CALI of EGFP-CP induces the formation of dorsal ruffles and filopodia only in the knockdown/rescue background. (A) DIC images of Rat2 EGFP-CP knockdown/rescue (KDR) cell line before and 3 min after CALI of EGFP-CP (Left) and fluorescent images of EGFP-CP before and immediately after CALI (Right); area of irradiation is indicated with a black circle. (Scale bar: 20 μm.) (B) Time-lapse images of the irradiated regions of EGFP-CP KDR cells (Upper) and cells expressing EGFP-CP without knockdown of endogenous CP (Lower; EGFP-CP no KD) before and after CALI. (Scale bar: 10 μm.) (C) DIC images (Left) and Sobel filtered binary images (Right; for details see Methods) before (Upper) and 2 min after (Lower) CALI. The white circle represents the 12-μm-diameter center of the irradiated area that was used to measure the local edge detection index. Raw edge index values are shown next to their respective images. (D) Plot showing the normalized edge detection values in the irradiated region over time for EGFP-CP KDR cells (n = 20), EGFP-CP no KD cells (n = 8), and cells expressing EGFP (n = 12); the break in each line represents when the movies were paused to perform the laser irradiation. Error bars are SEM.

CALI of EGFP-Mena in Mena/VASP-deficient cells induces more stable lamellipodial protrusions. (A–C) Phase contrast image of an MV^D7 cell (A), fluorescent image of EGFP-Mena immediately after laser irradiation (B), and kymograph of a representative EGFP-Mena CALI experiment (C). (A) Area of irradiation is indicated with a black circle; the line used to generate the kymograph in is indicated by a white line. (In C, horizontal scale bar: 3 min; vertical scale bar: 5 μm.) (D) Box and whisker plots displaying results from kymograph analysis. Kymographs were drawn in the irradiated regions of cells expressing either EGFP-Mena (Left) (34 protrusions before CALI, 38 protrusions 0–5 min after CALI, 34 protrusions 5–10 min after CALI, n = 11 experiments), or EGFP (Center) (39 protrusions before CALI, 49 protrusions after CALI, n = 9 experiments), and also in the nonirradiated regions of cells expressing EGFP-Mena (Right) (37 protrusions before CALI, 53 protrusions 0–5 min after CALI, n = 11 experiments). The top whisker indicates the 90th percentile, the top line of the box is the 75th percentile, the middle line of the box is the 50th percentile, the bottom of the box is the 25th percentile, the bottom whisker indicates the 10th percentile, the mean is indicated with the dotted line, and outlying points are shown as closed black circles. P values for EGFP-Mena experiments are Tukey's HSD post hoc test (persistence ANOVA P = 0.01, velocity ANOVA P = 0.02). P values for all other experiments are from Student's t test.

A lentiviral system for simultaneous knockdown and rescue with a functional EGFP-fusion complement. (A) Schematic of the lentiviral expression vector used for simultaneous knockdown of rat CPβ (rCPβ) and rescue with EGFP-human CPβ (EGFP-hCPβ). Open triangles, LoxP sites. (B) DIC images of a WT Rat2 fibroblast (Top) and Rat2 fibroblasts infected with only the knockdown portion of the vector (Middle) or the complete knockdown/rescue construct (Bottom). (Scale bar: 20 μm.) (C) Western blot of WT Rat2 fibroblasts (left lane) and a clonally derived Rat2 cell line infected with the knockdown/rescue vector.

CALI of EGFP-CP in KDR cells generates a local increase in barbed ends of actin filaments and dorsal F-actin. (A) DIC image (Left), fluorescent image of Alexa Fluor 568-labeled phalloidin from a deconvolved three-dimensional stack (Center), and overlay (Right) from an EGFP-CP KDR cell after CALI. Area of irradiation is represented by the red circle. (Scale bar: 20 μm.) (Lower) Zoomed versions of Upper. (B) Fluorescent image of EGFP-CP immediately after laser irradiation (Left) and a pseudocolored fluorescent image of actin filament barbed-ends labeled with Alexa Fluor 568-conjugated G-actin at 3 min post-CALI. (C) Graph showing the relative barbed end increase in the irradiated regions of Rat2 fibroblasts expressing the following constructs: EGFP (n = 12), EGFP-CP KDR (n = 19), EGFP-CP no KD (n = 7), EGFP-Mena (n = 9), and Mena/VASP null MEFs (MV^D7 cells) expressing EGFP-Mena (n = 5). Error bars are 95% confidence interval (C.I.). ∗, P < 0.01, compared with all other groups (Tukey's honestly significantly different (HSD) post hoc test; ANOVA P = 6.8 × 10⁻⁸).