Figure 4. The morphology and viability of cultured human neuroblastoma SHSY-5Y cells is affected upon PANK inhibi- tion. SHSY-5Y cells were cultured in medium supplemented with 0,5 mM HoPan. (A) After 6 days in culture the cell morphology was visualized with an inverted microscope. Representative images of control (mock treated) and HoPan treated cells are shown. (B) Cell viability after 0–6 days in culture with HoPan was assayed by a Trypan blue exclusion test. Viability of control untreated cells was set as 100% at each time point. doi:10.1371/journal.pone.0043145.g004 Finally, we tested whether the HoPan-induced phenotypes in SHSY-5Y cells could be reverted by the addition of pantethine to the cell culture medium. The rescue by pantethine was evident for all the phenotypes investigated (Figure 5B, F, G). In RA treated cultures, RA and HoPaN treated cultures and RA, HoPan and pantetehine treated cultures, all three classes of cells were present, however the amount of class 3 cells was strikingly different. Figure 5D, E, F shows representative images of the conditions used and quantification is presented in Figure 5G. These results strongly suggest that the observed defects are due to decreased levels of CoA and not due to some non-specific (and non-PANK related) effect of HoPan. These results indicate that accumulation of phosphorylated (and most likely inactive) form of cofilin under conditions of impaired CoA biosynthesis coincides with impair- ment of neurite formation in vitro –a process which is strongly dependent on actin remodeling. Discussion 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 Coenzyme A Levels Influence Phospo-Twinstar Levels 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 [38,39]. In Drosophila S2 cells F-actin rich lamellapodia are also being formed in response to insulin-induced Akt signaling [20]. 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 [40,41,42,43]. 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 [44] is linked to tumorigenesis. Mutations in the human LKB1 gene result in increased incidence of epithelial cancer [45], 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. 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 [4]. Previously we have established Drosophila and human cell line models for PKAN, in which PANK activity is impaired [5,6]. Here, we demonstrate that impaired CoA metabolism influences actin associated cellular events, such as the regulation of the cell shape. We show that inhibition of PANK activity results in changes in cell morphology in Drosophila as well as in human neuronal cell cultures. Further we identify a conserved mechanism of cofilin/Twinstar inactivation in response to decreased CoA levels. We show that 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 phosphoraylation. These results underscore previously published data that in general cofilin is an important regulator of actin dynamics (reviewed in [46]) and are in agreement with data showing that increased Tsr phosporylation is associated with the inability of S2 cells to form actin-based lammelipodia [20]. 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 dephosphorylat- ed. Finally, in our current work we show that not only Drosophila cells but also differentiated human neuronal cells show abnormal F-actin organization and an increase in phospho-cofilin levels upon impaired PANK function. Differentiated neuroblastoma SHSY-5Y cells undergo extensive morphological changes, which finally lead to the formation of neurites [34]. During these morphological changes, we observed that cofilin is maintained in a non-phosphorylated (active) state. The latter is most likely required for high actin filament dynamics, necessary to undergo the morphogenesis. Analogous involvement of cofilin in neuronal differentiation has also been reported both in primary cultures (such as isolated chicken neurons) and in other cell lines (such as PC12 cells) [28,29,30,31,32]. We show that the addition of PANK inhibitor, HoPan, to the medium not only inactivates cofilin but also affects the ability of differentiated SHSY-5Y cells to interact with tissue culture surface and the ability to form neurites. The physiological importance of actin regulation in neuronal function in vivo can be illustrated by a contribution of actin PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e43145