Mammalian LIM-kinases (LIMKs) phosphorylate cofilin and induce actin cytoskeletal reorganization. To elucidate the functional roles of LIMKs in vivo during developmental processes, the cDNA encoding a Drosophila homolog of LIMK (DLIMK) was isolated and two isoforms of DLIMK transcripts were identified coding for proteins with 1235 and 1257 amino acids, possessing the structure composed of two LIM domains, a PDZ domain, a protein kinase domain, and an unusual long C-terminal extension. In situ hybridization analysis in Drosophila embryos detected the uniformly distributed DLIMK mRNA in stages 2 to 5. In vitro kinase reaction revealed that DLIMK efficiently phosphorylates Drosophila cofilin (Twinstar) specifically at Ser-3, the site responsible for inactivation of its actin-depolymerizing activity. When expressed in cultured cells, wild-type DLIMK, but not its kinase-inactive form, induces changes in actin cytoskeletal organization. These observations suggest that the LIMK-cofilin signaling pathway for regulating actin filament dynamics is evolutionarily conserved between Drosophila and mammals (Ohashi, 2000).
The ADF (actin-depolymerizing factor)/cofilin family, represented by Twinstar in Drosophila is a stimulus-responsive mediator of actin dynamics. In contrast to the mechanisms of inactivation of ADF/cofilin by kinases such as LIM-kinase 1 (LIMK1: see Drosophila LIM-kinase1), much less is known about its reactivation through dephosphorylation. Slingshot (Ssh), a family of phosphatases that have the property of F actin binding, is described. In Drosophila, loss of ssh function dramatically increases levels of both F actin and phospho-cofilin (P cofilin) and disorganizes epidermal cell morphogenesis. In mammalian cells, human Ssh homologs (hSSHs) suppress LIMK1-induced actin reorganization. Furthermore, Ssh and the hSSHs dephosphorylate P cofilin in cultured cells and in cell-free assays. These results strongly suggest that the SSH family plays a pivotal role in actin dynamics by reactivating ADF/cofilin in vivo (Niwa, 2002).
Regulation of the dynamics of the actin cytoskeleton is fundamental in the construction and remodeling of a variety of polarized subcellular structures. The reorganization of the actin cytoskeleton is controlled at multiple levels. When focused on elongation of actin filaments, profilin (chickadee in Drosophila) promotes the elongation at barbed ends, and capping protein (CP: capping protein beta in Drosophila) stabilizes the barbed ends; a member of the ADF (actin-depolymerizing factor)/cofilin family accelerates depolymerization at pointed ends and severs long actin filaments (Niwa, 2002 and references therein).
Among actin binding proteins, ADF/cofilin is the most-characterized stimulus-responsive mediator of actin dynamics. In response to insulin and lysophosphatidic acid, LIM-kinases (LIMKs), activated by effectors of Rho family GTPases, phosphorylate ADF/cofilin specifically at Ser-3, and thereby inhibit filament-severing and monomer binding activities of ADF/cofilin proteins. Testicular protein kinases (TESKs) also phosphorylate Ser-3 of ADF/cofilin and inhibit its activity, although upstream pathways of TESKs activation appear to be separate from that of LIMKs. ADF/cofilin undergoes rapid dephosphorylation as well, in response to several extracellular stimuli, which could result from downregulation of the kinases, upregulation of a phosphatase(s), or both. In contrast to characterization of the kinases, however, much less is known about the phosphatases that reactivate ADF/cofilin (Niwa, 2002 and references therein).
Actin dynamics in vivo have been studied genetically in several systems, including Drosophila. Bristles and hairs on appendages are actin-based protrusions exposed on body surfaces; therefore, they are easily scored landmarks for isolating mutations that cause malformations. In fact, loci reported to be involved in such malformations include chickadee (chic) encoding Profilin, capping protein beta (cpb) encoding the subunit of CP, and twinstar (tsr) encoding ADF/cofilin. This study reports another locus, which has been named slingshot (ssh) after the bifurcation phenotypes of the bristles and hairs in these mutants. The ssh gene encodes a phosphatase that is conserved among several animal species. Loss of ssh function dramatically increases the level of actin filaments, which is similar to a phenotype of tsr mutant cells (Baum, 2000; Baum, 2001). In cultured mammalian cells, such a strong enhancement of actin polymerization is induced by overproduction of LIMKs or TESKs (Niwa, 2002 and references therein).
One hypothesis would be that Ssh and these kinases share the same substrate, ADF/cofilin, regulate its phosphorylation level, and consequently control its actin-depolymerizing activity. To pursue this possibility, multiple approaches were undertaken. Evidence is presented that loss of ssh function in Drosophila increases the level of phospho-cofilin (P cofilin) and that ssh genetically interacts with the Drosophila LIMK gene in actin-based cell morphogenesis. In cultured cells, expression of either of two human Ssh homologs (hSSHs) with LIMK1 or TESK1 suppresses actin reorganization induced by these kinases; and Ssh or hSSHs expression reduces the level of P cofilin. Finally, Ssh and the hSSHs dephosphorylate P cofilin as judged from the results of cell-free assays. These results suggest that the SSH family plays a critical role in controlling actin dynamics, presumably through dephosphorylating and thus reactivating cofilin in cellular and developmental contexts (Niwa, 2002).
In the course of genetic screening for loci that affect bristle number and/or morphology, a focus was placed on l(3)01207, which has one copy of PZ inserted into CG6238. Lethality of l(3)01207 was due to loss of CG6238 function, as shown by the facts that the lethality was recovered by remobilization of the P element and by CG6238 cDNA expression using a widely expressed GAL4 driver, daughterless (da)-GAL4. Through subsequent analysis, it was found that adult escapers of hypomorphic alleles had bifurcated and twisted bristles. This locus was renamed slingshot (ssh) (Niwa, 2002).
To study exactly how actin reorganization is affected by ssh mutations, ssh mutant clones were stained with dye-conjugated phalliodin. Loss of ssh function causes a dramatic increase in the level of F actin in a cell-autonomous manner. This phenotype is found in mutant clones in larval imaginal discs, pupal wings, and follicle epithelia of the egg chamber. Along the apico-basal cell axis, overaccumulation of actin filaments was not necessarily restricted to the apical subcellular region; basolateral positions were also brightly stained. At 30-36 hr after puparium formation (h APF) at 25°C, each epidermal cell in the wing localizes an assembly of actin bundles to its distal-most vertex, producing a single prehair in the wild-type. Prehairs of the mutant cells were much more intensely stained with dye-conjugated phalliodin than those of normal cells, indicating that individual mutant hairs contained more actin filaments. ssh null cells seem to show no obvious defect in proliferation or cell-cell adhesion when compared with cells in wild-type twin spots (Niwa, 2002).
A dramatic increase in the F actin level has also been reported in twinstar (tsr)/cofilin clones (Baum, 2000; Baum, 2001); in cultured mammalian cells, excessive actin polymerization is caused by overproduction of LIMKs or TESKs. Therefore, the possibility was examined that those kinases and the SSH family share the same substrate, ADF/cofilin, and thus control the level of phospho-ADF/cofilin in vivo (Niwa, 2002).
Whether loss of ssh function increases the level of phosphorylated Drosophila cofilin (P Dcofilin) in fly tissues was examined. For this purpose, binding and specificity of anti-P cofilin antibody to P Dcofilin was first characterized. Anti-P cofilin antibody was originally made against a phosphopeptide of mammalian cofilin (Toshima, 2001). When ssh1-11 clones were analyzed by staining or Western blotting with this anti-P cofilin antibody, the endogenous P Dcofilin level in ssh/ssh mutant cells was significantly elevated compared with that in the surrounding ssh/+ and +/+ wild-type cells, whereas the total amount of cofilin was not altered. This result is consistent with the hypothesis that a physiological expression level of Ssh is required for dephosphorylating P Dcofilin in vivo (Niwa, 2002).
Loss of ssh function and expression of Drosophila LIMK (DLIMK) exerts synergistic effects on wing hair morphogenesis and on the levels of F actin and P Dcofilin. Double-stranded RNA (dsRNA) of ssh sequences was expressed in dorsal cells of the wing. This phenocopied a hypomorphic ssh condition through a phenomenon called RNA interference (RNAi). DLIMK production on its own, whether the wild-type form (wt) or a kinase-inactive form (KI), does not give detectable phenotypes under the experimental conditions employed. The weak RNAi phenotype of ssh is strongly enhanced by expression of DLIMK-wt, but DLIMK-KI does not show this enhancement. Similarly, drastic increases in F actin and P Dcofilin levels result from coexpression of the dsRNA and DLIMK-wt. In those cells, unusually thick actin bundles are observed when images are collected with a low gain. Cells on ventral surfaces are not affected, where the transgene was silent (Niwa, 2002).
In addition to experiments using Drosophila, the approach of transfecting mammalian cell lines with hSSH plasmids was taken to test the following hypothesis: if hSSH dephosphorylates P cofilin, coexpression of hSSH together with LIMKs or TESKs is expected to counterbalance the promotion effect of those kinases on actin polymerization. To evaluate the degree of actin polymerization, F actin patterns were classified into three categories. In control cells expressing GFP alone, strong F actin signals were seen infrequently and most of the cells were scored as class 3, whereas LIMK1 or TESK1 expression made class 1 (strongest F actin signals) and class 2 (intermediate signal) predominant. Coexpression of hSSH-1L(wt) or hSSH-2(wt) with either kinase restored the cells to class 3 category; the CS phosphatase mutant forms, however, failed to counteract the kinases. These results support that the hSSH phosphatase activity inhibits LIMK1- or TESK1-dependent actin reorganization (Niwa, 2002).
The above coexpression results could be explained by the hypothesis that P cofilin is a direct target of the hSSHs; however, another interpretation is also possible. The kinase activity of LIMK1 is enhanced by phosphorylation at Thr-508 (Ohashi, 2000); therefore, the hSSHs might dephosphorylate LIMK1 and make it less active. This second hypothesis is less likely; a phosphomimetic form of LIMK1, LIMK1(T508EE), which is no longer phosphorylated at the residue of 508, induces actin polymerization. Nevertheless, hSSH-1L is still able to counteract LIMK1(T508EE) since it is LIMK1(wt) (Niwa, 2002).
Coexpression of hSSH-1L(CS) with LIMK1 evokes robust assembly of actin filaments in a way that is hardly induced by LIMK1 expression alone. When compared to F actin patterns in LIMK1-expressing cells, it looked as if almost all actin filaments in the coexpressing cell are assembled to build a limited number of thick bundles. These F-actin patterns were counted as class 2. These unusual structures were also induced by expressing hSSH-1L(CS) alone, but not at all by expressing the wild-type form. This abnormal assembly of the filaments is reminiscent of the consequence of coexpression of ssh-dsRNA and DLIMK in Drosophila epidermal cells, suggesting the possibility that hSSH-1L(CS) overexpression exerts a dominant-negative effect. hSSH-2(CS) also tends to induce actin reorganization, as shown by a slight expansion of the class 2 population when compared with the effect of its wild-type expression; however, unlike hSSH-1L(CS), hSSH-2(CS) can not generate the thick actin bundles (Niwa, 2002).
A straightforward prediction of the above results would be that expression of hSSH-1L(wt), hSSH-2(wt), or Ssh(wt) reduces the P ADF/cofilin level in cells. This was indeed found to be the case. Expression of hSSH-1L, hSSH-2, or Ssh causes a prominent decrease in the level of P cofilin or P Dcofilin; in contrast, hSSH-3(wt) expression is ineffective. Neither of the two CS forms nor MKP-5, which shows a limited sequence similarity to the catalytic domain of the SSH family, is able to decrease the P cofilin level. Complementary experiments have indicated that hSSH-1L is unable to dephosphorylate p38alpha and JNK2, which are efficiently inactivated by MKP-5. These results suggest that the SSH family displays distinct substrate specificity from the MKP family (Niwa, 2002).
Whether hSSH-1L, hSSH-2, and/or Ssh dephosphorylate P cofilin was directly assayed in cell-free systems. The hSSHs and cofilin were expressed in COS-7 cells; Ssh and Dcofilin were expressed in S2 cells. The immunoprecipitated enzymes and the purified substrates, which contain phosphorylated forms, were subject to in vitro reactions. Each wild-type form of the hSSHs or Ssh dephosphorylated P cofilin, but the CS forms do not, supporting the notion that cofilin is a substrate of hSSH-1L, hSSH-2, and Ssh. In the cell-free assay, 90% of P cofilin was dephosphorylated by Ssh after 15 min reaction (Niwa, 2002).
Under the experimental conditions used, hSSH-2 was reproducibly less active than hSSH-1L. When comparable amounts of hSSH-1L(wt) and hSSH-2(wt) were reacted with the same amount of P cofilin, over 99% of the substrate was dephosphorylated by hSSH-1L(wt), whereas only 60% was by hSSH-2(wt). ADF (also called destrin) is a close relative of cofilin in the vertebrate ADF/cofilin family and is also phosphorylated at Ser-3 by LIMKs and TESKs; hSSH-1L dephosphorylates phospho-ADF as well (Niwa, 2002).
In a transfection experiment, it was found that hSSH-1L(CS) predominantly colocalizes with F actin. This result prompted an investigation to see whether hSSHs and Ssh have the property of binding F actin. Tagged hSSHs and Ssh were incubated with actin filaments and centrifuged to examine whether each of them cosediment with F actin or not. Both wild-type and CS forms of hSSH-1L and Ssh preferentially cosedimented with actin filaments, whereas hSSH-2(wt), hSSH-2(CS), and hSSH-3(wt) exhibit weaker binding capabilities. A fraction of hSSH-3(CS) precipitates in the absence of actin filaments; thus, whether hSSH-3(CS) binds F actin or not was difficult to address with this method. Consistent with the result of the cosedimentation assay, Ssh(wt) and Ssh(CS) appear to colocalize with cortical actin in wing epithelia at 30 h APF (Niwa, 2002).
These studies using the whole animal and cell lines have demonstrated that Ssh and two human homologs (hSSH-1L and -2) prevent excessive actin polymerization and these SSH family members reduce the P cofilin level in cells. These studies have also shown in cell-free assays that Ssh and the hSSHs dephosphorylate cofilin efficiently and bind to actin filaments. All of these results are consistent with the hypothesis that Ssh and the hSSHs control reorganization of actin cytoskeleton by reactivating cofilin. Both the cofilin-phosphatase activity and the F actin binding ability of hSSH-2 are weaker than those of hSSH-1L under the experimental conditions used. Extensive domain-swapping between hSSH-1L and -2 may reveal whether the two activities are related to one another (Niwa, 2002).
In genomes of S. cerevisiae, C. elegans, and Arabidopsis, appear to have no ortholog of genes encoding LIMK, TESK, and Ssh, although cofilin in all of these species has a Ser-3 or equivalent serine residue. This is suggestive of a model in which the actin-depolymerizing activity of cofilin in those species may be regulated in vivo by a set of enzymes structurally distinct from LIMK, TESK, and Ssh. Alternatively, the activity may not be regulated by serine phosphorylation, but by other means such as PIP2 binding or intracellular pH changes. The latter possibility is supported in S. cerevisiae and D. discoideum by the report that P cofilin is not detected in vegetatively growing cells (Niwa, 2002).
The analysis of ssh mutants shows that a critical role of Ssh is in the building of wing hair, bristle, and arista, presumably through reactivating P cofilin. These cellular extensions in insects share a number of structural features with those found throughout the animal kingdom, such as the brush border of intestinal epithelial cell. Formation of all those protrusions on apical cell surfaces requires packing of parallel filaments through actin-bundling proteins. Therefore, thickening and/or splitting phenotypes in ssh mutants are most likely due to overaccumulated filaments that were not arranged in a normal array. Ssh does not appear to be required for cell proliferation or viability, which provides a contrast to an effect of loss of a generic enzyme PP1 or PP2A. All these results are consistent with the hypothesis that cofilin is the major substrate of Ssh and that Ssh does not display broad substrate specificity (Niwa, 2002).
It is worth noting that mutations of some other loci cause phenotypes similar to or reminiscent of those of ssh and twinstar (tsr)/cofilin. In act up (acu)/caplet (capt) clones in imaginal discs and follicle cells, the level of actin filaments is elevated as in ssh clones, and bristle malformation occurs. acu/capt encodes cyclase-associated protein (CAP), and CAP limits filament formation catalyzed by Enabled at apical cell junctions. The splitting or branching of wing hair, bristle, and arista is caused by loss of tricornered (trc) or furry (fry). The Trc protein is an evolutionally conserved kinase; Fry is also conserved among species, but its biochemical function is unknown. It would be intriguing to explore whether trc or fry phenotypes are associated with altered levels of actin filaments and P cofilin (Niwa, 2002).
The current studies did not focus on pursuing whether or not Ssh regulates cofilin functions in cell behaviors other than epithelial cell morphogenesis. For example, one of the important functions of ADF/cofilin is the dynamic regulation of the cortical ring at the cleavage furrow to complete cytokinesis (Nagaoka, 1995; Abe, 1996). During ana/telophase in tsr mutants, aberrantly large actin-based structures appear at the site of contractile ring formation and fail to disassemble at the end of telophase (Gunsalus, 1995). The apparent dispensability of Ssh in mitosis in the wing could be explained by the possibility that only a small fraction of active cofilin molecules are required for dynamics of the contractile ring and/or that a cofilin-phosphatase(s) other than Ssh is responsible for the progression of cytokinesis (Niwa, 2002).
In response to extracellular stimuli, some cells are known to undergo rapid dephosphorylation of ADF/cofilin (Moon, 1995); all of these stimuli result in cytoskeletal reorganization, possibly by way of reactivation of ADF/cofilin. During semaphorin-3A (Sema-3A)-induced growth cone collapse, a rapid increase and subsequent decrease in P cofilin occurs in growth cones within 5 min after exposure to Sema-3A (Aizawa, 2001). This supports a mechanism of local and transient regulation of phosphorylation-dephosphorylation cycling. In some systems, stimulation of cortical actin dynamics occurs without a net change in the ratio of P to non-P ADF/cofilin. Instead, what may be crucial is the turnover rate of the P ADF/cofilin pools or differential distributions of phosphorylation and dephosphorylation spots within cells (Meberg, 1998; Yang, 1998). The studies reported here raise an obvious question to be addressed, that is, whether the Ssh family is involved in the above stimuli-driven actin reorganizations. It is thus necessary to explore whether the activity of the SSH family is regulated in response to the stimuli, and if so, to determine the components of such regulatory mechanisms (Niwa, 2002).
To examine how cofilin phosphorylation is regulated, genetic studies were performed on the cofilin phosphatase encoded by ssh (Niwa, 2002). Neuroblast clones homozygous for null alleles of ssh (ssh1–63 and ssh1–11) did not have any obvious reduction of cell numbers. Therefore, unlike cofilin, Ssh is not required for cell proliferation. However, all ssh neuroblast clones exhibited axon growth or growth and guidance defects. In "severe" cases (approximately 10%), ssh-/- axons failed to extend beyond the peduncle. Guidance defects were also detected either at the peduncle, where a large accumulation of axons led to the disorganization of the peduncle, or from the dendrite region. Approximately 90% of ssh-/- neuroblast clones exhibited only axon growth defects, which were categorized as "strong" or "weak". Developmental studies indicate that, at least for γ and alpha/ß neurons, these defects are a direct result of axon extension failure (Ng, 2004).
No obvious axon growth defects were detected in ssh-/- single-cell γ clones. This is possibly because axon growth is more sensitive to tsr than to ssh mutations or because of perdurance effects (although homozygous mutant for the ssh mutation, clones may inherit enough wild-type protein or mRNA from heterozygous parental neural precursors to support Ssh function for some time after clone generation). To compare mutant phenotypes of ssh and tsr in axon growth, ssh-/- alpha/ß-only neuroblast clones were examined. The ssh-/- alpha/ß axons display axon growth defects similar to those observed in tsr-/- alpha/ß clones (Ng, 2004).
Given the similarity of phenotypes between ssh and tsr, whether Ssh regulates axon growth through cofilin dephosphorylation was investigated. First whether Ssh phosphatase activity is required for axon growth was tested. Overexpression of UAS-ssh WT or UAS-ssh CS (a point mutation in the phosphatase domain that renders it inactive; Niwa, 2002) in MB neurons did not result in gross axon defects. Expression of UAS-ssh WT, but not UAS-ssh CS, in ssh-/- neuroblast clones rescues the ssh mutant defects, indicating that Ssh phosphatase activity in MB neurons is essential for axon growth (Ng, 2004).
Transgenic suppression experiments were performed by expressing different cofilin transgenes in ssh-/- clones. Strikingly, expression of S3A or wild-type cofilin in ssh-/- clones almost completely suppresses the ssh-/- defects. In contrast, expression of S3E cofilin produces a very weak effect. The suppression of the ssh phenotype by the overexpression of active cofilin indicates that the major function of Ssh in axon growth is to dephosphorylate cofilin (Ng, 2004).
To determine whether regulation of cofilin by Ssh is required for axon growth in other neurons, ssh-/- axon projections of contralateral projecting neurons of the optic lobe (OL) and the antennal lobe (AL) were examined. In both cases, highly penetrant axon growth phenotypes were found in ssh-/- neuroblast clones. These phenotypes were rescued by expressing wild-type, but not phosphatase inactive, Ssh in ssh-/- neuroblast clones, indicating that Ssh regulates axon growth in these neurons cell-autonomously in a phosphatase-dependent manner. Furthermore, expression of active cofilin also suppresses these ssh growth defects. Therefore, cofilin dephosphorylation by Ssh most likely plays a general role in axon growth of many neuronal types (Ng, 2004).
Previous biochemical studies have established cofilin as a direct target of LIM kinase. To determine if Drosophila LIMK1 is the cofilin kinase in MB neurons, LIMK1 was overexpressed in all MB neurons, which resulted in axon growth defects. In 'severe' cases, all axon lobes were truncated. In addition, there were axon guidance defects at the peduncle, where axons formed a ball-like structure. In cases of 'strong' phenotypes, no guidance defects were detected, but both dorsal and medial axons were truncated. In cases of 'weak' phenotypes, axon growth defects were detected only in the dorsal lobes, while the medial lobes appeared normal. The expression level of individual transgenic lines caused the variability in the extent of axon defects. As determined from the epitope tag (HA) staining, stronger expression lines were associated with highly penetrant, severe axon growth and guidance defects, while weaker expression lines were associated with a mixture of severe, strong, weak, and, in some cases, no axon defects. Overexpression of kinase-inactive forms of LIMK1 (UAS-LIMK1 KI) at comparable levels (as determined by anti-HA staining where LIMK1 KI is similarly localized to axons as wild-type LIMK1) resulted in normal axon projections. This indicates that the axon growth defects caused by LIMK1 overexpression depend on its kinase activity (Ng, 2004).
Developmental studies indicate that the LIMK1 overexpression phenotype is similar to the ssh-/- phenotype. For at least γ and alpha/ß neurons, these phenotypes are also a result of axon extension failure. To test whether endogenous LIMK1 plays a role in MB morphogenesis, LIMK1 RNAi was expressed in MB neurons. This results in axon guidance and some axon growth defects. Therefore, LIMK1 is likely to play an endogenous role in MB morphogenesis (Ng, 2004).
The similarities between the ssh-/- and LIMK1 overexpression phenotypes suggest that LIMK1 and Ssh exert their effects by regulating a common substrate, cofilin. To genetically test whether LIMK1 overexpression leads to the inactivation of cofilin through phosphorylation, either Ssh or cofilin were coexpressed in a LIMK1 overexpression background. These experiments focussed on the transgenic line UAS-LIMK1 WT F4, which has an intermediate level of expression, allowing sensitive genetic interactions to be detected. Consistent with the idea that LIMK1 and Ssh regulate the phosphorylation of a common substrate, coexpression of wild-type Ssh fully suppresses the LIMK1 phenotype. In addition, coexpression of either wild-type or S3A cofilin also suppressed the LIMK1 overexpression phenotype. These experiments support the model that axon growth defects caused by LIMK1 overexpression are in large part a result of cofilin inactivation (Ng, 2004).
So far, this work has demonstrated that axon growth requires cofilin and that cofilin activity is regulated positively by Ssh phosphatase and negatively by LIM kinase. How is this cytoskeletal pathway directed by Rho GTPases in vivo? The role of the Rho-Rok pathway in regulating LIMK was examined first. Since MB axon growth is sensitive to the level of LIMK1 activity, genetic interaction studies were performed, either reducing the gene dose of endogenous Rho signaling components by half or overexpressing components of the Rho pathway in a LIMK1 overexpression background. Removing one copy of Rho1 (also called RhoA) strongly suppresses the axon growth phenotype caused by LIMK1 overexpression. Halving the gene dose of the Drosophila Rho effector kinase Rok (also called Drok) also suppresses the LIMK1 overexpression phenotype. However, this effect is much weaker, possibly because the Rok level is not as dose sensitive (Billuart, 2001). In contrast, coexpressing Rok or Rho1 with LIMK1 strongly enhances the axon growth defects, even though overexpressing either Rho1 or Rok alone does not disrupt axon growth. These results suggest that Rho1 and Rok positively regulate LIMK1 activity (Ng, 2004).
The possible involvement of upstream regulators of Rho1 was also tested. Introducing one mutant copy of RhoGEF2 or pebble, two guanine nucleotide exchange factors known to positively regulate Rho1 or overexpression of the Rho1 inhibitor p190 RhoGAP, all resulted in suppressing the LIMK1 overexpression phenotype. Taken together with known biochemical activities of these signaling components, these genetic interaction data suggest that Rho1 activates Rok, which then directly activates LIMK1. In turn, this pathway is likely to be regulated positively by at least two RhoGEFs (RhoGEF2 and Pbl) and negatively by p190 RhoGAP in MB neurons in vivo (Ng, 2004).
While LIMK1 overexpression in MB neurons provides a sensitive assay for genetic interactions, the model was also tested using endogenous components. According to the model, overexpression of LIMK1 results in cofilin hyperphosphorylation, which is phenocopied in ssh mutants. Therefore, modulating the level of Rho1 signaling should also modify the ssh-/- phenotype in a similar manner. Indeed, loss of one copy of either Rho1 or RhoGEF2 also markedly suppresses the ssh-/- defects (Ng, 2004).
Biochemical studies have shown that LIM kinase is activated by Pak, a downstream effector kinase for Rac and Cdc42. Whether Pak activation could affect the LIMK pathway in axon growth was tested. In similar genetic interaction experiments, it was found that introducing three independently generated Drosophila Pak mutant alleles suppresses the LIMK1 overexpression phenotype. In addition, Pak overexpression also results in axon growth and guidance defects similar to those seen with LIMK1 overexpression, consistent with the model in which Pak activates LIMK1, leading to cofilin hyperphosphorylation. As predicted from this model, coexpression of active versions of cofilin with Pak results in a partial suppression of the growth and guidance defects (Ng, 2004).
Next it was determined which Rho GTPase pathway (Cdc42, Rac1/Rac2, or Rho1) regulates Drosophila Pak. It was found that loss of one copy of Cdc42 results in a strong suppression of the Pak overexpression phenotypes. Similar reduction of Rac1 and Rac2 (Rac1J10, Rac2Δ) results in weaker suppression. In contrast, reducing Rho1 results in a slight enhancement of the Pak overexpression phenotype. This suggests that the Pak overexpression phenotype specifically reflects the positive signaling input from Cdc42 and Rac in vivo (Ng, 2004).
Next it was tested whether mutations in the Cdc42 or Rac genes (Rac1, Rac2, Mtl, and the different combinations) would modify the LIMK1 overexpression phenotype. Reducing the Cdc42 activity leads to the suppression of the LIMK1 phenotype, suggesting that Cdc42 acts through Pak to activate LIMK1. Loss of one copy of both Rac2 and Mtl does not alter the LIMK1 phenotype. However, the LIMK1 phenotype is very sensitive to the levels of endogenous Rac1, since introducing one copy of a strong hypomorphic allele of Rac1 (Rac1J10) significantly suppresses the LIMK1 phenotype. This suppression is further enhanced when one copy of a null allele of Rac2 is introduced. These results suggest that Rac signaling (in particular, Rac1 and Rac2) also acts to activate LIMK1. This suggestion was also supported by the overexpression experiment. Overexpression of wild-type Rac1 (UAS-Rac1 WT) results in a weak LIMK1-like phenotype. However, when Rac1 and LIMK1 are coexpressed, axon growth defects were enhanced. Together with previous biochemical studies, these results suggest that Cdc42 and Rac act via Pak to activate LIMK1 (Ng, 2004).
To verify these genetic interactions with endogenous components, whether reducing either Cdc42 or Rac activity would modify the ssh-/- phenotype was examined. As with Rho1, reducing Cdc42 or Rac activity also partially suppresses the ssh-/- defects. Thus, these data indicate that Rho1, Cdc42, and Rac can all act through distinct downstream kinases to activate LIMK1 (Ng, 2004).
Surprisingly, reducing Rac GTPase activity can also result in enhancing the LIMK1 overexpression phenotype. For instance, the suppression effect of Rac1J10 Rac2Δ/+ was reverted by heterozygosity of Mtl (Rac1J10 Rac2Δ MtlΔ/+). This enhancement is more evident when one copy of the strongest allele of Rac1 was introduced into the LIMK1 overexpression background (Rac1J11/+). This effect is not due to dominant effects of this Rac1 allele, since in the same genetic background Rac1J11/+ animals did not display LIMK1-like growth defects without the LIMK1 expression transgene. Introduction of another hypomorphic allele of Rac1 (Rac1J6) together with Rac2 (Rac1J6 Rac2Δ/+) also strongly enhances the LIMK1 overexpression phenotype (Ng, 2004).
One interpretation of these results is that, while Rac can activate LIMK1 (via Pak), there is an alternative pathway downstream of Rac, acting antagonistically to LIMK1, that promotes axon growth. This is consistent with the finding that loss of Rac GTPase activity leads to axon growth defects in MB neurons (Ng, 2002). This pathway is likely to be Pak independent, since the Rac axon growth-promoting activity does not require direct binding to Pak (Ng, 2002), and Pak activation leads to axon growth inhibition. To further verify the existence of a Pak-independent pathway in regulating axon growth, use was made of a well-described Rac1 effector domain mutant. In mammalian fibroblasts, Rac1 activates Pak1 and, through an independent downstream pathway, promotes lamellipodia formation. A Y40C mutation in the effector binding domain of Rac1 results in the loss of Pak1 activation, but the lamellipodia promoting activity is maintained. Transgenic overexpression of Rac1 Y40C alone at levels comparable to those of wild-type Rac did not result in gross axon phenotypes. However, in contrast to wild-type Rac1 that strongly enhances LIMK1, coexpression of Rac1 Y40C strongly suppresses the LIMK1 overexpression phenotypes. These results suggest that Rac1 activates a Pak-independent pathway to counteract the effects of LIMK1 activity on axon growth (Ng, 2004).
Whether upstream activators of Rac (RacGEFs), could regulate these distinct axon growth pathways was tested. Trio encodes a RacGEF essential for axon guidance in MB neurons. Introducing one mutant copy of trio significantly suppressed the LIMK1 overexpression phenotype, suggesting that Trio acts to activate LIMK1. This was further verified by the overexpression experiments in which overexpression of wild-type Trio (UAS-trio) alone results in a mild LIMK1-like phenotype, while coexpression with LIMK1 results in a strong enhancement of the axon growth defects. Trio has two GEF domains—GEF1 is specific for Rac1, Rac2, and Mtl in vitro and in vivo, and GEF2 can activate Rho1/RhoA in vitro. Overexpression of the isolated Trio GEF1 domain (UAS-trio GEF1) in MB neurons results in severe axon growth defects, whereas overexpression of the isolated Trio GEF2 domain (UAS-trio GEF2) does not result in any gross defects. These results support the model that, in MB neurons, Trio, via its GEF1 domain, acts through Rac and Pak to activate LIMK1. These findings are also consistent with those of previous studies in which Trio acted via Rac/Pak to regulate Drosophila photoreceptor axon guidance (Ng, 2004).
In contrast to Trio, loss of one copy of still life (sif), encoding a different RacGEF, markedly enhances the LIMK1 phenotype. In overexpression experiments, UAS-sif alone does not result in gross axon defects. However, coexpression of Sif resulted in a strong suppression of the LIMK1 phenotype. These experiments suggest that Sif activates the pathway that acts antagonistically to LIMK1 (Ng, 2004).
The data suggest that Rac promotes axon growth via a pathway antagonistic to Pak and LIMK1. How does this pathway act to promote axon growth? One strong possibility is that Rac stimulates actin polymerization to promote axon growth. Therefore a number of candidate genes known to promote actin polymerization were tested. The following genetic criteria were establised by which these candidate pathways should work: (1) like Rac, loss of the candidate gene should result in axon growth defects; (2) genetic interactions with LIMK1, either through loss- or gain-of-function analyses, should show that they act antagonistically to LIMK1 (Ng, 2004).
The role of the actin nucleation factor SCAR-Arp2/3 complex was tested. Rac has been shown to promote de novo actin polymerization through interactions via SCAR and the Arp2/3 complex. Activation of the SCAR-Arp2/3 complex is required to establish cell protrusions during lamellipodia and filopodia formation, making it a good candidate pathway for promoting axon growth. This hypothesis was tested by making MARCM clones in MB neurons using null alleles of SCAR, WASp (a protein related to SCAR, also called Wsp), double mutants for SCAR and WASp, or Arpc1 (Flybase-Sop2), an essential component of the Arp2/3 complex. No axon growth defects were detected in single-cell or neuroblast clones. In addition, reduction of SCAR or Sop2 levels did not modify the LIMK1 overexpression phenotype. Furthermore, overexpression of SCAR did not suppress, but mildly enhanced, the LIMK1 overexpression phenotype. Taken together, these results suggest that the SCAR/WASp-Arp2/3 pathway does not play an essential role in axon growth of MB neurons, and it is unlikely that this pathway contributes to the Rac pathway that promotes axon growth (Ng, 2004).
Several other known regulators of actin polymerization were tested for their contribution to MB axon growth. The actin polymerization stimulators profilin (encoded by chickadee or chic) and Enabled (Ena) have been shown to be essential for axon growth and guidance in Drosophila. In addition, genetic interaction studies suggest that these proteins may be involved in Rac GTPase signaling. When assayed for MB axon growth, both ena-/- and chic-/- single-cell and neuroblast clones exhibited drastic axon growth defects. Interestingly, when examined at higher resolutions, neither ena nor chic axons displayed filopodia-like and lamellipodia-like protrusions at the axon termini characteristic of tsr-/- neurons. These genetic interaction experiments found no evidence that ena or chic act antagonistically to LIMK1. In fact, Ena overexpression strongly enhances the LIMK1 overexpression phenotype. Thus, although both Ena and profilin are essential for MB axon growth, they do not appear to constitute the Rac-mediated axon growth-promoting pathway (Ng, 2004).
Finally, the Formin-class protein Diaphanous (Dia) was tested, since it has been implicated in regulating actin polymerization downstream of Rho GTPases. dia-/- MB neurons do not exhibit axon growth defects in single-cell clones or in neuroblast clones, which exhibit strong cell proliferation defects. Interestingly, reducing Dia activity appears to suppress the LIMK1 overexpression phenotype, suggesting that Dia can act in a pathway that enhances, but does not antagonize, LIMK1 (Ng, 2004).
Lim Kinase (Limk) belongs to a phylogenetically conserved family of serine/threonine kinases, which have been shown to be potent regulators of the actin cytoskeleton. Despite accumulating evidence of its biochemical actions, its in vivo function has remained poorly understood. The association of the Limk1 gene with Williams Syndrome indicates that proteins of this family play a role in the nervous system. To unravel the cellular and molecular functions of Limk, the Limk gene in Drosophila has been either knocked out or activated. At the neuromuscular junction, loss of Limk leads to enlarged terminals, while increasing the activity of Limk leads to stunted terminals with fewer synaptic boutons. In the antennal lobe, loss of Limk abolishes the ability of p21-activated kinase (Pak) to alter glomerular development. In contrast, increase in Limk function leads to ectopic glomeruli, a phenotype suppressible by the coexpression of a hyperactive Cofilin gene. These results establish Limk as a critical regulator of Cofilin function and synapse development, and a downstream effector of Pak in vivo (Ang, 2006).
Biochemical experiments have shown that the Drosophila Limk specifically phosphorylates the serine 3 amino acid of Cofilin, a key regulator of actin turnover. In human, phosphorylation of serine 3 of Cofilin has been shown to shut down its actin depolymerizing activity. Indeed, mutation of the serine 3 amino acid of Cofilin to an alanine leads to a constitutively activated Cofilin protein. It was therefore hypothesized that Limk down-regulates Cofilin function during glomerular development. To test this hypothesis, Limkkd was expressed either alone, with wild-type Cofilin, or with CofilinS3A in ORNs. Expression of Limkkd results in disruption of glomerular structures and ectopic glomeruli at the midline in 100% of the ALs. Coexpression with wild-type Cofilin did not modify the Limkkd phenotype. In contrast, coexpression with CofilinS3A leads to the reappearance of distinct glomerular structures and the loss of ectopic midline structures in 83% of the ALs. These results support the idea that Limk negatively regulates Cofilin function by acting through the serine-3 amino acid during glomerular development (Ang, 2006).
Coenzyme A (CoA) is a pantothenic acid-derived metabolite essential for many fundamental cellular processes including energy, lipid and amino acid metabolism. Pantothenate kinase (PANK), which catalyses the first step in the conversion of pantothenic acid to CoA, has been associated with a rare neurodegenerative disorder PKAN. However, the consequences of impaired PANK activity are poorly understood. This study used Drosophila and human neuronal cell cultures to show how PANK deficiency leads to abnormalities in F-actin organization. Cells with reduced PANK activity are characterized by abnormally high levels of phosphorylated cofilin, a conserved actin filament severing protein. The increased levels of phospho-cofilin coincide with morphological changes of PANK-deficient Drosophila S2 cells and human neuronal SHSY-5Y cells. The latter exhibit also markedly reduced ability to form neurites in culture--a process that is strongly dependent on actin remodeling. The results reveal a novel and conserved link between a metabolic biosynthesis pathway, and regulation of cellular actin dynamics (Siudeja, 2012).
The actin cytoskeleton mediates a variety of essential biological functions in all eukaryotic cells. The molecular mechanisms responsible for actin remodeling have been extensively studied. However, relatively little is known how cellular metabolism influences cell morphology and cytoskeleton dynamics. Recent studies convincingly show that these events are closely linked. For example, it is well recognized that insulin and growth factor signaling induces actin filament remodeling promoting membrane ruffling in diverse mammalian cell types. In Drosophila S2 cells F-actin rich lamellapodia are also being formed in response to insulin-induced Akt signaling. Further, studies using Drosophila and mammalian systems suggest that energy metabolism is directly linked to cell structure regulation via the Lkb1/AMP-activated protein kinase (AMPK) signaling pathway. The physiological importance of this crosstalk between metabolism and the cytoskeleton remains poorly understood but relevant to investigate because it may contribute to the understanding of specific pathological conditions. For example Lkb1, which plays a central role in energy metabolism and actin filaments assembly is linked to tumorigenesis. Mutations in the human LKB1 gene result in increased incidence of epithelial cancer, which suggests that loss of Lkb1 in epithelia may contribute to the tumorigenic process through effects on the actin cytoskeleton. Disturbance of the tight link between the cellular metabolic status and regulation of the actin cytoskeleton may also underlie a number of metabolic diseases with an unresolved pathophysiology (Siudeja, 2012).
PKAN is a severe neurodegenerative disease caused by mutations in the human pantothenate kinase 2 (PANK2)-encoding gene and the molecular basis of this devastating disease are largely unknown. Previous studies have established Drosophila and human cell line models for PKAN, in which PANK activity is impaired. This study has demonstrate that impaired CoA metabolism influences actin associated cellular events, such as the regulation of the cell shape. Inhibition of PANK activity results in changes in cell morphology in Drosophila as well as in human neuronal cell cultures. A conserved mechanism was identified of cofilin/Twinstar inactivation in response to decreased CoA levels. In Drosophila the inability of CoA-deficient S2 cells to form actin rich lamellipodia, correlates with an increase in phospho-Tsr levels, and Cdi kinase and slingshot phosphatase are involved in this increased phosphorylation. These results underscore previously published data that in general cofilin is an important regulator of actin dynamics and are in agreement with data showing that increased Tsr phosporylation is associated with the inability of S2 cells to form actin-based lammelipodia. It is currently unclear whether decreased levels of CoA influence kinase activity of Cdi or phosphatase activity of Slingshot. Alternatively, Tsr itself could be modified in the presence of decreased CoA levels in such a way that it is being more efficiently phosphorylated or less efficiently dephosphorylated (Siudeja, 2012).
The Rac-Cofilin pathway is essential for cytoskeletal remodeling to control axonal development. Rac signals through the canonical Rac-Pak-LIMK pathway to suppress Cofilin-dependent axonal growth and through a Pak-independent non-canonical pathway to promote outgrowth. Whether this non-canonical pathway converges to promote Cofilin-dependent F-actin reorganization in axonal growth remains elusive. This study demonstrates that Sickie, a homolog of the human microtubule-associated protein neuron navigator 2, cell-autonomously regulates axonal growth of Drosophila mushroom body (MB) neurons via the non-canonical pathway. Sickie was prominently expressed in the newborn F-actin-rich axons of MB neurons. A sickie mutant exhibited axonal growth defects, and its phenotypes were rescued by exogenous expression of Sickie. Phenotypic similarities and genetic interactions were observed among sickie and Rac-Cofilin signaling components. Using the MARCM technique, distinct F-actin and phospho-Cofilin patterns were detected in developing axons mutant for sickie and Rac-Cofilin signaling regulators. The upregulation of Cofilin function alleviated the axonal defect of the sickie mutant. Epistasis analyses revealed that Sickie suppresses the LIMK overexpression phenotype and is required for Pak-independent Rac1 and Slingshot phosphatase to counteract LIMK. It is proposed that Sickie regulates F-actin-mediated axonal growth via the non-canonical Rac-Cofilin pathway in a Slingshot-dependent manner (Abe, 2014).
During brain development, neurons undergo multiple morphological changes to form an elaborate neural network. The Drosophila mushroom body (MB), which forms bilaterally symmetric and dorsomedially bifurcated axonal lobe structures in the central brain, has been well studied as a model of neuronal development. Among various regulators of neuronal morphogenesis, ADF/Cofilin and Rac GTPase (Rac) are key molecules in controlling cytoskeletal remodeling in axonal development. Cofilin [Twinstar (Tsr) in Drosophila] plays an essential role as a regulator of axonal growth by severing and depolymerizing F-actin. Because Cofilin is activated by dephosphorylation by the Slingshot (Ssh) phosphatase and is inactivated by phosphorylation by LIMK, the loss of Ssh or excessive activation of LIMK results in an axonal growth defect. In Drosophila, Rac has been proposed to act as a bidirectional switch for signaling cascades. One signaling event is the canonical Rac-Pak-LIMK pathway to suppress Cofilin-dependent axonal growth. The overexpression of Pak, a downstream effector of Rac, induces axonal growth defects similar to those observed with LIMK overexpression. In addition, introducing one mutant copy of Rac or Pak suppresses the axonal defect induced by LIMK overexpression. Another signaling event is the Pak-independent non-canonical pathway to positively regulate axonal growth. Rac mutant animals show multiple MB axonal defects, but the axonal growth defect is alleviated by the exogenous expression of Rac1Y40C, which lacks the ability to activate Pak but does not affect lamellipodia formation. Furthermore, Rac1Y40C overexpression remarkably suppressed the LIMK overexpression phenotype (Abe, 2014).
Although several pieces of evidence have suggested the importance of the non-canonical pathway and predicted the existence of its mediator, whether this pathway finally converges upon the downstream Cofilin pathway and subsequent F-actin reorganization remains unclear. Moreover, many biochemical studies have assessed the regulation of Cofilin function and F-actin states using in vitro systems; the endogenous changes in F-actin and Cofilin phosphorylation appear not to have been analyzed simultaneously with an internal control in developing brain. To address these issues, a novel factor was sought that interacts with Rac-Cofilin signaling components and positively regulates MB axonal growth. It was observed that Sickie, which has a calponin homology (CH) actin-binding domain and shares structural similarities with the human microtubule-associated protein (MAP) neuron navigator 2 (NAV2), showed prominent expression in F-actin-rich newborn MB axons and genetically interacted with Rac-Cofilin signaling regulators. Although Sickie was originally identified by genome-wide RNAi screening in Drosophila S2 cells and the report proposed the involvement of Sickie in the innate immune response (Foley, 2004), in this report focus was placed on the function of Sickie in the regulation of Cofilin-mediated F-actin remodeling and propose an expanded model of regulatory mechanisms during axonal development (Abe, 2014).
By combining the MARCM technique with epistatic analysis, this study demonstrated that Sickie regulates the axonal growth of Drosophila MB neurons via the non-canonical Rac-Cofilin pathway. The following model is proposed. In wild type, Sickie relays the non-canonical pathway signal to Ssh to facilitate F-actin-mediated axonal growth by counteracting the canonical signal. In a sickie mutant, mediation of the non-canonical pathway is defective, which causes an imbalance in the regulation of Cofilin activity. Because neurons are morphologically polarized and the amount of actin is limited in each cell, the growing axons may efficiently control actin recycling by facilitating F-actin turnover by balancing between the non-canonical and canonical pathways. Consistently, a stronger axonal growth defect was found with increased P-Cofilin in the LIMKWTM6 ssh1-63 and sickieΔ LIMKKD double-mutant animals than in the single mutants ssh1-63, sickieΔ and LIMKKD. Cofilin activity might be decreased in the developing axons of these double mutants by the preponderance of the canonical pathway. If so, these results highlight an essential role of the non-canonical pathway to balance Cofilin activity in axonal growth (Abe, 2014).
Unlike the clear elevation of P-Cofilin levels in the ssh1-63 mutant, constitutive activation of LIMK did not result in a similar increase in P-Cofilin despite F-actin elevation. This apparent paradox might be explained by considering the positive regulation of Ssh by F-actin. The phosphatase activity of SSH-1L is F-actin dependent, and the addition of F-actin dramatically increases its phosphatase activity. In the LIMKKD mutant axons, endogenous Ssh may be activated by a large amount of F-actin and subsequently dephosphorylates Cofilin. Consistently, highly elevated signals of both F-actin and P-Cofilin were detected in the LIMKWTM6 ssh1-63 double-mutant clones. In this mutant, Cofilin activity was severely reduced by high phosphorylation levels due to constitutive LIMK activation and a lack of Ssh phosphatase activity, resulting in the posterior arrest severe axonal defect, similar to the cofilin knockdown mutant. In addition, relatively moderate increases in P-Cofilin signal were detected in the developing axons of the sickieΔ LIMKKD double mutant. These results also support the model that Sickie functions in the same pathway as Ssh to positively regulate Cofilin function by counteracting the canonical Rac-Pak-LIMK pathway. Ssh might be downregulated in the sickie mutant axons due to defects in the mediation of Pak-independent Rac1 function or in the interaction among Ssh and F-actin by the loss of Sickie. The similar increases in the P-Cofilin and F-actin signals and the similar posterior arrest phenotype in the LIMKWTM6 ssh1-63 double-mutant clone and those of the PakMyr mutant clone are also consistent with results of in vitro studies that showed that SSH-1L is inactivated by Pak4. Thus, in the current model, Pak concurrently inactivates Ssh and activates LIMK in axonal growth (Abe, 2014).
Whereas ssh or cofilin mutants are embryonic lethal and their mutant clones display developmental defects in non-neuronal tissues, sickie mutants are not embryonic lethal, and conspicuous phenotypes are found only in the substructures of the central brain, such as MB and EB, implying that more elaborate mechanisms involving Sickie function are required for ensuring their proper development. Given that MB neurons exhibit a densely bundled axonal morphology, the growing MB axons might require Sickie to smoothly extend their neurites within the lobe core region by coordinating the dynamics of actin and microtubules (MTs). Sickie and human neuron navigator proteins (NAVs) have conserved EB1-binding motifs, and Sickie shows a genetic interaction with MT components. Double RNAi of sickie and EB1 or β-tubulin both resulted in synergistic increases in the axonal defects. In addition, a recent cell biological study demonstrated a functional link between Cofilin and MTs. Through its interaction with EB1, Sickie might act as a navigator for the plus-end of MTs to link to the F-actin complex and thereby ensure elaborate neuronal wiring. To further elucidate the signaling mechanism, the relationships with other components of the Ssh-dependent Cofilin pathway need to be studied. Recent studies have revealed that PKD, 14-3-3 protein and Pak4 play key roles in suppressing Ssh function (Abe, 2014).
Finally, preliminary data suggest a post-developmental role for Sickie. Adult stage-specific knockdown of Sickie in MBs impairs olfactory memory. Moreover, recent mammalian studies have suggested the possible involvement of NAVs and Cofilin in neurodegenerative disease. In Alzheimer's disease brains, the NAV3 transcript level is elevated, and Cofilin-actin rod-shaped inclusions, which are formed by the hyperactivation of Cofilin, are enriched. Further studies are required to understand the wide variety of contributions of sickie and the general importance of this evolutionarily conserved gene in brain development and function (Abe, 2014).
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