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.