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nevertheless For bacterial expression of 6His tagged fascin 1, cDNAs encoding wild type, S39A, or S39D human fascin 1 from the pEGFP fascin 1 plasmids were cloned between the NotI and XhoI restriction sites of pET 30a. The reading frame was adjusted by subsequent digestion and blunting of the NotI site. BIM, Y27632, ML7, BDM and blebbistatin were obtained from Calbiochem. C3 endotoxin and RhoA G LISA kit were from Cytoskeleton Inc. Antibodies used included mouse monoclonal antibody to fascin Inhibitors,Modulators,Libraries 1. to LIMK1, LIMK2 and phos phoLIMK12 and to Rho, ROCK I and II. Cell extracellular matrix adhesion and Immunofluorescence C2C12 cells were maintained in DMEM containing 20% fetal calf serum, and SW480 cells were maintained in DMEM with 10% FCS.

For transient transfections, cells were plated at 30% to 40% confluency, and trans fected with transfection reagentsin accordance with the manufacturers instruc Inhibitors,Modulators,Libraries tions. At 48 hours post transfection, cells were re plated as single cell suspensions onto glass surfaces coated with 50 nmolL FN or Engelbreth Holm Swarm laminin under serum free Inhibitors,Modulators,Libraries conditions, as described pre viously. In experiments involving pharmacological inhibitors, cells were pretreated with the agent for 30 minutes prior to the adhesion assays being set up, and the agent was maintained in the medium throughout the assay. The concentrations of inhibitors to be used were established in pilot experiments, and the lowest concentrations that affected cell morphology and actin organization reproducibly without evidence of cytotoxi city, as determined by Trypan blue exclusion, were cho sen for the experiments.

After 1 or 2 hours at 37 C to allow adhesion and initiation of ran dom cell migration, non adherent cells were removed by rinsing in Tris buffered saline and adherent cells were fixed in 2% paraformaldehyde and permea bilized in TBS with 0. 5% Triton X100 for staining Inhibitors,Modulators,Libraries with tetramethyl rhodamine isothiocyanate phalloidin, phalloidin 633, or antibodies to vinculin or phosphotyrosine. For fascin 1 staining, cells were fixed in absolute methanol, and for LIMK12 staining, they were fixed in 4% paraformaldehyde. Digi tal images were taken at room temperature under the 63 objective of a laser scanning confocal microscope using confocal acquisition software.

Morphometric analysis of adherent C2C12 cells was carried out by measuring cell areas and the numbers and lengths of individual Inhibitors,Modulators,Libraries peripheral quality control fascin or F actin bundles from calibrated digital images using the software Improvision Openlab. Fluorescence resonance energy transferfluorescence lifetime imaging microscopy FLIM was performed cells transfected with specified constructs, with fixation and data analyzed as described previously. Details of time domain FLIM performed with a multiphoton microscope system have been described previously. FLIM capability was provided by time correlated single photon counting electronics.

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