Similar to fascin 1, the cofilin pathway has been linked to breast cancer metastasis. It was recently things shown that cofilin associates transiently with retracting filopodia, and that its actin severing activ ity is enhanced on actin bundles crosslinked by fascin 1. The relationship between the fascin 1LIMK12 complex and cofilin in the dynamics of filopodia are questions Inhibitors,Modulators,Libraries of future interest. It will Inhibitors,Modulators,Libraries be important to estab lish if fascin 1 competes with cofilin for LIMK1 kinase availability, thereby modulating actin dynamics directly, or whether LIMK12 act directly or indirectly as a scaf fold to maintain fascin 1 in proximity to F actin. Alterna tively, fascin 1 binding might inhibit the kinase activity of LIMK12, thereby affecting actin dynamics indirectly, as previously shown for nischarin.

LIMK1 contributes to the transcriptional activity of serum response factor, and it will be interesting to determine if this activity is modulated by fascin 1. Conclusions In this study, we have identified Inhibitors,Modulators,Libraries a novel activity of the small GTPase Rho in promoting the interaction of fas cin 1 with actin in ECM adherent, migratory cells. Rho, Rho kinases, and LIMK12 were identified as key effec tors, and activated LIMK1 or LIMK2 act as novel bind ing partners of fascin 1. These findings have many implications for our understanding of how fascin 1 pro Inhibitors,Modulators,Libraries tein complexes and actin dynamics are coordinated in contractile and protrusive actin based structures in intact cells, and may be particularly relevant to carci noma cell migration and metastasis.

In the course of Inhibitors,Modulators,Libraries the study, we developed a novel method for analysis of the fascin 1actin interaction by FRETFLIM. This method has potential general applicability for analyzing the activities of actin binding proteins in intact cells. Methods Cell lines and materials C2C12 mouse skeletal myoblast and SW480 human colon carcinoma cells were from the American Type Culture Collection. GFP fascin 1 and PKCg mRFP con structs were prepared as previously described. PKCa mRFP was a gift from T. Ng, and the GFP tagged forms of LIMK1, LIMK2, ROCKI, and ROCKII were gifts from G. Jones. GFP caldesmon and the deletion mutant GFP caldesmon 445 were a gift from Alexander Bershadsky. The Ras bind ing domain glutathione S transferase plasmid was the gift of M. Schwartz. GFP lifeact was a gift from R. Wedlich Soldner.

The mRFP fascin 1S39A and mRFP fascin 1S39D con structs were prepared by subcloning cDNAs for human fascin 1S39A and fascin 1S39D into pcDNA3 mRFP using the PCR primers shown in Table 1. mRFP fascin 1 cDNA was generated from GFP fascin 1WT by subclon ing into pCR Blunt, using the PCR primers shown in Table 1, and then subcloned into pcDNA3 mRFP using Oligomycin A solubility HindIII and EcoRI restriction sites. A vector encoding GFP fascin 1 under control of a truncated CMV promoter was generated by replacing the CMV promoter by the speckled promoter from a GFP paxillin plasmid using AseI and NheI restriction sites.