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Drosophila myosin phosphatase and its role in dorsal closure

Tomoaki Mizuno1,2, Kyoko Tsutsui1 and Yasuyoshi Nishida1,*

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



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  		Fig. 1. (A) Schematic representations of basic structural features of DMBS-L (long) and DMBS-S (short) proteins and a comparison with human MBS. The N-terminal ankyrin repeats are indicated in red, and the C-terminal leucine-zipper motif in blue. The sequence specifically found in DMBS-L is shown in yellow. The position of the threonine residue phosphorylated by DRho-kinase is indicated above the rectangles (P), and the conserved region around the phosphorylation site is indicated in green. Human MBS (MYPT2 type a) (Fujioka et al., 1998Go) is schematically represented above DMBS-L, and the percentages of identity/similarity of the amino acid sequences in the three conserved regions are indicated between the two upper rectangles. The GenBank accession numbers for DMBS-L and –S cDNAs are AY048675 and AY048676, respectively. (B) A comparison of the amino acid sequence around the putative phosphorylation site of DMBS with those of the vertebrate MBSs. The amino acid residues with positional identity are in white type on black, and the residues with similarity to the vertebrate sequences are shaded gray. The asterisk indicates the threonine residue, which has been demonstrated to be phosphorylated by Rho-kinase in vertebrates. (C) Phosphorylation of DMBS by DRho-kinase and identification of its phosphorylation sites. The HA-tagged DRho-kinase expressed in 293 cells was immunoprecipitated, and the precipitate was used for in vitro kinase assay with GST-fused DMBS-L proteins as substrates. Lanes 1 to 3; GST-DMBS-L, lanes 4 to 6; GST-DMBS-L T594A, in which Ala was substituted for Thr594. The wild-type DRho-kinase (lanes 2 and 8), kinase-dead DRho-kinaseK116A (lanes 3 and 6), and vector alone (lanes 1 and 4) were used for the reactions. The arrow indicates the position of DMBS-L and the arrowhead the position of the autophosphorylated DRho-kinase. Portions of the immunoprecipitates were also tested for amounts of kinase by immunoblotting with an anti-HA antibody (lower panel). Lane 7, vector only; lane 8, wild-type DRho-kinase; and lane 9, DRho-kinaseK116A.

 


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  		Fig. 2. Expression of DMBS during development. Antisense (A-C) or sense RNA (D) probes were used to detect the transcripts. (A,D) Blastoderm embryos, (B) germband-elongated embryo, and (C) germband-retracted embryo. Anterior is to the left and dorsal to the top.

 


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  		Fig. 3. Identification of the DMBS mutations. (A) The genomic structure at the DMBS locus. The exon/intron structure is shown with columns and lines under the genomic map. The coding and untranslated regions are indicated as red and blue columns, respectively. The DMBS-L-specific exon is represented by a yellow column. The sites of the P-element insertions are indicated above the map. (B) Reduction in the amounts of the DMBS proteins in DMBS mutants. Protein blots were probed with either an anti-DMBS-C1 antibody (upper panel) or an anti-Pnut antibody (lower panel). Extracts were prepared from late third instar larvae of the following genotypes; wild type (lane 1), Df(3L)th117/DMBSP2r31 (lane 2), DMBSE1/DMBSP2r31 (lane 3), and DMBSP1/DMBSP2r31 (lane 4). Arrows indicate the positions of DMBS bands. (C) Increase of phospho-MRLC in DMBS mutant larvae. The extracts were prepared as described (Winter et al., 2001Go) from the larvae as in B, except for lane 5 for which sqhAX3; GFP-Sqh larvae were used. Protein blots were probed with an anti-phospho-MRLC antibody (upper panel) or an anti-Sqh antibody (lower panel). The band corresponding to the GFP-Sqh fusion protein is not included. (D) Reduction in the amounts of DMBS proteins in the DMBS mutant females. Protein blots were probed with either an anti-DMBS-C1 antibody (upper panel) or an anti-Pnut antibody (lower panel). The extracts were made from adult females of the following genotypes; wild type (lane 1), Df(3L)th117/DMBSP2 (lane 2), DMBSE1/DMBSP2 (lane 3), and DMBSP1/DMBSP2 (lane 4). Arrows indicate the positions of the DMBS bands.

 


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  		Fig. 4. Defects in dorsal closure in embryos both maternally and zygotically affected in DMBS mutants and in embryos overexpressing DRhk+. (A,D) Wild type embryos, (B,E) DMBS mutant embryos, and (C,F) embryos overexpressing DRhk+. (A-C) Embryos viewed with dark-field optics. Anterior is to the left and dorsal up. The arrow indicates the dorsal hole. (D-F) Dorsal views of the lethal embryos without dorsal holes. Arrows indicate the dorsal midline. Anterior is to the left.

 


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  		Fig. 5. Distribution of nonmuscle myosin II during dorsal closure. (A-C) Lateral views of embryos before elongation of the lateral epidermis, (D-F) Lateral views of embryos during elongation of the lateral epidermis, and (G-I) Dorsal views of embryos during fusion of the lateral epidermis. Embryos were stained with an anti-myosin heavy chain antibody (A,D,G) or an anti-phospho-MRLC antibody (C,F,I). MRLC was visualized by expression of the GFP-Sqh transgene (B,E,H). Arrows indicate the leading edge of the lateral epidermis in A-F, and the dorsal midline in G-I.

 


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  		Fig. 6. Cellular aberrations of dorsal closure in DMBS mutant embryos and in embryos overexpressing DRhk+. The wild-type embryos (A,D,G,I), DMBS mutant embryos (B,E,H,J) and embryos overexpressing DRhk+ (C,F) were stained with an anti-phospho-tyrosine antibody (A-C), phalloidin (D-F), an anti-myosin heavy-chain antibody (G,H), and an anti-phospho-MRLC antibody (I,J). The DMBS mutant embryos were obtained from a mating between DMBSP2/Df(3L)th117 females and DMBSE1/TM3, P[ry+t7.2=HZ2.7]DB2 males, and the mutant embryos were identified by use of the blue balancer. DRhk+ was driven by 69B-GAL4. The aberrantly distributed phospho-MRLC is indicated with arrowheads in J. Polygonally shaped leading edge cells (B,C) and the leading edge cells with aberrant accumulation of F-actin (E,F) are boxed.

 


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  		Fig. 7. Lethality in the DMBS mutant embryos (A) and in the embryos overexpressing DRhk+ (B), and the suppression of lethality by zipEbr. Embryonic lethality in the crosses indicated on the left. The maternal (left) and paternal (right) genotypes are given.

 


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  		Fig. 8. DMBS genetically interacts with zip and with mutations in the components of the Rho signaling pathway. A partial defect of zip causes the malformed adult wing phenotype with a gradation in severity. (A) A wild type wing. (B,C) Wings of the zipEbr/zip02957 transheterozygotes. (D) Percentages of malformed wings in the flies with genotypes indicated on the left side of the panel. Heterozygosity for DMBSE1 significantly suppressed wing defects. (E) A model for the pathways regulating nonmuscle myosin II activation. See the text for the detail.

 

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