Mitochondrial Fragmentation Due to Inhibition of Fusion Increases Cyclin B through Mitochondrial Superoxide Radicals

<div><p>During the cell cycle, mitochondria undergo regulated changes in morphology. Two particularly interesting events are first, mitochondrial hyperfusion during the G₁-S transition and second, fragmentation during entry into mitosis. The mitochondria remain fragmented between late G₂- and mitotic exit. This mitotic mitochondrial fragmentation constitutes a checkpoint in some cell types, of which little is known. We bypass the ‘mitotic mitochondrial fragmentation’ checkpoint by inducing fragmented mitochondrial morphology and then measure the effect on cell cycle progression. Using <i>Drosophila</i> larval hemocytes, <i>Drosophila</i> S2R⁺ cell and cells in the pouch region of wing imaginal disc of <i>Drosophila</i> larvae we show that inhibiting mitochondrial fusion, thereby increasing fragmentation, causes cellular hyperproliferation and an increase in mitotic index. However, mitochondrial fragmentation due to over-expression of the mitochondrial fission machinery does not cause these changes. Our experiments suggest that the inhibition of mitochondrial fusion increases superoxide radical content and leads to the upregulation of cyclin B that culminates in the observed changes in the cell cycle. We provide evidence for the importance of mitochondrial superoxide in this process. Our results provide an insight into the need for mitofusin-degradation during mitosis and also help in understanding the mechanism by which mitofusins may function as tumor suppressors.</p></div

Mitochondrial fusogens regulate cell cycle across cell types.

<p>(A) Punctae of phosphoHistone H3 counted in the proliferating wing pouch region of <i>Drosophila</i> third instar larval wing disc. Mitofusin depleted wing pouch (<i>scallopedGAL4</i>><i>mitofusin</i> RNAi) contains more phosphoHistoneH3 punctae than control wing pouch (<i>scallopedGAL4</i>><i>OR47</i> RNAi, top). (B) Control wing pouches contain 54 ± 7 punctae. Depletion of mitofusin increases the number of punctae to 77 ± 6 and the depletion of mitoPLD increases this to 65 ± 7. Values are mean ± SEM from 3 larave per genotype and the increase in number is significant (<i>p<0</i>.<i>05</i>, Mann-Whitney test). (C) Overexpression of GFP-Drp1 in the wing pouch (<i>scallopedGAL4</i>><i>GFP-Drp1</i>) does not change the number of phosphoHistone H3-positive spots compared to controls overexpressing mCD8-GFP (<i>scallopedGAL4</i>><i>mCD8-GFP</i>). The overexpression of GFP itself can increase the number of punctae. (D) S2R⁺ cells are classified as tubular or fragmented, depending on the predominant kind of mitochondria. Cells with almost equal proportions of fragments and tubules are classified as intermediate. (E) S2R⁺ cells treated with mock dsRNA (targeting GFP) are predominantly tubular (48.77 ± 4.83%), a minority display fragmented mitochondria (19.31 ± 4.71%), while the remaining are classified as intermediate (31.93 ± 1.9%). S2R⁺ cells treated with dsRNA targeting mitofusin are fragmented (98.1 ± 1.2% fragmented). Numbers are mean ± SEM from three experiments with at least 100 cells per treatment per experiment, color scheme as in D. Fragmentation is significant (<i>p<0</i>.<i>01</i>, t-test). (F) The depletion of mitofusin increases the proportion of cells in the G₂/M phase of cell cycle by 8%, compared to controls (gfp dsRNA treated cells). (G) The depletion of mitofusin increases the mitotic index of these cells from 0.025 ± 0.007 (gfp dsRNA treated controls) to 0.125 ± 0.021 and (H) also increases the cell number from 1.1 x 10⁶ ± 0.035 x 10⁶ to 1.35 x 10⁶ ± 0.13 x 10⁶. Differences are significant (F, <i>p</i><0.05, Mann-Whitney test, G, H, <i>p<0</i>.<i>05</i>, <i>t</i>-test).</p

Depletion of mitochondrial fusion proteins increases cell number and mitotic index.

<p>(A) <i>Drosophila</i> homolog of mitofusin or of mitoPLD was depleted by expressing the UAS-RNAi construct in larval hemocytes using <i>collagenGAL4</i>, mitochondrial morphology was imaged by MitoTracker DeepRed staining and classified (as illustrated in Fig 2). In the controls, i.e., cells depleted of the unrelated gene OR47 using <i>collagenGAL4</i>, 64.4 ± 2.3% cells have tubular mitochondrial morphology and 35.6 ± 2.3% cells have fragmented mitochondrial morphology. Upon depleting mitofusin 95.3 ± 2.7% cells, and on depleting mitoPLD 88.2 ± 2.7% cells display fragmented mitochondrial morphology. (B) The number of circulating hemocytes per larva was estimated using a Neubauer chamber. Control larvae contain 5300 ± 900 circulating hemocytes per larva. Depletion of mitofusin (<i>collagenGAL4>marf RNAi</i>) increases this number to 11480 ± 1480, while depletion of mitoPLD (<i>collagenGAL4>mitoPLD RNAi</i>) increases it to 10490 ± 1360 circulating hemocytes per larva. Histogram represents data from 3 independent experiments with 2 to 6 larvae per genotype per experiment. The differences are significant by t-test, <i>p<0</i>.<i>01</i>. (C) Third instar larvae excessively depleted of mitofusin (<i>collagenGAL4/collagenGAL4>marf RNAi/ marf RNAi</i>, right) displayed melanotic masses (arrowhead) consistent with an increase in cell number, while control larvae (CS-Bz, left) did not show such masses. Inset shows a magnified view of one of these melanotic masses. (D) Mitotic hemocytes were detected by phospho (Serine-10) histone H3 labeling, and the stage within mitosis detected by relative arrangement of the tubulin spindle and the chromatin- (i, iv) metaphase, (ii, v) anaphase, (iii, vi) telophase. (E) Correlating with the number of circulating hemocytes, controls have low mitotic index (0.27 ± 0.13). Depletion of mitofusin in the hemocytes increased the mitotic index by 5-fold to 1.73 ± 0.41, while depletion of mitoPLD increased it by 3-fold to 0.8 ± 0.19. The increases are significant (<i>p<0</i>.<i>05</i>, <i>t</i>-test). (F, G) <i>Drosophila</i> larval hemocytes overexpressing GFP-Drp1 (green) have fragmented mitochondria (MitoTracker DeepRed, red). (H) <i>Drosophila</i> larval hemocytes possessing fragmented mitochondria due to Drp1 overexpression have the same mitotic index as cells from control larvae (overexpressing mCD8-GFP) and containing tubular mitochondria. Thus, fragmenting mitochondria by increased fission does not increase mitotic index.</p

Inhibition of mitochondrial fusion influences mitosis through cyclin B.

<p>(A) Doublet-discrimination module operating on all events to identify single cells with unreplicated and replicated DNA on the basis of pulse area and pulse width using the DNA stained by propidium iodide to generate a signal. Gate identifies single cells used for further analysis. (B) Negative control for the cyclin B staining, shows S2R⁺ cells treated with secondary antibody but not the primary anti-cyclin B. Fluorescence detected (Y-axis) is non-specificty, and is used to determine the gate for the cyclin B positive population. (C) Controls (S2R⁺ cells treated with gfp dsRNA) contain 12.2% cyclin B positive cells. (D) In S2R⁺ cells depleted of mitofusin, the population of cyclin B positive cells increases to 18.8%. (E, F) The content of cyclin B in the positive population (gates in C, D) increases from 66.3 a.u (controls, E) to 71.7 a.u. upon depletion of mitofusin (F).</p

Kinetics of cell cycle progression.

<p>Kinetics of cell cycle progression.</p

Mitochondrial superoxide governs cell number.

<p>(A) Pseudo-colored images of <i>Drosophila</i> larval hemocytes derived from control (<i>collagenGAL4>OR47 RNAi</i>) and mitofusin depletion (<i>collagenGAL4>marf RNAi</i>) and stained with dihydoethidium to monitor superoxide content. Warmer colors in the images corresponding to higher intensities in the LUT bar match greater dihydroethidium fluorescence indicating greater superoxide. (B) Depletion of mitofusin increases the total fluorescence per cell from 2.5 x 10⁷ ± 1.3 x 10⁷ a.u. to 6.3 x 10⁷ ± 3.5 x 10⁷ a.u. Values are mean ± SD from a single experiment with 30 cells per genotype. (C) Control larvae contain 4350 ± 647 circulating hemocytes. Depleting the mitochondrial MnSOD increases this number to 9716 ± 240 circulating hemocytes per larva, however depleting cytosolic SOD (SOD1) does not change the circulating hemocyte number significantly (5766 ± 1186). Values are mean ± SEM from 3 experiments, containing 2–5 larvae per genotype per experiment. Increase in cell number for MnSOD RNAi is significant (<i>p<0</i>.<i>05</i>, t-test). (D) Mitofusin depleted larvae, also expressing RFP, contain 11488 ± 2630 circulating hemocytes. Depletion of MnSOD along with depletion of mitofusin does not increase the cell numbers further (9240 ± 2100 circulating hemocytes per larva). However, the overexpression of MnSOD in larval hemocytes depleted of mitofusin restores the cell number (3675 ± 650) to wild-type levels. Values are mean ± SEM from 3 experiments, containing 2–5 larvae per genotype per experiment. Decrease in cell number for MnSOD overexpression is significant (<i>p<0</i>.<i>05</i>, t-test). (E) Depletion of mitofusin and overexpression of MnSOD decreases the total superoxide content (as inferred from dihydroethidium fluorescence), from 8 x 10⁸ a.u. ± 1.5 x 10⁸ a.u. (the depletion of mitofusin alone) to 5.4 x 10⁸ ± 0.7 x 10⁸. Values are mean ± SD from a three experiments with 50 cells per genotype.</p