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Overlapping roles of Drosophila Drak and Rok kinases in epithelial tissue morphogenesis.

Neubueser D, Hipfner DR - Mol. Biol. Cell (2010)

Bottom Line: Drak activity is largely redundant with that of the Drosophila ROCK orthologue, Rok, such that it is essential only when Rok levels are reduced.The lethality of drak/rok mutants can be rescued by restoring Sqh activity, indicating that Sqh is the critical common effector of these two kinases.These results provide the first evidence that DAPK family kinases regulate actin dynamics in vivo and identify Drak as a novel component of the signaling networks that shape epithelial tissues.

View Article: PubMed Central - PubMed

Affiliation: Institut de recherches cliniques de Montréal, Montreal, QC, Canada.

ABSTRACT
Dynamic regulation of cytoskeletal contractility through phosphorylation of the nonmuscle Myosin-II regulatory light chain (MRLC) provides an essential source of tension for shaping epithelial tissues. Rho GTPase and its effector kinase ROCK have been implicated in regulating MRLC phosphorylation in vivo, but evidence suggests that other mechanisms must be involved. Here, we report the identification of a single Drosophila homologue of the Death-associated protein kinase (DAPK) family, called Drak, as a regulator of MRLC phosphorylation. Based on analysis of mutants, we find that Drak broadly promotes proper morphogenesis of epithelial tissues during development. Drak activity is largely redundant with that of the Drosophila ROCK orthologue, Rok, such that it is essential only when Rok levels are reduced. We demonstrate that these two kinases synergistically promote phosphorylation of Spaghetti squash (Sqh), the Drosophila MRLC orthologue, in vivo. The lethality of drak/rok mutants can be rescued by restoring Sqh activity, indicating that Sqh is the critical common effector of these two kinases. These results provide the first evidence that DAPK family kinases regulate actin dynamics in vivo and identify Drak as a novel component of the signaling networks that shape epithelial tissues.

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Drak and Rok have partly redundant functions during development. (A and B) Dorsal views of drakdel,rok2/drakdel (A) and wild-type (B) third instar larvae. The bilaterally symmetrical main dorsal trunks of the tracheal system are indicated by yellow arrowheads. Portions of the dorsal tracheal trunks are frequently absent in drakdel,rok2/drakdel mutants (A, red arrows). (C and D) Wings imaginal discs from wild-type (w1118) (C) and drakdel,rok2/drakKO (D) third instar larvae. Red arrowhead indicates distorted wing pouch. (E–H) Third instar wing imaginal discs from wild-type (E), drakdel/drakKO (F), rok2/+ heterozygous (G), and drakdel,rok2/drakKO (H) animals. Apoptotic cells are stained with an antibody recognizing activated Caspase-3 (green), F-actin is stained with phalloidin (red), and Armadillo is stained blue. (I–L) Anterior regions of wild-type (I), drakdel (J), rok2/Y (K), and drakdel,rok2/Y (L) embryonic cuticles.
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Figure 4: Drak and Rok have partly redundant functions during development. (A and B) Dorsal views of drakdel,rok2/drakdel (A) and wild-type (B) third instar larvae. The bilaterally symmetrical main dorsal trunks of the tracheal system are indicated by yellow arrowheads. Portions of the dorsal tracheal trunks are frequently absent in drakdel,rok2/drakdel mutants (A, red arrows). (C and D) Wings imaginal discs from wild-type (w1118) (C) and drakdel,rok2/drakKO (D) third instar larvae. Red arrowhead indicates distorted wing pouch. (E–H) Third instar wing imaginal discs from wild-type (E), drakdel/drakKO (F), rok2/+ heterozygous (G), and drakdel,rok2/drakKO (H) animals. Apoptotic cells are stained with an antibody recognizing activated Caspase-3 (green), F-actin is stained with phalloidin (red), and Armadillo is stained blue. (I–L) Anterior regions of wild-type (I), drakdel (J), rok2/Y (K), and drakdel,rok2/Y (L) embryonic cuticles.

Mentions: To identify the redundant functions of Drak and Rok, we analyzed drak−/−,rok+/− animals. drak−/−,rok+/− embryos hatched to form grossly normal looking larvae. However, at later time points it became apparent that more than half of the larvae were missing random segments of the tracheal tree (Figure 4A). These defects were not observed in wild-type, rok+/−, or drak mutant larvae (Figure 4B and data not shown). The tracheal tree is formed by epithelial cells which undergo a series of changes in cell shape, migrations, and rearrangements (Cabernard et al., 2004). The tracheal defects suggested that drak and rok may be redundantly required for one or more of these processes.


Overlapping roles of Drosophila Drak and Rok kinases in epithelial tissue morphogenesis.

Neubueser D, Hipfner DR - Mol. Biol. Cell (2010)

Drak and Rok have partly redundant functions during development. (A and B) Dorsal views of drakdel,rok2/drakdel (A) and wild-type (B) third instar larvae. The bilaterally symmetrical main dorsal trunks of the tracheal system are indicated by yellow arrowheads. Portions of the dorsal tracheal trunks are frequently absent in drakdel,rok2/drakdel mutants (A, red arrows). (C and D) Wings imaginal discs from wild-type (w1118) (C) and drakdel,rok2/drakKO (D) third instar larvae. Red arrowhead indicates distorted wing pouch. (E–H) Third instar wing imaginal discs from wild-type (E), drakdel/drakKO (F), rok2/+ heterozygous (G), and drakdel,rok2/drakKO (H) animals. Apoptotic cells are stained with an antibody recognizing activated Caspase-3 (green), F-actin is stained with phalloidin (red), and Armadillo is stained blue. (I–L) Anterior regions of wild-type (I), drakdel (J), rok2/Y (K), and drakdel,rok2/Y (L) embryonic cuticles.
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Related In: Results  -  Collection

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Figure 4: Drak and Rok have partly redundant functions during development. (A and B) Dorsal views of drakdel,rok2/drakdel (A) and wild-type (B) third instar larvae. The bilaterally symmetrical main dorsal trunks of the tracheal system are indicated by yellow arrowheads. Portions of the dorsal tracheal trunks are frequently absent in drakdel,rok2/drakdel mutants (A, red arrows). (C and D) Wings imaginal discs from wild-type (w1118) (C) and drakdel,rok2/drakKO (D) third instar larvae. Red arrowhead indicates distorted wing pouch. (E–H) Third instar wing imaginal discs from wild-type (E), drakdel/drakKO (F), rok2/+ heterozygous (G), and drakdel,rok2/drakKO (H) animals. Apoptotic cells are stained with an antibody recognizing activated Caspase-3 (green), F-actin is stained with phalloidin (red), and Armadillo is stained blue. (I–L) Anterior regions of wild-type (I), drakdel (J), rok2/Y (K), and drakdel,rok2/Y (L) embryonic cuticles.
Mentions: To identify the redundant functions of Drak and Rok, we analyzed drak−/−,rok+/− animals. drak−/−,rok+/− embryos hatched to form grossly normal looking larvae. However, at later time points it became apparent that more than half of the larvae were missing random segments of the tracheal tree (Figure 4A). These defects were not observed in wild-type, rok+/−, or drak mutant larvae (Figure 4B and data not shown). The tracheal tree is formed by epithelial cells which undergo a series of changes in cell shape, migrations, and rearrangements (Cabernard et al., 2004). The tracheal defects suggested that drak and rok may be redundantly required for one or more of these processes.

Bottom Line: Drak activity is largely redundant with that of the Drosophila ROCK orthologue, Rok, such that it is essential only when Rok levels are reduced.The lethality of drak/rok mutants can be rescued by restoring Sqh activity, indicating that Sqh is the critical common effector of these two kinases.These results provide the first evidence that DAPK family kinases regulate actin dynamics in vivo and identify Drak as a novel component of the signaling networks that shape epithelial tissues.

View Article: PubMed Central - PubMed

Affiliation: Institut de recherches cliniques de Montréal, Montreal, QC, Canada.

ABSTRACT
Dynamic regulation of cytoskeletal contractility through phosphorylation of the nonmuscle Myosin-II regulatory light chain (MRLC) provides an essential source of tension for shaping epithelial tissues. Rho GTPase and its effector kinase ROCK have been implicated in regulating MRLC phosphorylation in vivo, but evidence suggests that other mechanisms must be involved. Here, we report the identification of a single Drosophila homologue of the Death-associated protein kinase (DAPK) family, called Drak, as a regulator of MRLC phosphorylation. Based on analysis of mutants, we find that Drak broadly promotes proper morphogenesis of epithelial tissues during development. Drak activity is largely redundant with that of the Drosophila ROCK orthologue, Rok, such that it is essential only when Rok levels are reduced. We demonstrate that these two kinases synergistically promote phosphorylation of Spaghetti squash (Sqh), the Drosophila MRLC orthologue, in vivo. The lethality of drak/rok mutants can be rescued by restoring Sqh activity, indicating that Sqh is the critical common effector of these two kinases. These results provide the first evidence that DAPK family kinases regulate actin dynamics in vivo and identify Drak as a novel component of the signaling networks that shape epithelial tissues.

Show MeSH
Related in: MedlinePlus