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.

Retrospective imaging demonstrates ER dynamics that occur on acetylated MTs. (A–D, left) ER sliding events were captured during live cell imaging of cells expressing GFP–Sec61-β and mCherry–α-tubulin at the times indicated (in seconds). (right) Cells were then fixed and stained with antibody to α-tubulin and acetylated α-tubulin and reimaged. Merged images are shown with the indicated colors. (A and B) Examples of ER sliding on MTs with homogeneous acetylation staining. Sliding event progression during live imaging is marked with arrows for the starting position (white) and for end position (yellow). (C) An example of ER sliding from starting position (white arrows) to a patch of acetylation (yellow arrows) and retracting to a second acetylation patch (blue arrows). (D) An example of a sliding event, followed by an ER dynamic that leads to a ring formation (dashed triangles) around an acetylated MT patch. Bars, 1 µm.

Acetylated MTs and ER sliding events correlate with MT curvature. (A) Immunofluorescence staining of α-tubulin and acetylated α-tubulin on untreated cells. (B) Diagram of curvature. (C, left) Graph of curvature of acetylated (n = 37) or nonacetylated MTs (n = 37). Acetylated MTs were picked by drawing a line through the cell at a point containing the maximum number of acetylated MTs. α-Tubulin staining of MTs that were not acetylated were picked along that line if they were isolated at the line. The mean curvature of the MT for 2.5 µm on either side of the line (or 5 µm total) was determined by “imodcurvature.” (right) The frequency of MT curvature in groups of indicated values for nonacetylated MTs or acetylated MTs. (D) Curvature measurements for dynamic MTs (labeled with EB3; n = 37), TAC MTs (labeled with YFP-STIM1; n = 16), and ER sliding MTs (GFP–Sec61-β; n = 41). Right panel shows frequency of each event. (C and D) Error bars show standard deviation. P-values were determined by unpaired t test: **, P = 3.7 × 10⁻⁷ (C); and **, P < 0.001 (D). Bar, 10 µm.

Hyperacetylation of MTs by drug treatment increases ER sliding but not TAC events. (A) COS-7 cells were fixed and immunofluorescently labeled with α-tubulin and acetylated α-tubulin after treatment with 125 nM TSA for 0, 30, 60, and 90 min. (B) COS-7 whole-cell lysate from cells treated for 0, 30, 60, and 90 min with 125 nM TSA were immunoblotted (IB) with antibodies to acetylated α-tubulin and α-tubulin. (C) Number of sliding events longer than 1 µm in a 10 × 10–µm region during 5-min period before and 30–35 and 60–65 min after treatment with 125 nM TSA (n = 4 different GFP–Sec61-β–expressing cells; see Videos 4 and 5). (D) Number of TAC events, as in C, in four different YFP-STIM1–expressing cells. (E) Graph of the curvature of ER sliding events (GFP–Sec61-β) from 60–90 min after 125 nM TSA treatment (n = 30) as compared with ER sliding curvature from Fig. 3 and the curvature of a representative population of MTs from a cell treated for 60 min with TSA (n = 35). Error bars show standard deviation. P-value was determined by unpaired t test: **, P < 0.001. (F) As in A, cells were treated for 0, 1, and 6 h with TSA. (G–I) As in A–C for cells treated with 10 µM tubacin. Bars, 10 µm.

ER and mitochondria dynamics are coupled, and mitochondrial localization is enriched on acetylated MTs. (A) Merged image of a COS-7 cell showing colocalization of mito-dsRed and GFP–Sec61-β (left), mito-dsRed and immunolabeled for acetylated α-tubulin (middle), and immunolabeled for α-tubulin and acetylated α-tubulin (right). Dashed boxes indicate mitochondria imaged in C. (B) Time-lapse of ER–mitochondria interactions in cells expressing GFP–Sec61-β and mito-dsRed at the times indicated (see Videos 6–8). Left panel shows an ER tubule moving behind a mitochondrion. Middle panel shows a mitochondrion moving behind an ER tubule. Right panel shows massive co-rearrangement of both. Arrows indicate the position of mitochondrion (white) or ER (yellow) at each time point. (C, left) As in B. (right) Cells were fixed, immunolabeled with antibodies to α-tubulin and acetylated α-tubulin, and reimaged. Images were merged and shown with indicated colors. (D) As in C, except cells were imaged between t = 8 and t = 10 min after 5 µM nocodazole treatment (see Videos 9 and 10). (top) Arrows mark an ER tubule moving behind a dynamic mitochondrion; after fixation it colocalizes with acetylated MTs. (bottom) Arrows mark where a dynamic mitochondria tubule grows and retracts, followed by a growing ER tubule on an acetylated MT. (E) Percentage of mitochondria that retrospectively colocalize to sites of MT acetylation in untreated cells (77% of 57 total, 72% of 29 static, and 82% of 28 dynamic mitochondria from 10 cells; left) and cells treated for 10 min with 5 µM nocodazole (71% of 48 total, 59% of 22 static, and 81% of 26 dynamic mitochondria from 8 cells; right). (F) Total number of mitochondria from the experiment described in D that are dynamic before (dark gray; n = 36) versus after nocodazole treatment (light gray; n = 28). Bars: (A) 10 µm; (B–D) 1 µm.

Early endosomes that interact with the ER are not enriched on acetylated MTs. (A) Merged image of a COS-7 cell showing colocalization of mCherry-Rab5 and GFP–Sec61-β. Dashed boxes indicate early endosomes imaged in B. (B) Time-lapse images of ER–endosome interactions in cells expressing GFP–Sec61-β and mCherry-Rab5 at the times indicated. Arrows indicate the position of the endosome at each time point. (C) Merged images of a fixed COS-7 cell expressing GFP–Sec61-β and mCherry-Rab5 and immunolabeled for α-tubulin and acetylated α-tubulin. Images shown are merges for the indicated labels. (D) Table indicating localization of endosomes to ER and MTs. (E) Percentage of MT-localized endosomes that colocalize to acetylated MTs. Left bar is total MT localized (n = 61 of 238; 26%), middle bar is ER and MT localized (n = 47 of 162; 29%), and right bar is MT and non-ER localized (n = 14 of 76; 18%) from five cells. (F) Percentage of endosomes that colocalize with acetylated MTs before (dark gray) and 10 min after (light gray) 5 µM nocodazole (NZ) treatment. Left bars are all endosomes (untreated n = 61 of 426 [14%] from five cells; nocodazole-treated n = 47 of 443 [11%] from five cells). Right bars are ER localized (untreated n = 47 of 266 [18%]; nocodazole-treated n = 29 of 198 [15%]). Bars: (A and C) 10 µm; (B) 1 µm.