Genetic Evidence for Antagonism Between Pak Protein Kinase and Rho1 Small GTPase Signaling in Regulation of the Actin Cytoskeleton During Drosophila Oogenesis

Abstract

During Drosophila oogenesis, basally localized F-actin bundles in the follicle cells covering the egg chamber drive its elongation along the anterior-posterior axis. The basal F-actin of the follicle cell is an attractive system for the genetic analysis of the regulation of the actin cytoskeleton, and results obtained in this system are likely to be broadly applicable in understanding tissue remodeling. Mutations in a number of genes, including that encoding the p21-activated kinase Pak, have been shown to disrupt organization of the basal F-actin and in turn affect egg chamber elongation. pak mutant egg chambers have disorganized F-actin distribution and remain spherical due to a failure to elongate. In a genetic screen to identify modifiers of the pak rounded egg chamber phenotype several second chromosome deficiencies were identified as suppressors. One suppressing deficiency removes the rho1 locus, and we determined using several rho1 alleles that removal of a single copy of rho1 can suppress the pak phenotype. Reduction of any component of the Rho1-activated actomyosin contractility pathway suppresses pak oogenesis defects, suggesting that Pak counteracts Rho1 signaling. There is ectopic myosin light chain phosphorylation in pak mutant follicle cell clones in elongating egg chambers, probably due at least in part to mislocalization of RhoGEF2, an activator of the Rho1 pathway. In early egg chambers, pak mutant follicle cells have reduced levels of myosin phosphorylation and we conclude that Pak both promotes and restricts myosin light chain phosphorylation in a temporally distinct manner during oogenesis.

A Pak-regulated cell intercalation event leading to a novel radial cell polarity is involved in positioning of the follicle stem cell niche in theDrosophilaovary

“…A requirement for Pak in basal stalk cell intercalation is consistent with its established roles in regulating the actin cytoskeleton during cell shape change and motility (Bokoch, 2003). We have shown that Pak regulates actomyosin contractility during development of the follicular epithelium, and it is possible that misregulation of contractility underlies the Pak basal stalk phenotype (Vlachos and Harden, 2011). We assessed the status of actomyosin contractility in basal stalks by staining for phosphorylated myosin light chain ( pMLC), which reveals active myosin.…”

“…Normally, this cannot be addressed owing to the degradation of all Pak chambers at around stage 10 (Conder et al, 2007); however, as we previously demonstrated, heterozygosity for any component of the Rhoactivated actomyosin contractility pathway suppresses this degradation (Vlachos and Harden, 2011). Heterozygosity for an allele of Rho1 enabled a small number of Pak mutant paired-egg chambers to develop into fused mature eggs, complete with dorsal appendages (Fig.…”

“…The group I p21-activated kinases (Paks) are important regulators of the F-actin cytoskeleton, and we have been studying the function of Pak in development of the follicular epithelium in the Drosophila ovary, where Pak can affect cell shape through the regulation of actomyosin contractility (Conder et al, 2007;Vlachos and Harden, 2011). Here, we characterize a unique phenotype in Pak mutant ovarioles bearing age-matched, side-by-side fused egg chambers.…”

Drosophila egg chamber elongation

“…Genetic screens, on the other hand, could be used to either identify genes, which when mutated in follicle cells, lead to round eggs, or genes, which when mutated in combination with a previously identified round egg gene, lead to the suppression or enhancement of the round egg mutant phenotype. Both of these genetic screen approaches have been recently used to identify additional proteins that contribute to egg chamber elongation, although detailed characterization of the roles of most of these genes has not yet been performed 44 , 73 . Even with the completion of these recent screens, it is unlikely that all of the genes that contribute to egg chamber elongation have been identified.…”

“…Since these fibers are anchored at either end by focal adhesions that connect them to the ECM, their contraction results in the generation of traction forces which help pull the cell forward 79 . In follicle cells, the activated form of non-muscle Myosin II is present at the basal surface starting at stage 7 44 , 80 . This suggests that contraction of the basal actin filaments may be involved in generating the force necessary to mediate follicle cell migration during egg chamber rotation.…”