Coenzyme A Levels Influence Phospo-Twinstar Levels Figure 3. Cdi and Ssh signaling is involved in the regulation of Twinstar in response to decreased levels of CoA. (A) Drosophila LIM kinases, dLimk, Center divider (Cdi), and Slingshot phosphates (Ssh) regulate levels of p-Tsr in S2 cells. Cells were treated with dsRNA against dLimk, Cdi, Ssh or with control dsRNA and the levels of Tsr and p-Tsr were assayed using Western blot analysis. Tubulin was used as a loading control. (B) dLimk, Cdi or Ssh were down-regulated for 3 days after which cells were subcultured and left untreated or HoPan was added to the cell culture medium. After additional 48 hr cell lysates were assayed for levels of Tsr and levels of p-Tsr. (C) Quantification of the levels of p-Tsr as illustrated in B. Fold change after HoPan addition is indicated above the bars, n.s: not significant. doi:10.1371/journal.pone.0043145.g003 PANK inhibition inactivates mammalian cofilin and affects neuritogenesis in vitro In the experiments as shown above we demonstrate that interfering with the pantothenate kinase activity in Drosophila S2 cells results in the failure to form actin-based lamella and in the accumulation of phosphorylated (and most likely inactive) Tsr – the Drosophila ortholog of cofilin. In humans mutations in the PANK2 gene cause PKAN – a severe neurodegenerative disorder with a largely unresolved pathophysiology. The regulation of cytoskeletal dynamics plays a fundamental role in neuronal function and it is especially important in the process of neuritogenesis [26,27]. Furthermore, cofilin (together with Lim kinases and Slingshot) appears to be a key regulator of actin cytoskeleton dynamics during growth cone formation and neurite extension [28,29,30,31,32]. Therefore, we investigated whether inactivation of PANK activity in human neuronal cells leads to changes in the phosphorylation status of cofilin and whether this correlates with altered neuronal morphology. First, we cultured undifferentiated neuroblastoma SHSY-5Y cells during four doubling times with addition of HoPan in the cell culture medium in order to block the enzymatic activity of PANK. Under these conditions of HoPan treatment reduced cell counts and abnormal morphology was observed (Figure 4A and B). In addition, HoPan also induced a detachment of the treated cells from the culture dishes. This clearly indicates that, similarly to Drosophila S2 cells, impaired CoA biosynthesis affects survival and morphology of dividing human neuroblastoma cells. Further we used retinoic acid (RA) to differentiate SHSY-5Y cells [33,34] and to investigate the effects of HoPan on neurite formation. First we examined cofilin phosphorylation levels upon differentiation of SHSY-5Y cells. Western blot analysis revealed that treatment with RA resulted in a significant decrease in phospho-cofilin levels as compared to mock treated cells (Figure 5A). Further, we compared the levels of phospho-cofilin in differentiated SHSY- 5Y cells non-treated or treated with HoPan. Similarly to our results obtained with Drosophila cells, HoPan induced an accumu- lation of the phosphorylated cofilin in human SHSY-5Y cells (Figure 5B). Finally, we investigated the effect of HoPan treatment on the morphology and differentiation of RA treated SHSY-5Y cells (see also Figure S3). Firstly, control SHSY-5Y cells were differentiated with RA, plated on poly-lysine and analyzed after 24 hours and 48 hours. Morphology was investi- gated by F-actin staining and differentiation status was analyzed by using b -III-Tubulin – a neuronal specific isoform of tubulin [35,36,37] as a marker for differentiation. In RA treated cultures (at 24 and 48 hours) three classes of cells were observed. The first class of cells displayed a round shape; they were b-III-Tubulin negative, with low F-actin and only short (if any) filopodia. These cells were classified as not-differentiated (arrowheads Figure 5C; Figure S3A). The second class of cells had a spread, angular appearance. Strong, filamentous actin fibers were present and a low expression of b-III-Tubulin could be detected (Figure S3B, C). The third class of cells, mainly detected 48 hours after seeding had an elongated shape with rather small cell body, accompanied by high levels of F-actin. They showed strong neurite growth and high expression of b-III-Tubulin (Figure 5C, arrows). They were scored as fully differentiated neurite-bearing cells. No b-III- Tubulin expressing cells (class 2 and 3) were observed in the SHSY-5Y cell culture not treated with RA. 48 hours after RA treatment, 60% of the control cells were fully differentiated and showed neurite growth and a high expression of b-III-Tubulin (class 3, Figure 5C, D). In contrast HoPan treated cells showed a low (less than 10%) percentage of cells with high b- III-Tubulin expression and neurite formation (Figure 5E). PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e43145