Figure 7
A serine phosphorylation site in Mena11a regulates its function.
(A) Alignment of Mena11a protein sequences across species. Blue: conserved serine 3 in the 11a insertion sequence. (B–H): MTLn3 cells, stably expressing Mena11a and Mena11a S > A mutant stimulated with 5 nM EGF. (B) DIC images of membrane protrusion during stimulation. Arrowheads indicate dampened membrane protrusions in Mena11a cells, lamellipodial protrusions in Mena11a S > A mutant cells. (C) (Top): Membrane protrusion kinetics of >195 cells. (Bottom): At t = 180 seconds after stimulation. Unpaired t-test **p < 0.01. (D) Kymographs from time-lapse DIC movies of MTLn3 cells stably expressing Mena11a and the Mena11a S > A mutant, and stimulated with 5 nM EGF for 300 seconds. Kymographs demonstrate lamellipodial activity; ascending contours of edge represent protrusion events, descending contours of edge represent withdrawal events. (E) Box and whisker plots of time, protrusion persistence, and velocity of individual protrusion events for MTLn3 cells stably expressing Mena11a and the Mena11a S > A mutant while being stimulated with 5 nM EGF. Center-line of box indicates the median, top indicates the 75th quartile, bottom indicates the 25th quartile. Whiskers represent 10th and 90th percentiles. Data from 90 events of protrusion; Unpaired t-test ***p < 0.005, n.s.: not significant. (F) Barbed end incorporation in cells after stimulation for 60 seconds. Barbed ends and F-actin visualized with rhodamine-G-actin and phalloidin labeling, respectively. Scale bar, 10 μm. Insets at 38X magnification show barbed end distribution at leading edge. (G) Quantification of relative number of barbed ends at leading edge after stimulation for 60 seconds in >20 cells. Unpaired t-test, n.s. not significant. (H) Normalized pixel intensities of relative number of barbed ends plotted as a function of distance from the cell edge (mean ± SEM) of cells after stimulation for 60 seconds. (C,E,G): Results done in triplicates, error bars represent SEM.
