Figure 1.
Mitochondria localize to the cytokinetic cleavage furrow in dividing HeLa cells.
(A) Spinning disk confocal time-lapse images of HeLa cells stained with 40 nM MitoTracker Red to visualize mitochondria. The time points are representative of five stages of division from metaphase to late-cytokinesis. Shown are the following: a single focal plane from the center of the confocal stack (upper row), a maximum projection of the full Z-stack (middle row) and a merge of the DIC image with the mitochondrial single focal plane (bottom row). The full time-lapse can be seen in Movie S1. Note the enrichment of mitochondria in the region of the cleavage furrow and reduction at the cell poles as division progresses. Yellow arrowheads indicate the position of the cleavage furrow. Time is given in minutes after anaphase onset. Bar, 10 µm. (B) Quantification of the distribution of mitochondria from cell pole to equator at each representative stage of division. An overlay of all five stages is also shown (last panel). The normalized distance from cell pole to equator is displayed on the x-axis and the average mitochondrial fluorescence intensity is displayed on the y-axis. Data are represented as mean +/– SEM (25 cells, N = 100) and lines fitted by non-linear regression.
Figure 2.
Mitochondria localize to the cleavage furrow in C2C12, Vero and Ptk2 cells.
Fixed images of dividing C2C12, Vero and Ptk2 cells in early cytokinesis (C2C12), mid cytokinesis (Vero) and late cytokinesis (Ptk2). Cells were co-stained for mitochondria (MitoTracker Red, red), actin (Phallodin-488, green) and DNA (DAPI, blue). Bar, 10 µm.
Figure 3.
Mitochondrial localization in cytokinesis is distinct from the endoplasmic reticulum, Golgi and lysosomes.
Fixed images of HeLa cells in late cytokinesis co-stained for mitochondria (MitoTracker Red, red), DNA (DAPI, blue) and endoplasmic reticulum (anti-GRP-170), Golgi (anti-Golgin-97) or lysosomes (anti-Lamp-1) shown in green. Bar, 10 µm.
Figure 4.
Mitochondria localize to the cleavage furrows in multipolar and monopolar cytokinesis.
(A) Live-imaging of an aberrant HeLa cell with a tripolar spindle undergoing cytokinesis stained with MitoTracker Red to visualize mitochondria. Three frames are shown from early to late cytokinesis. (B) Live-imaging of HeLa cells stained with MitoTracker Red that were treated with Monastrol to generate monopolar spindles then induced into cytokinesis with Purvalanol A. The time points shown are representative of five stages of monopolar division. The full time-lapse can be seen in Movie S2. Time is given in minutes after the first visible sign of chromosome polarization (indicating the onset of monopolar division). (C) Same as (B), but cells were fixed and stained for mitochondria (MitoTracker Red, red), microtubules (anti-alpha-tubulin, green), actin (Phalloidin-488, magenta) and DNA (DAPI, blue). Yellow arrowheads indicate the position of the cleavage furrow. Bar, 10 µm.
Figure 5.
Mitochondrial recruitment to the cleavage furrow is not dependent on F-actin.
(A) Live-imaging of HeLa cells treated with DMSO (upper row), Latrunculin A (middle row) or Jasplakinolide (bottom row) and stained with MitoTracker Red to visualize mitochondria. Five representative frames are shown from metaphase to late cytokinesis. The full time-lapse can be seen in Movie S3. Yellow arrowheads indicate the position of the cleavage furrow. Bar, 10 µm. (B) Quantification of the distribution of mitochondria from cell pole to equator at each stage of division in cells treated with DMSO (left panel; 11 cells, N = 44), Latrunculin A (middle panel; 8 cells, N = 32) and Jasplakinolide (right panel; 8 cells, N = 32). The normalized distance from cell pole to equator is displayed on the x-axis and the average mitochondrial fluorescence intensity is displayed on the y-axis. Data are represented as the mean and lines fitted by non-linear regression (see also Figure S3).
Figure 6.
Mitochondrial recruitment to the cleavage furrow is dependent on microtubules.
(A) Live-imaging of HeLa cells treated with DMSO (top row), Early Nocodazole (second row), Late Nocodazole (third row) or Taxol (bottom row) and stained with MitoTracker Red to visualize mitochondria. Five representative frames are shown from metaphase to late cytokinesis. Treatments were added to live cells either 2 (DMSO, Noco Early, Tax) or 4 minutes (Noco Late) after anaphase onset. Note the mislocalized mitochondria at the cell poles (blue arrows) in drug-treated cells compared with DMSO-treated control cells. Red asterisks indicate the first time point following drug-addition and yellow arrowheads indicate the position of the cleavage furrow. The full time-lapse can be seen in Movie S4. Bar, 10 µm. (B) Quantification of the distribution of mitochondria from cell pole to equator at each stage of division in cells treated with DMSO (top left; 8 cells, N = 32), Early Nocodazole (top right; 8 cells, N = 32), Late Nocodazole (bottom left; 10 cells, N = 40) and Taxol (bottom right; 6 cells, N = 24). The normalized distance from cell pole to equator is displayed on the x-axis and the average mitochondrial fluorescence intensity is displayed on the y-axis. Data are represented as the mean and lines fitted by non-linear regression (see also Figure S4). (C) Quantification of mitochondrial inheritance by daughter cells in control (Untreated and DMSO) and drug-treated (Noco Late and Taxol) cells. Data are represented by a box and whisker plot showing maximum, minimum, upper and lower quartiles and sample median. n.s = not significant.