Impairment of Ecad cis dimerization alters the actin dynamics of cells spread on Ecad-Fc. Cells coexpressing wt Ecad-GFP or cis-Ecad-GFP and LifeAct-Ruby were seeded on Ecad-Fc substrates for 2 h and then subjected to spinning disk live-cell imaging for 3 min at a frequency of one image per 500 ms. (A) Still Images of LifeAct-Ruby distribution. Bar, 25 µm. The actin retrograde flow was quantified by kymograph analysis (yellow lines 1–3, 1 pixel width, perpendicular to the cell membrane in Ecad dense region). Superimposed on the kymographs are the means of actin retrograde flow speed for wt Ecad (n = 156 kymographs from 26 cells) and cis-Ecad (n = 192 kymographs from 32 cells) cells. The actin retrograde flow was significantly faster for cis-Ecad expressing cells than for cells expressing wt Ecad (P ≤ 0.0002, Student’s t test). (B) Similar kymographs of the LifeAct-Ruby signal extending on a longer time window revealed the cyclic protrusion of the edge of wt Ecad and cis-Ecad expressing cells. (C) Quantification of the maximum amplitude and frequency of membrane protrusions (mean values ± SEM; n = 100 kymographs from 26 cells for wt Ecad cells and n = 130 kymographs from 32 cells for cis-Ecad expressing cells). ****, P ≤ 0.0001; ***, P ≤ 0.005, Student’s t test.
Impairment of Ecad cis dimerization alters the actin dynamics of cells spread on Ecad-Fc. Cells coexpressing wt Ecad-GFP or cis-Ecad-GFP and LifeAct-Ruby were seeded on Ecad-Fc substrates for 2 h and then subjected to spinning disk live-cell imaging for 3 min at a frequency of one image per 500 ms. (A) Still Images of LifeAct-Ruby distribution. Bar, 25 µm. The actin retrograde flow was quantified by kymograph analysis (yellow lines 1–3, 1 pixel width, perpendicular to the cell membrane in Ecad dense region). Superimposed on the kymographs are the means of actin retrograde flow speed for wt Ecad (n = 156 kymographs from 26 cells) and cis-Ecad (n = 192 kymographs from 32 cells) cells. The actin retrograde flow was significantly faster for cis-Ecad expressing cells than for cells expressing wt Ecad (P ≤ 0.0002, Student’s t test). (B) Similar kymographs of the LifeAct-Ruby signal extending on a longer time window revealed the cyclic protrusion of the edge of wt Ecad and cis-Ecad expressing cells. (C) Quantification of the maximum amplitude and frequency of membrane protrusions (mean values ± SEM; n = 100 kymographs from 26 cells for wt Ecad cells and n = 130 kymographs from 32 cells for cis-Ecad expressing cells). ****, P ≤ 0.0001; ***, P ≤ 0.005, Student’s t test.
