Locostatin Impairs Migration Figure 3. Locostatin has no effect on EGF-induced MAPK activity. (A, B) HeLa cells expressing control or depleted RKIP or (C, D) MEFs expressing wild-type RKIP (RKIP+/+) or (E, F) MEFs not expressing RKIP (RKIP2/2) were serum starved overnight and then pre-treated for 30 min with DMSO or locostatin (concentrations as indicated). EGF (10 ng/ml) was added in the final 5 minutes of incubation. Whole cell lysates (30 mg) were separated on SDS-PAGE gels (12.5%), transferred to nitrocellulose and analysed by immunoblotting with anti-phospho ERK and anti-total ERK antibodies (A, C and E). ERK phosphorylation was assessed by normalizing phospho-ERK levels to total ERK levels as depicted in bar graphs (B, D and F). The results shown are representative of three independent experiments. doi:10.1371/journal.pone.0006028.g003 to 50 mM locostatin which impaired wound healing at all time points measured (Fig. 4B). Locostatin (50 mM) when compared to vehicle (0.25% DMSO) treatment (24 h time point) reduced migration by 4065% (n = 8 separate wounds) in RKIP+/+ MEFs (Fig. 4B). Surprisingly, depletion of RKIP (depletion described here refers to short-hairpin RNA interference with RKIP in MEFs) further reduced migration to 7763% at this concentration of locostatin (Fig. 4D). To confirm that remaining RKIP in the depleted MEFs was not responsible for the observed effects, we also tested RKIP deficient MEFs (RKIP2/2). Total loss of RKIP almost completely inhibited migration in the presence of 50 mM locostatin, 9665% at 24 h when compared to RKIP+/+; p,0.01; compare Fig. 5B and D). Furthermore, the rate of migration in the presence of vehicle was not significantly affected by the presence or absence of RKIP. Therefore, these results indicate that 50 mM locostatin is sufficient to inhibit migration, and RKIP confers significant protection to cells within this concentration range. Locostatin impairs cytoskeletal organization, disrupts the mitotic spindle and promotes nuclear fragmentation Since locostatin has been previously reported to inhibit cell migration, its physiological effects are likely mediated, at least in part, via cytoskeletal interactions. To investigate the cytoskeleton we pretreated MEFs of varying genotypes with locostatin (50 mM) and stained them with phalloidin to locate actin filaments within the cell. Previous studies [17] reported that locostatin did not affect the formation of new filamentous actin bundles in MDCK cells post wounding. By contrast, we observed a time dependent induction of cytoskeletal reorganization by locostatin in MEFs (Fig. 6). In vehicle control MEFs (RKIP+/+, depleted and RKIP2/2), cells stained positive for filamentous F-actin with an even distribution of F-actin around the nucleus as well as along stress fibers within the cell (Fig. 6). In the presence of locostatin (50 mM) we observed a slight decrease in actin staining after 2 h of treatment, and by 6 h actin was predominantly localized to the perinuclear region of the cell (Fig. 6). Similarly, RKIP depleted cells displayed a locostatin effect; however, by 6 h it was evident that locostatin caused the collapse of the cytoskeleton as evidenced by near absent actin staining throughout the cell (Fig. 6). Likewise, locostatin caused reduced actin staining in RKIP deficient (RKIP2/2) MEFs (Fig. 6, lower panel). As observed with migration, the effect of locostatin was more pronounced in RKIP2/2 MEFs compared to RKIP+/+. By 6 h, treatment with locostatin in all genotypes exhibited disrupted actin cytoskeleton as detected by phalloidin. However, following 2 h treatment with locostatin the RKIP+/+ cells looked similar to vehicle control cells, whereas the RKIP depeleted or RKIP2/2 cells already displayed defects in the actin cytoskeleton. Thus, RKIP appears to transiently protect cells from locostatin mediated disruption of the actin cytoskeleton. The mitotic spindle is composed of microtubules that are highly sensitive to both polymerizing and depolymerizing agents, and therefore could serve as a sensor for microtubule-dependent cytoskeletal changes. To test this possibility, we determined whether locostatin affects mitotic spindle structure. RKIP+/+ MEFs or RKIP-depleted MEFs were either untreated or treated with locostatin (20 mM) for the indicated time (Fig. 7A) and cells were fixed and stained with anti-a tubulin antibody. As shown in Fig. 7A locostatin caused a time dependent loss of spindle fiber tension in RKIP+/+ and RKIP-depleted cells. Both RKIP+/+ and RKIP2/2 MEFs were treated for 6 h with locostatin (20 mM) which showed defects in mitotic spindle formation and chromo- some organization (Fig. 7B). Although abnormal spindles were observed after 2 h treatment with 20 mM locostatin in all cell types, there were fewer abnormal spindles following 1 h treatment in RKIP expressing cells (supplemental Fig. S1). We also observed at this dose of locostatin a disruption in chromatin condensation in a manner reminiscent of Taxol, a drug that causes loss of spindle fiber tension (Fig. 7B). This effect was independent of RKIP expression, indicating that locostatin targets cytoskeletal elements through a mechanism that does not involve RKIP binding or inhibition of RKIP function. Discussion In this study, we have demonstrated that locostatin inhibits migration in MEFs independent of RKIP expression and Raf/ MAPK signaling. The locostatin precursor binds to RKIP’s ligand-binding pocket but has no effect on Ser-153, the known site for PKC phosphorylation of RKIP that leads to dissociation of RKIP from Raf-1 [4]. Similarly, increasing concentrations of locostatin and its precursor had very little effect on RKIP phosphorylation by PKC or RKIP/Raf-1 association. Finally, locostatin in RKIP-depleted and RKIP-deficient cell lines (HeLa and MEFs) had no effect on the EGF-induced activity of MAPK, a downstream signaling target of Raf. Furthermore, our data shows that locostatin is not specific to RKIP and has off target effects. In particular, locostatin reorganizes the actin cytoskeleton, disrupts the mitotic spindle and inhibits cell migration via a mechanism that is independent of the MAPK pathway. The protective effect of RKIP toward locostatin disruption of the cytoskeleton and inhibition of migration indicates that RKIP can influence locostatin action but not necessarily as a mediator. Our results raise the possibility that RKIP may function to trap locostatin, sequestering the toxic drug within the cell. Our results add a different perspective to our understanding of locostatin interaction with RKIP. Previous studies have shown that locostatin prevents RKIP from co-precipitating with Raf-1. However, it is possible that the chemically reactive nature of locostatin may have caused other interactions that sterically hinder Raf access to RKIP. The deleterious effects of locostatin on cytoskeletal structures and cell migration even in cells lacking RKIP suggest that locostatin has other targets that may not have been previously identified. One puzzling observation is that locostatin inhibits spindle organization and cell migration at 20– 50 mM but the precursor binds to RKIP at millimolar concentra- tions. The reduced apparent affinity of the precursor may be attributable to the different modes of interaction between the two compounds. Locostatin acts as a suicide inhibitor that covalently attaches itself to RKIP, while the precursor lacks a chemically reactive group and binds RKIP in a reversible manner [13]. An alternative explanation for these differences may be the size of locostatin compared to its precursor, which may also cause PLoS ONE | www.plosone.org 6 June 2009 | Volume 4 | Issue 6 | e6028