Figure 1. CoA deficient Drosophila S2 cells fail to form actin- based lamella. S2 cells were seeded on concanavalin A to induce cell spreading and lamella formation. Actin was visualized with rhodamine- phalloidin staining. (A,B) Representative images of the Ctrl; (C,D) dPANK/Fbl RNAi; (E,F) dPANK/Fbl RNAi treated cells supplemented with pantethine; (M,N) Twinstar RNAi; and (O,P) Twinstar (Tsr) RNAi treated cells supplemented with pantethine are shown. (G–L) S2 cells were seeded in concanavalin A coated glass bottom culture dishes and imaged during cell spreading and lamella formation in phase contrast with an inverted Zeiss confocal microscope using a 636 glycerol- immersion lens. Frames were acquired every 20 seconds for 20 min- utes. Individual images from Ctrl (G), dPANK/Fbl RNAi (H), dPANK/Fbl RNAi treated cells supplemented with pantethine (I) are shown. Images Coenzyme A Levels Influence Phospo-Twinstar Levels are representative of at least 5 movies taken in different regions of each dish. The white box in all three images depicts the area used to create the kymographs in J–L. (J) Kymographs of Ctrl cells; (K) dPANK/Fbl RNAi; (L) dPANK/Fbl RNAi treated cells supplemented with pantethine. The kymographs were generated from an area transecting the cell membrane (outlined by a white box in G–I) and correspond to the movies from which the images were taken (for movies see supplemen- tary Figure S4, S5, S6, S7). (Q) Quantification of the percentage of spread (lamella forming) cells as illustrated in A–L. Scale bars represent 10 mm. Ctrl = control/non-treated cells; Pan = pantethine treatment; dPANK = dPANK/fbl RNAi treated cells; tsr = twinstar RNAi treated cells. doi:10.1371/journal.pone.0043145.g001 neurite formation – processes which are strongly dependent on actin remodeling. Results dPANK/Fbl downregulation leads to actin abnormalities in Drosophila S2 cells In Drosophila pantothenate kinase is encoded by a single gene dPANK/fbl [8]. Decreased expression of dPANK/Fbl protein leads to a dramatic reduction in the levels of total CoA both in Drosophila dPANK/fbl mutant flies and Schneider’s S2 cells [6]. Interestingly, dPANK/fbl mutant flies show actin abnormalities during sper- matogenesis and oogenesis [8,9]. To elucidate the link between CoA metabolism and actin cytoskeleton, we first investigated whether RNAi mediated downregulation of dPANK/Fbl in S2 cells also leads to F-actin abnormalities. Drosophila Schneider’s cells have been successfully used to identify new players involved in actin cytoskeleton dynamics and regulation of cell morphology [16,17]. Hence, we plated control and dPANK/Fbl downregulat- ed S2 cells on concanavalin A-coated glass coverslips to induce cell flattening and spreading [16]. As revealed by phalloidin staining, control cells showed the characteristic morphology with lamella formation, whereas CoA deprived cells failed to undergo morphological changes under these conditions (Figure 1A, B, C, D, for quantification see Figure 1Q). Impaired function of various proteins involved in formation of actin-based lamella is associated with specific morphological changes in S2 cells [16]. We have compared the morphology of CoA deficient cells with these reported abnormalities and noticed similarities between dPANK/ Fbl RNAi-treated cells and cells depleted of the Drosophila ADF/ cofilin homolog, Twinstar (Tsr) (Figure 1M,N). Next we assayed the dynamics of the lamella by making time-lapse recordings using phase contrast microscopy (for movies see Movie S1, S2, S3, S4) (Rogers et al., 2003). Control cells plated on concanavalin A show dynamic behavior of lamella (Figure 1G,J) whereas dPANK/Fbl RNAi-treated cells do not form the lamella (Figure 1H,K). Further, we asked whether observed actin abnormalities where indeed the consequence of decreased cellular CoA levels or were due to the absence of dPANK/Fbl protein. Previously we showed that addition of pantethine to the cell culture medium restored CoA levels in the presence of strongly reduced levels of dPANK/ Fbl protein [6](Siudeja et al., 2011). Pantethine supplementation reversed the morphological abnormalities of dPANK/Fbl depleted cells (Figure 1E,F,I,L for quantification see Figure 1Q), confirming that decreased CoA levels are strongly associated with a failure of S2 cells to spread on concanavalin A coated surface and are strongly associated with abnormal actin based lamella. Addition of pantethine to Tsr RNAi-treated cells did not rescue the phenotype (Figure 1O,P), further proving that the beneficial effect of pantethine in dPANK/Fbl RNAi cells is due to the restoration of CoA levels in these cells, and not due some general PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43145