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Fig. 6. mDia2 maintains free filament barbed ends at focal adhesions. Barbed-end actin incorporation (green) and fluorescent phalloidin staining of F-actin (red) in a control cell (A) and mDia2 antibody-injected cell (anti-mDia2; B). To find mDia2 antibody-injected cells after permeabilization, mDia2 antibody was co-microinjected with fluorescent tubulin that can be seen incorporated into microtubules that are easily distinguishable from actin structures. Arrows indicate plaques at the termini of actin bundles (arrowheads). (C) Intensity ratio of fluorescent actin incorporation marking free barbed filament ends relative to total F-actin (phalloidin) at the terminal 2 µm of actin bundles (n=10 cells per treatment, 
5-10 bundles/cell). (D) Intensity ratio of fluorescent actin incorporation marking free barbed filament ends relative to total F-actin (phalloidin) (± s.e.m.) from the leading-edge into the cell center is not altered by 
mDia2 inhibition, n=10 cell per treatment, three regions per cell. (E,F) Fluorescent phalloidin, barbed-end actin incorporation, and paxillin immunofluorescence in a control cell (E) and an mDia2 antibody-injected cell (F). In a control cell, actin incorporates at focal adhesions that are positive for paxillin (arrowheads), but this does not occur in mDia2 antibody-injected cells. Note the altered focal adhesion morphology in cells injected with mDia2 antibody as seen by paxillin immunofluorescence (F), suggesting that antibody inhibition renders focal adhesions labile to the pre-permeabilization procedure required for localization of free barbed ends.
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