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Fig. 3. Actin assembly mechanisms. (A) G- and F-actin pool were isolated from keratinocytes treated with 0.2 µM latrunculin, or left untreated, for 5 minutes. After separation using SDS-PAGE and Coomassie staining, bands were quantified. After latrunculin treatment, there were increased levels of G-actin, with a concomitant reduction in the amount of F-actin. (B) Global levels of G-actin and F-actin pools were quantified by immunofluorescence during a time course after induction of cell-cell contacts. Keratinocytes were stained with DNAse1 (G-actin) and phalloidin (F-actin) and the whole optical field quantified for each fluorophore. No significant changes were observed in the relative concentration of either actin pool during the time course examined by staining or SDS-PAGE (data not shown). Values are expressed as percentage of total actin. (C) Quantification of immunofluorescence of G- and F-actin pools at the junctional-actin region. Only junctions where junctional actin was clearly separated from thin bundles were examined. At time zero, areas of close apposition of neighbouring membranes were quantified. Data in A-C are representative of two independent experiments. Error bars represent standard deviation. *P<0.05. (D) Polymerization of actin at cell-cell junctions occurs preferentially via the barbed end. After formation of new contacts for different amounts of time, cells were permeabilized for 2 minutes in cytoskeleton buffer containing labelled Alexa Fluor 568-G-actin (0.45 µM), in the presence or absence of 2 µM cytochalasin B (see text for details). After fixation, E-cadherin, total actin (phalloidin) and incorporated actin were visualized. Results are representative of at least three independent experiments. Arrows indicate labelled actin incorporation at junctions. Bar, 50 µm.
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