Signaling Downstream of Rho

Drosophila Rho-associated kinase (Drok), an effector of RhoA, is essential for transducing signals to the actin cytoskeleton in wing cells (Winter, 2001). Since the effector domain mutant analysis of RhoA suggests that a cytoskeletal pathway is important for axon retraction, tests were performed to see if the Drok pathway is involved. Carboxy-terminal truncation of mammalian Rho-kinase/ROCK results in its constitutive activation. Expression of an analogous activated form (Drok-CAT; Winter, 2001) in MB neurons led to truncated dorsal lobes similar to the phenotypes of p190 RNAi and weak RhoA activation. A presumptive kinase-dead point mutation (Drok-CAT.KG; Winter, 2001) has no effect, indicating that Drok signaling is dependent on its kinase activity. Developmental studies indicate that the Drok-CAT phenotypes also results from axon retraction, as does the p190 RNAi phenotype (Billuart, 2001).

Neuroblast clones homozygous for Drok2 (Winter, 2001) do not show apparent defects in cell proliferation, because the adult clones contain dorsal axon lobes contributed by later born neurons. Close examination of Drok2 neuroblast clones reveals that 10 of 17 contain at least one axon that extends significantly further than the heterozygous neurons within the same MB. Although this phenotype is subtle, it is not seen in 19 control clones, the parental chromosome for the Drok2 mutant, nor in many other genotypes studied. Thus, it is concluded that Drok is required to limit dorsal axon extension (Billuart, 2001).

Biochemical and genetic evidence indicates that a key output for Drok signaling in vivo is the regulation of phosphorylation of myosin regulatory light chain (MRLC) encoded by spaghetti squash (sqh) (Winter, 2001). To test if endogenous MRLC is part of the axon retraction pathway regulated by p190, genetic interaction experiments were performed by reducing the dose of endogenous sqh in the context of the p190 dsRNA expression. Marked suppression of the phenotype was observed in flies heterozygous for a null mutation of sqh (sqhAX3). In contrast, expression of a phosphomimetic mutant, Sqh-E20E21, markedly enhanced the p190 phenotype, whereas analogous expression of a nonphosphorylable form (Sqh-A21) had no effect. Further, truncation of the medial lobe was frequently observed when Sqh-E20E21 was expressed with the intermediate p190 RNAi line. This is evident from the FasII staining, showing that the medial ß axons (strongly FasII positive) only extend a fraction of the length of the medial lobe. This phenotype was only observed in the strongest p190 RNAi lines, never in the intermediate line alone. Taken together, these results strongly suggest that Drok and phosphorylation of Drosophila MRLC participate in mediating axon retraction as a result of p190 inactivation (Billuart, 2001).

Signaling Upstream of Rho

Since rat p190 can rescue the Drosophila RhoGAP (p190) loss-of-function phenotypes, tests were performed to see if upstream regulators of mammalian p190 could interact with Drosophila p190 to regulate MB axon morphogenesis. The Src family of tyrosine kinases phosphorylate mammalian p190. Tests were performed to see if the Drosophila Src homolog, Src64, regulates p190 activity. Heterozygosity for two Src64 alleles significantly suppresses the p190 RNAi phenotype, with the strength of suppression correlating with the strength of the alleles used. This result is consistent with the notion that Src64 negatively regulates p190 (Billuart, 2001).

Tests were performed to see if integrin could regulate p190 activity in MB neurons, since mammalian studies suggest potential links between integrin and p190, as well as integrin and Src. Integrins function as heterodimers of one alpha and one ß subunit. Drosophila has five genes for integrin alpha subunits, including one, volado (vol), also called scab, which is preferentially expressed in MB neurons and, when mutated, results in a short-term memory defect. No significant modification of the p190 RNAi phenotype was observed in flies heterozygous for a null mutation of vol (vol4). This may be because vol is not dosage sensitive or because it functions redundantly with other integrin alpha subunits in regulating p190 activity. Only two genes encode integrin ß subunits in the fly, ßPS and ßnu; ßnu may not associate with an alpha subunit. The myospheroid (mys) gene, encoding ßPS, shows robust genetic interaction with p190. In flies with one wild-type copy of mys, the p190 RNAi phenotype is markedly suppressed for each of the three mys alleles tested, suggesting that p190 is negatively regulated by integrin (Billuart, 2001).

Finally, MB neuroblast clones homozygous for mys1 were examined using the MARCM system. Twelve of 52 neuroblast clones exhibited obvious dorsal lobe axon overextension not seen in control flies. These include overextension of thin axon bundles near the tip of dorsal lobe similar to those seen in Drok2 clones, or overextension of a large portion of the dorsal axons similar to those seen in MB neurons overexpressing p190. This experiment indicates that integrins are essential negative regulators of axon extension in MB neurons (Billuart, 2001).


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RhoGAP: Biological Overview | Evolutionary Homologs | Regulation

date revised: 25 February 2009

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