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doi: 10.1242/10.1242/dev.00298

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Roles of myosin phosphatase during Drosophila development

¹ Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA
² Howard Hughes Medical Institute, 200 Longwood Avenue, Boston, MA 02115, USA

View larger version (20K): [in a new window]   Fig. 1. The Drosophila MYPT gene. (A) Genomic organization of DMYPT. Exons are shown in purple and introns in blue. The start and stop codons of DMYPT, as well as the location of the two P-insertions, DMYPT03802 and DMYPT3727, are indicated. The sequence of DMYPT cDNA differs from the annotation of CG5891 in several places. Compared to the original annotation, we find that DMYPT has: (1) a three base pair (bp) deletion at the beginning of exon 5 (numbered after the annotation); (2) no exon 6; (3) a 48 bp insertion after exon 10; (4) a 24 bp deletion at the end of exon 15; and (5) four additional exons at the 3' end. At the amino acid level, DMYPT (GenBank accession number AF500094) is similar to CG5891 over the first 337 residues and is unrelated thereafter. (B) BLAST alignment of DMYPT with three human MYPT isoforms: 2/a, 2/b, and 1. Homologous regions are shown in red with percentages of amino acid identity and similarity indicated. DMYPT contains four ankyrin repeats highlighted in green at the N terminus of the protein. The overall homology is higher between DMYPT and HMYPT2 than HMYPT1. (C) Amino acid sequence around the inhibitory phosphorylation site threonine (asterisk in B and C). (D) A schematic drawing of the chromosome arm and deficiencies around the DMYPT locus. Deficiencies that fail to complement with DMYPT are shown in red and those that do complement are shown in green. Regions deleted in the deficiencies are marked with dashed line. The uncertain breakpoints are shown in blue.

View larger version (113K): [in a new window]   Fig. 2. Dorsal closure defects associated with DMYPT mutations. Cuticles of (A) wild-type embryo, (B,C) DMYPT03802 homozygous embryos with different sized dorsal holes; (D) homozygous Df(3L)th102 embryo, (E) Df(3L)th102/DMYPT03802 embryo, and (F) homozygous DMYPT7-51 embryo. DMYPT7-51 is one of the imprecise excision derivatives of DMYPT3727. Anterior is to the left and dorsal is up.

View larger version (161K): [in a new window]   Fig. 3. Cell shape is mildly perturbed in DMYPT mutant embryos. (A) Phosphotyrosine immunostaining of the ectoderm and amnioserosa of wild-type (wt) and DMYPT mutant (DMYPT2-199) embryos at an early stage of dorsal closure (top panels) and midway through dorsal closure (bottom panels). The cell shape, organization and polarity of the mutants is comparable to wild type. (B) Immunostaining for the ectodermal marker, fasciclin III, reveals orderly cell elongation at the leading edge of wild-type embryonic epidermis (arrowhead). DMYPT mutant embryos (DMYPT2-199, DMYPT03802) show occasional disruption of cell shape and elongation toward the end of dorsal closure (lower left panel, arrowheads). All panels are lateral views of embryos with anterior to the left.

View larger version (117K): [in a new window]   Fig. 4. DMYPT is required for oogenesis. (A,C) Morphology of eggs derived from WT (OreR) (A) and from DMYPT03802 homozygous GLCs (C) (dorsal view, anterior is up). Eggs derived from DMYPT03802 GLCs are about 25% the size of wild-type ones. (B,D) Stage 13-14 egg chambers of WT (B) and DMYPT03802 GLCs (D) (lateral view, anterior is up-left). Loss of DMYPT activity blocks the dumping of the nurse cell cytoplasm into the oocyte. Arrows indicate the dorsal appendages of stage 13/14 eggs.

View larger version (153K): [in a new window]   Fig. 5. DMYPT is required for ring canal growth. Filamentous actin staining was used to reveal ring canals of egg chambers. (A,B) Stage 8 egg chambers from (A) wild type (OreR) and (B) DMYPT03802 GLCs. Notice that the ring canals (arrows) in wild-type egg chamber are much larger than those in DMYPT03802 GLCs. The insets are higher magnification views of regions of stage 10 egg chambers. (C) Egg chambers of wild type (stages 3, 5, 7 and 9). (D) Egg chambers of DMYPT03802 GLCs (stages 3, 8 and 10). Note that in wild type, the ring canals grow as the egg chambers progress through oogenesis. This does not occur in DMYPT03802 GLCs.

View larger version (74K): [in a new window]   Fig. 6. Effects of DMYPT mutation on protein localization. Stage 10 egg chambers from (left panels) wild-type flies, and (right panels) DMYPT03802 GLCs. Immunolocalization of Kelch (A), Hts (B), Zip (C) and phosphotyrosine (D). Loss of DMYPT activity has little effect on the overall distribution of Kelch, Hts and Zip. Note the ectopic accumulation of phosphotyrosine-containing proteins in DMYPT03802 GLCs (arrows in D).

View larger version (112K): [in a new window]   Fig. 7. DMYPT is a negative regulator of Rho/myosin II signaling in vivo. Heterozygosities for loss-of-function mutations in RhoA (D), Drok (E), zip (F), and the non-phosphorylatable sqh mutation (H), suppress the rough eye phenotype caused by GMR-Rac (B). In contrast, loss-of-function mutations in DMYPT (C) and ck (G) enhance the GMR-Rac phenotype. (A) OreR (wild type, +), (B) GMR-Rac7A/+, (C) GMR-Rac7A/DMYPT03802, (D) RhoA720/+;GMR-Rac7A/+, (E) Drok2/+;GMR-Rac7A/+, (F) zip1/+;GMR-Rac7A/+, (G) ckP13/+;GMR-Rac7A/+ and (H) sqh[A20A21]/+;GMR-Rac7A/+.

View larger version (193K): [in a new window]   Fig. 8. Genetic interactions in the eye. Scanning electron micrographs of (A) OreR (wild type, +), (B) GMR-RhoA/+, (C) GMR-RhoA/+; DMYPT03802/+, (D) GMR-Rac7A/+, (E) sqh[E20E21]/+;GMR-Rac7A/+, and (F) sqh[A20A21]/+;GMR-Rac7A/+. Heterozygosity for DMYPT03802 inhibits the formation of bristles in GMR-RhoA eyes, but has little effect on the overall eye size. In addition, the phospho-mimicking sqh mutation enhances, while the non-phosphorylatable sqh mutation suppresses, the rough eye phenotype associated with GMR-Rac.