Supplementary Materials Supplemental Materials supp_26_12_2279__index. IIA (NMMIIA), which was confirmed by immunoprecipitation. GFP-FliI colocalized with NMMIIA at cell protrusions. Purified FliI containing gelsolin-like domains (GLDs) 1C6 capped actin filaments efficiently, whereas FliI GLD 2C6 did not. Binding assays showed strong interaction of purified FliI protein (GLD 1C6) with the rod domain of NMMIIA ((Campbell gene cause defects in indirect flight muscles; consequently, they cannot fly. In mammalian cells, FliI regulates cell migration (Cowin = 3, at least 50 cells per group; # 0.02 using ANOVA and comparing antibody with control (irrelevant antibodyCtreated) cells. (E) Projected surface area of spreading FliI KND cells is twofold higher than FliI OE cells on fibrillar or monomeric collagen. Data are reported as mean SD, = 3, with at least 40 cells/group. * 0.05 using ANOVA and comparisons between OE with WT or KND cells. (F) (i) Representative images showing round (a), spreading (b), or spread cells (c) as a function of time after plating. Rounded cells have twice the diameter of spreading cells. (ii, iii) FliI OE, WT, and KND cells plated on fibrillar or monomeric collagen. At 60 min, the initial spreading process Spinorphin was largely complete, and there were lower percentages of spread FliI OE cells than WT or FliI KND cells. (G) FLiI OE, WT, and KND cells immunostained for endogenous Myo10 show localized staining at tips of filopodia in FliI OE cells and punctate staining at peripheries of KND cells. (H) Fascin-immunostained cells show localization of fascin to filopodial cell extensions in FliI OE cells. (I) (i) Quantitative analysis of circularity index indicates that KND and WT cells are more circular and are less irregular on their contours than are OE cells (* 0.05 by ANOVA; = 3 experiments/group, with 40 cells analyzed/group). (ii, iii) Histograms show mean SD of length and number of cell extensions in OE, WT, and KND cells (= 3 experiments/group, with at least 40 cells analyzed/group.* 0.05 are comparisons of OE with WT and KND cells by ANOVA. We examined whether the phenotype observed in FliI KND cells was caused by off-target, nonspecific effects of the short hairpin RNA (shRNA). We reintroduced FliI by retroviral introduction of a mutated, shRNA-resistant yet functional FliI into FliI KND cells, and these cells showed extension formation (Figure 1C, i and ii). We examined the expression of integrins specific for collagen binding by immunoblotting of whole-cell lysates. We found similar expression levels of the 1, 2, 11, 10, and 1 integrins and the fibrillar collagen receptor discoidin domain receptor 1 (DDR1; Supplemental Figure S1A). Spinorphin Surface expression of the 1 integrin (measured by flow cytometry of nonpermeabilized cells with the KMI16 antibody) was higher ( 0.05) in FliI OE than in WT and KND cells (Supplemental Figure S1B). These results were consistent with our previous data showing that FliI KND cells are less adhesive to collagen and migrate more quickly over collagen than WT cells (Mohammad 0.02). Because spreading cells form abundant protrusions (Dubin-Thaler 0.05). We quantified the number of round, spreading, and spread cells as a function of time after plating. We defined a spread cell as one that exhibited a greater than twofold increased radius compared with the mean radius of an initially plated cell (Figure 1Fi). At 30 min, there were lower percentages of FliI OE Spinorphin spreading cells than FliI KND or WT spreading cells ( 0.05). The initial spreading process was largely complete by 60 min for all cell types, and at 60 min, there were lower percentages of spread FliI OE cells than WT or GPX1 FliI KND cells (Figure 1F, i and ii). The dynamics of cell spreading for the three different cell types were similar on monomeric collagen and fibrillar collagen. Spreading on extracellular matrix substrates involves actin filament assembly at the leading edge (Pollard and Borisy, 2003 ), which can manifest as the generation of membrane extensions such as filopodia. Several proteins, such as the small GTPase Cdc42 (Nobes and Hall, 1995 ), N-WASP (Pollard and Borisy,.