Asterisks indicate a statistical difference (** < 0.01, *** < 0.001, obtained using Dunnetts test against wild-type ezrin). 2.5. to a significant buildup of actin fibers, a decrease in nuclear volume, and an increase in cytoskeletal stiffness. Finally, cell transfection with the dominant negative ezrin FERM domain induces significant morphological and nuclear changes and affects actin, microtubules, and the intermediate filament vimentin, resulting in cytoskeletal fibers that are longer, thicker, and more aligned. Collectively, our results suggest that ezrins phosphorylation state and its intracellular localization plays a pivotal role in cell migration, modulating also biophysical properties, such as membraneCcortex linkage, cytoskeletal and nuclear organization, and the mechanical properties of cells. < 0.05, ** < 0.01, *** < 0.001, obtained using Dunnetts test against wild-type ezrin). 2.2. Subcellular Distribution of Ezrin Mutants From the time-lapse videos, we observed that ezrins intracellular distribution patterns during cell migration were different for different mutants (Figure 2A). Thus, we aimed to identify the relationship between ezrins intracellular distribution and the previously observed biophysical properties. Accordingly, we defined the polarization ratio and peak front-to-back ratio separately to describe the intracellular distribution during migration. The polarization ratio describes the spread of the fluorescence intensity within the cell area, with 1 meaning a full homogeneous spread and 0 concentrated at one point. The peak front-to-back ratio identifies the averaged intracellular location where most protein is found with respect to the direction of cell movement, with 1 representing the cell front and 0 the cell rear. Active ezrin T567D was the most highly localized mutant with the lowest polarization ratio of 0.51 (Figure 2B). Furthermore, its localization was preferentially at the cell rear, displaying the smallest value measured from all mutations for the peak front-to-back ratio (Figure 2C). Conversely, inactive ezrin T567A formed a well-localized ring around the nucleus (Figure 2B). Wild-type ezrin and dominant negative FERM domain displayed the broadest distribution through the cell cytoplasm, yielding the highest values for the polarization ratio of 0.54 (Figure 2B). Open in a separate window Figure 2 The subcellular distribution of ezrin and its mutations during migration. (A) Example fluorescent images of transfected cells obtained from the time-lapse videos. The example cell for ezrin T567D showed clear persistent directional migration, indicated by the arrow. The other example cells showed no clear directional migration. Scale bar 50 m. Box plots show the results of the polarization ratio (B) and peak front-to-back ratio (C). Box plots extend from the 10th to the 90th percentile, whiskers from the 5th to the 95th. The plot shows the relationship between the cell migration velocity and the polarization ratio (D) and peak front-to-back ratio (E), error bars indicate SD. A total of n = 21 (ezrin), n = 45 (ezrin T567D), n = 52 (ezrin T567A), ITGB2 and n = 60 (FERM) cells were analyzed from n = 4 repeats. Asterisks indicate a statistical difference (*** < 0.001, obtained using Dunnetts test against wild-type ezrin). Since cell migration is a dynamic process, the values of the cell migration speed, polarization ratio, and peak front-to-back ratio for each individual cell change during the course of a time-lapse experiment. Therefore, we assessed whether there was a relationship between the instantaneous cell migration SB-568849 speed and intracellular protein distribution patterns. To do so, we pooled together the results from all frames in all videos, and plotted the instantaneous migration speed against the instantaneous protein distribution parameters. We found that there was a strong linear SB-568849 relationship between the migration speed and polarization ratio and peak front-to-back ratio for active ezrin T567D, that is, when active ezrin T567D accumulated at the cell rear, cells migrated faster SB-568849 (Figure 2D,E). Together, these results suggest that active ezrin T567D enhances cell migration by preferentially SB-568849 localizing at the cell rear while the presence of ezrin (in any phosphorylation state) in the vicinity of the nucleus tends to hinder cell migration. 2.3. The Effect of.