hartshorne et al., 1998; Wissmann et al., 1999
).
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Thr594 may correspond to the major phosphorylation site in vertebrate MBS (Fig. 1B). The threonine residue was replaced with an alanine, and this recombinant DMBS was used as a substrate. As shown in Fig. 1C, the level of phosphorylation was significantly reduced, indicating that Thr594 is the major site phosphorylated by DRho-kinase. It has been reported that mammalian MBS is phosphorylated at several sites by Rho-kinase (Kawano et al., 1999), and there presumably are other phosphorylation sites in DMBS as well.
Expression of DMBS during development
The pattern of the expression of DMBS during development was analyzed by in situ hybridization using DMBS-L as a probe (Fig. 2). A significant amount of DMBS mRNA was uniformly detected in blastoderm stage embryos (Fig. 2A), and it would be mostly of maternal origin. DMBS is expressed ubiquitously throughout embryogenesis (Fig. 2B,C). In the imaginal discs from third instar larvae, the DMBS transcript was uniformly detected (data not shown). Tissue- and stage-specificity of the expression for each isoform remain to be analyzed.
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A polyclonal antibody against a synthetic polypeptide corresponding to the carboxy-terminal region of DMBS was developed, producing a major band of about 95 kDa and several minor bands on the immunoblot (Fig. 3B). Correspondence of these bands to DMBS-L and -S is not certain at this moment. The amounts of the DMBS proteins were analyzed in the mutants. Mutants heterozygous between P2r31 and the strong alleles or Df(3L)th117, which deletes the region including DMBS, survived to third instar larvae, and the amount of DMBS was greatly reduced in the extracts prepared from these larvae (Fig. 3B). The result indicates that the mutations are correlated to the amount of DMBS. MRLC is encoded by spaghetti-squash (sqh) (Karess et al., 1991), and the levels of its phosphorylated form in the mutants were also examined. As shown in Fig. 3C, the levels of phospho-MRLC are significantly elevated in the mutants, indicating that the activity of myosin phosphatase is decreased in the mutants.
To further confirm that the mutations are at the DMBS 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 complemented the lethality of both l(3)72Dd03802 and l(3)72Dd3 (Table 1). This may suggest that, despite the expression of multiple isoforms of DMBS, 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.
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Defects in dorsal closure in embryos lacking DMBS
The animals homozygous for or transheterozygous between the strong DMBS alleles are larval lethal and embryonic development seems to proceed normally (Fig. 7A). This would be because of the maternal contribution of DMBS+ activity, a notion consistent with the observation that a significant amount of maternal mRNA is present in early-stage embryos (Fig. 2A). To analyze the function of DMBS during embryogenesis, we tried to reduce the maternal contribution.
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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 (Fig. 4B). In the remaining lethal embryos, the pattern of dorsal hairs was disturbed along the dorsal midline (Fig. 4E). These phenotypic variations would be due to the residual activity of maternal DMBS derived from the weak allele, DMBSP2. The results indicate that DMBS is required in the process of dorsal closure.
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Before the onset of the lateral epidermis elongation, the heavy chain of nonmuscle myosin II and MRLC outline the inner surface of the plasma membrane of the lateral epidermal cells at low levels, and they are concentrated at moderate levels along the leading edge of the lateral epidermis (Fig. 5A,B). The phosphorylated form of MRLC is localized similarly (Fig. 5C). During the course of extensive elongation, the heavy chain and MRLC accumulate at high levels along the leading edge (Fig. 5,D,E). Phosphorylated MRLC is also detected at a high level along the leading edge (Fig. 5F). After the meeting of the two lateral epidermal sheets, both the heavy chain and MRLC at the leading edge become diffuse (Fig. 5G,H), and the phosphorylated form of MRLC disappears at the site of fusion (Fig. 5I). These observations indicate that the distribution and phosphorylation of nonmuscle myosin II are dynamically regulated during dorsal closure.
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To examine whether defects in the dorsal closure in the embryos lacking DMBS or overexpressing DRhk+ 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. As already described, 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 (Fig. 7A). We expected that a reduction in the gene dosage of zip+ would suppress the defects in the DMBS mutant or DRhk+-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 DMBS should be heterozygous for zipEbr. As expected, the embryonic lethality was reduced to nearly half that of the corresponding cross (Fig. 7A). Similarly, the heterozygosity for zipEbr considerably suppressed lethality due to ectopic DRhk+-expression (Fig. 7B). These results strongly suggest that either loss of DMBS+ or overexpression of DRhk+ causes hyperactivation of nonmuscle myosin II through increasing the levels of phosphorylation of MRLC.
zipEbr is a point mutation reported to be highly sensitive to genetic backgrounds (Halsell and Kiehart, 1998; Halsell et al., 2000
). About 70% of the flies transheterozygous between zipEbr and zip02957 have malformed wings with varying degrees of severity (Fig. 8B-D). 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 (Fig. 8D) (Halsell et al., 2000
). A half reduction of Drok, which encodes DRho-kinase (Winter et al., 2001
), also dominantly enhanced zipEbr. This indicates the involvement of the Rho signaling pathway and its effector, DRho-kinase, in the myosin function of adult wing morphogenesis (Halsell et al., 2000
). When the flies are also heterozygous for DMBSE1, wing malformation is significantly suppressed (Fig. 8D), suggesting that DMBS functions antagonistically to the Rho signaling pathway (Fig. 8E) and is also involved in adult morphogenesis.
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DISCUSSION |
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Interactions with the Rho signaling pathway
The phenotype produced by overexpression of DRhk+ resembles that of DMBS mutant embryos, and both phenotypes can be suppressed by reducing the gene dosage of zip+. The results suggest that these phenotypes are the result of the hyperactivation of nonmuscle myosin II, and that myosin phosphatase and DRho-kinase function antagonistically toward each other in regulating nonmuscle myosin II (Fig. 8E). A similar observation has been reported for let-502 and mel-11 of C. elegans, which encode the homolog of Rho-kinase and MBS, respectively. The genes have been demonstrated to function in the hypodermal cell-shape change associated with the elongation of the embryo in C. elegans (Wissmann et al., 1997), thus indicating the conservation of the regulatory mechanisms of nonmuscle myosin II in morphogenesis. The genetic link between zip and the mutations in the Rho signaling pathway has been demonstrated (Halsell et al., 2000
) (Fig. 8A-D), and this indicates the regulation of nonmuscle myosin II by the Rho signaling pathway. The suppression of both phenotypes generated by double heterozygosity for zip and the mutations in the components of the Rho-signaling pathway by DMBS demonstrates that DMBS functions antagonistically toward the Rho-signaling pathway in the regulation of nonmuscle myosin II (Fig. 8E).
Roles of DMBS in dorsal closure
During dorsal closure, an extensive cell shape change in the lateral epidermis takes place. One of the major forces underlying the morphogenetic process is provided by the constriction of a supracellular purse-string revealed by the high level accumulation of F-actin and the heavy chain of nonmuscle myosin II along the leading edge of the lateral epidermis (Young et al., 1993) (Fig. 6D,G). We found that MRLC is also highly accumulated along the leading edge (Fig. 5D,E; Fig. 6G). Small quantities of these components of actomyosin are also detected along the inner surface of the plasma membrane. Phosphorylated MRLC was detected in significant amounts along the leading edge of the lateral epidermis (Fig. 5F and Fig. 6I), indicating that nonmuscle myosin II is persistently activated along the leading edge during extensive epidermal spreading.
In DMBS mutant embryos, the defects in dorsal closure seem to be confined to the leading edge cells, and these cells fail to fully elongate. In contrast, the lateral epidermal cells located more ventrally elongate more or less normally. It has been reported that dorsal closure is driven by multiple forces and that it can proceed in the absence of an intact contractile purse-string at the leading edge (Young et al., 1993; Kiehart et al., 2000
). These lateral epidermal cells are under tension during dorsal closure (Kiehart et al., 2000
), and they themselves may produce the forces to elongate.
It should be noted that the accumulation of nonmuscle myosin II in the leading edge cells was essentially not affected. Activation of nonmuscle myosin II takes place along the leading edge as in normal embryos, since the phosphorylated MRLC was detected there in the mutant embryos (Fig. 6H). In addition to the distribution along the leading edge, a significant accumulation of phosphorylated MRLC was detected also on the dorsal side of the boundaries between the leading edge cells in the mutant embryos (Fig. 6J). This may indicate the role of myosin phosphatase in inactivating nonmuscle myosin II in this subcellular location to coordinate elongation of the leading edge cells.
The results suggest the localized activation of myosin phosphatase during the normal course of dorsal closure. One possible explanation for this localized activation is that there is a subcellular distribution of myosin phosphatase itself in this region. However, a suitable antibody would have to be raised against DMBS to determine if this was so. Another possible explanation is the localization of an activator of myosin phosphatase in this region. Thus, there must be a subcellular localization of mechanisms for the regulation of nonmuscle myosin II. It has been demonstrated that the cellular polarity of the leading edge cells is altered from basolateral to apical in the leading edge during elongation (Ring and Martinez-Arias, 1993; Woods and Bryant, 1993
; Fehon et al., 1994
). It would be of interest to learn how cellular polarity affects the pattern of regulation of the nonmuscle myosin II in the leading edge cells. The results obtained in this study demonstrate the importance of both positive and negative regulation of nonmuscle myosin II in morphogenesis.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
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.[Abstract]
Amano, M., Ito, M., Kimura, K., Fukata, Y., Chihara, K., Nakano, T., Matsuura, Y. and Kaibuchi, K. (1996). Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J. Biol. Chem. 271, 20246-20249.
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.
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401-415.
Brown, N. H. and Kafatos, F. C. (1988). Functional cDNA libraries from Drosophila embryos. J. Mol. Biol. 203, 425-437.[Medline]
Fehon, R. G., Dawson, I. A. and Artavanis-Tsakonas, S. (1994). A Drosophila homologue of membrane-skeleton protein 4.1 is associated with septate junctions and is encoded by the coracle gene. Development 120, 545-557.
Frangioni, J. V. and Neel, B. G. (1993). Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal. Biochem. 210, 179-187.[Medline]
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.[Medline]
Gotwals, P. J. and Fristrom, J. W. (1991). Three neighboring genes interact with the Broad-Complex and the Stubble-stubbloid locus to affect imaginal disc morphogenesis in Drosophila. Genetics 127, 747-759.
Halsell, S. R., Chu, B. I. and Kiehart, D. P. (2000). Genetic analysis demonstrates a direct link between rho signaling and nonmuscle myosin function during Drosophila morphogenesis. Genetics 155, 1253-1265.
Halsell, S. R. and Kiehart, D. P. (1998). Second-site noncomplementation identifies genomic regions required for Drosophila nonmuscle myosin function during morphogenesis. Genetics 148, 1845-1863.
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.[Medline]
Jordan, P. and Karess, R. (1997). Myosin light chain-activating phosphorylation sites are required for oogenesis in Drosophila. J. Cell Biol. 139, 1805-1819.
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.[Medline]
Karess, R. E., Chang, X. J., Edwards, K. A., Kulkarni, S., Aguilera, I. and Kiehart, D. P. (1991). The regulatory light chain of nonmuscle myosin is encoded by spaghetti-squash, a gene required for cytokinesis in Drosophila. Cell 65, 1177-1189.[Medline]
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.
Kiehart, D. P., Galbraith, C. G., Edwards, K. A., Rickoll, W. L. and Montague, R. A. (2000). Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila. J. Cell Biol. 149, 471-490.
Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., Yamamori, B., Feng, J., Nakano, T., Okawa, K. et al. (1996). Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273, 245-248.[Abstract]
Martinez-Arias, A. (1993). Development and patterning of the larval epidermis of Drosophila. In The development of Drosophila melanogaster. pp. 517-608. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Matsumura, F., Ono, S., Yamakita, Y., Totsukawa, G. and Yamashiro, S. (1998). Specific localization of serine 19 phosphorylated myosin II during cell locomotion and mitosis of cultured cells. J. Cell Biol. 140, 119-129.
Mizuno, T., Amano, M., Kaibuchi, K. and Nishida, Y. (1999). Identification and characterization of Drosophila homolog of Rho-kinase. Gene 238, 437-444.[Medline]
Neufeld, T. P. and Rubin, G. M. (1994). The Drosophila peanut gene is required for cytokinesis and encodes a protein similar to yeast putative bud neck filament proteins. Cell 77, 371-379.[Medline]
Noselli, S. (1998). JNK signaling and morphogenesis in Drosophila. Trends Genet. 14, 33-38.[Medline]
Ring, J. M. and Martinez-Arias, A. (1993). puckered, a gene involved in position-specific cell differentiation in the dorsal epidermis of the Drosophila larva. Development, Suppl. 251-259.
Sisson, J. C., Field, C., Ventura, R., Royou, A. and Sullivan, W. (2000). Lava lamp, a novel peripheral Golgi protein, is required for Drosophila melanogaster cellularization. J. Cell Biol. 151, 905-917.
Staehling-Hampton, K., Hoffmann, F. M., Baylies, M. K., Rushton, E. and Bate, M. (1994). dpp induces mesodermal gene expression in Drosophila. Nature 372, 783-786.[Medline]
Strutt, D. I., Weber, U. and Mlodzik, M. (1997). The role of RhoA in tissue polarity and Frizzled signalling. Nature 387, 292-295.[Medline]
Tan, J. L., Ravid, S. and Spudich, J. A. (1992). Control of nonmuscle myosins by phosphorylation. Ann. Rev. Biochem. 61, 721-759.[Medline]
Winter, C. G., Wang, B., Ballew, A., Royou, A., Karess, R., Axelrod, J. D. and Luo, L. (2001). Drosophila rho-associated kinase (drok) links frizzled-mediated planar cell polarity signaling to the actin cytoskeleton. Cell 105, 81-91.[Medline]
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.[Abstract]
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, 111-127.[Medline]
Woods, D. F. and Bryant, P. J. (1993). Apical junctions and cell signalling in epithelia. J. Cell Sci. Suppl. 17, 171-181.[Abstract]
Young, P. E., Richman, A. M., Ketchum, A. S. and Kiehart, D. P. (1993). Morphogenesis in Drosophila requires nonmuscle myosin heavy chain function. Genes Dev. 7, 29-41.[Abstract]