Fig. 5.

ER tubule junction dynamics is reduced in ATL-depleted cells. (A) COS7 were transfected with ss-mCherry-KDEL, treated with scrambled siRNAs (SCR), and subjected to live cell imaging. Movies with 1-s frame rate were acquired, and the ER was segmented as described for fixed cells in Fig. 1 A and B. Example frames of the movie are shown at the indicated time points. (Scale bar, 10 µm.) (B) As in A but the cells were treated with siRNAs to ATL2 and ATL3. (C) The skeletonized ER network obtained after processing the images of the movie shown in A was superimposed at the indicated time points. (D) As in C but the ER skeletons were obtained from movies shown in B. (E) The total 3WJ number density per ER skeleton length (n) was determined in each frame of the movies and plotted over time for the indicated conditions (SCR, control; ATLdKD). (Right) Quantification of the mean 3WJ number of all movies analyzed. (N = 13 SCR cells, and 14 ATLdKD cells *** indicates statistical significance P < 0.001 determined by Welch's test.) (F) As in E but the equilibration rate was determined for each movie by calculating the ratio between the number of 3WJs and the mean over all time points. (Right) The mean coefficient of variation (CV) was determined over all movies analyzed. (N = 13 SCR cells and 14ATLdKD cells. ** indicates statistical significance P < 0.01 determined by Welch's test.) (G) Example frames from 10-min-long movies demonstrating the difference in network dynamics in newly forming ER areas between SCR and ATLdKD cell. (Scale bar, 10 µm.)