Figure 5. 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.
Figure 5.

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.

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