(CoA-unrelated) positive effects of pantethine on the actin cytoskeleton. Twinstar phosphorylation is increased in CoA deficient cells The similarity in morphology between dPANK/Fbl depleted and Tsr depleted cells prompted us to hypothesize that Tsr could be involved in the actin abnormalities in the CoA deficient background. Tsr is essential for actin remodeling in Drosophila, both in vivo and in vitro [10,16,18,19]. The actin binding and severing activity of Tsr is inhibited by phosphorylation at the conserved serine residue 3. Thus, using specific antibodies [20], we investigated levels of phopsho-Twinstar (p-Tsr) in CoA-deficient S2 cells. Western blot analysis revealed a significant increase in dPANK/Fbl RNAi-treated cells as compared to control cells (Figure 2A). Further, we used a non-genetic approach to block the CoA biosynthesis pathway by using the pantothenate kinase inhibitor HoPan [21]. HoPan treatment resulted also in an increase in p-Tsr levels in S2 cells (Figure 2B). Increased phosphorylation of Tsr is already observed after 4 days dPANK/ fbl RNAi treatment (Figure S1). The increase in Tsr phosphor- Figure 2. Inhibition of dPANK activity in S2 cells induces an increase in phospho-Twinstar levels. Immunoblots of whole cell extracts were incubated with an antibody against Tsr and p-Tsr. Tubulin was used as a loading control. (A) Cells were treated with control dsRNA or dsRNA to downregulate dPANK/Fbl protein levels and medium was supplemented with pantethine when indicated. The graph illustrates the quantified levels of p-Tsr relative to untreated control cells. (B) S2 cells were cultured in the presence of pantethine, HoPan or both compounds. The graph illustrates the quantified levels of p-Tsr relative to untreated control cells. doi:10.1371/journal.pone.0043145.g002 Coenzyme A Levels Influence Phospo-Twinstar Levels ylation after 4 days dPANK/Fbl RNAi was rescued by the addition of pantethine (during the following 3 days), demonstrating that in S2 cells Tsr phosphorylation levels are responsive to changes in CoA levels and do not respond to the levels of the PANK protein itself (Figure 2). These results also indicate that increased phosphorylation of Tsr in response to decreased CoA levels is reversible upon restoration of CoA levels. Increased phosphorylation of Tsr upon HoPan treatment is also rescued by pantethine (Figure 2B). These data, together with our morphological studies (Figure 1) strongly suggest that decreased CoA levels are associated with hyperphosphorylation (and inhibition) of Tsr, leading to the observed actin abnormalities. Next we investigated whether the increased levels of p-Tsr are a general response to metabolic stress. Hereto, we serum starved the cells and we blocked ATP synthesis with oligomycin. Increased phosphorylation of AMP-activated kinase confirmed these stress conditions [22,23] (Figure S2). Under these metabolic stress conditions, levels of p-Tsr did not change (upon serum starvation) or showed a decrease (upon incubation with oligomycin) (Figure S2). In addition serum starvation did not induce changes in actin lamellipodia homeostasis in Drosophila S2 cells [20]. These results demonstrate that by decreasing CoA levels a specific cellular response is provoked, different from a general reaction induced by starvation. Cdi kinase and Slingshot phosphatase are involved in Twinstar hyperphosphorylation associated with decreased CoA levels Having established that CoA deficiency correlates with defects in F-actin organization and with an increase in accumulation of the phosphorylated form of Tsr, we further aimed to identify upstream mediators of this response. Cofilin phosphorylation is catalyzed by a family of LIM kinases [13,15]. In Drosophila two kinases belonging to this group have been characterized, dLimk - the ortholog of human LIM kinase [24] and Center divider (Cdi) - orthologus to human testicular kinase 1 (TESK1) [25]. Further, the reactivation of cofilin is achieved by dephosphorylation catalyzed by Slingshot (Ssh) phosphatases [14]. Hence, by using an RNAi-approach we tested whether the accumulation of p-Tsr in a CoA deficient background is mediated by the above- mentioned players. Consistent with previous reports [20], RNAi induced silencing of either dLimk or Cdi resulted in a decrease in basal Tsr phosphorylation levels, whereas a knockdown of Ssh phosphatase induced the opposite effect (Figure 3A). Interesting- ly, cells expressing reduced levels of dLimk still showed an increase in p-Tsr levels in response to decreased CoA levels caused by HoPan treatment. Conversely, cells expressing reduced levels of Cdi kinase or Ssh did not show any change in the levels of p-Tsr upon incubation with HoPan (Figure 3B and C). These results implicate that normal levels of both Cdi kinase and Slingshot phosphatase are required for increased p-Tsr levels in response to impaired CoA metabolism. The two-fold induction in Tsr phosphorylation in the presence of reduced levels of dLimk indicate that either the observed phosphorylation is dLimk independent or reduced levels of dLimk are still sufficient to induce Tsr phosphorylation. All together these results suggest that in Drosophila S2 cells Tsr regulation in response to fluctuating CoA levels is mediated by pathways, which include Cdi kinase and Slingshot phosphatase activity, both of which are known regulators of the cofilin phospho-status. PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e43145