ER sliding events occur on a nocodazole-resistant population of MTs, which is consistent with MT acetylation. (A) Merged image of COS-7 cell expressing GFP–Sec61-β at t = 0 (green) and t = 30 s (red). Arrows indicate an unchanged ER position (yellow), a new sliding event (white), or a position of ER rearrangement (blue; see Video 1). (B) Examples of a TAC and sliding event (top and bottom panel, respectively). Dynamic events were captured by coexpressing mCherry–α-tubulin (red) and either YFP-STIM1 (TAC; green) or GFP–Sec61-β (sliding; green). Images shown are 10 s or 40 s apart for TAC versus sliding, respectively. Arrows indicate ER movement. (C) Graph of speed of TAC (n = 10) versus sliding events (n = 30). **, P = 4.3 × 10−10; unpaired t test. (D) Number of sliding events longer than 1 µm in a 10 × 10–µm region during 5 min before and 15–20 min after 5 µM nocodazole treatment (n = 4 cells; see Videos 2 and 3). (E) Number of TAC events as in D in four different YFP-STIM1–expressing cells. (F) Example of an ER sliding event in a GFP–Sec61-β– and mCherry–α-tubulin–expressing cell 15 min after nocodazole treatment. Arrows depict the movement of an ER tubule along an MT. (G) Immunofluorescence staining of α-tubulin and acetylated tubulin in cells treated for 0, 5, 10, and 20 min with 5 µM nocodazole (NZ) before fixation. (H) Graph of the percentage of MTs containing acetylation staining over half of its length or >5 µm of its length at various times of nocodazole treatment (n = 3 cells per time point). (C and H) Error bars show standard deviation. Bars: (A and G) 10 µm; (B and F) 1 µm.
