Drosophila Fwd and mammalian PI4Kβ rescue the cytokinesis defect of fwd mutant males. (A) Phase–contrast micrographs of early round haploid spermatids from wild-type, fwd mutant (fwd/Df), and fwd mutant flies expressing GFP-Fwd or GFP-FwdKD. Nuclei appear as light-colored discs, mitochondrial derivatives as dark-colored organelles. Multinucleate spermatids containing four haploid nuclei accompanied by an enlarged mitochondrial derivative (arrowheads) indicate failure of cytokinesis during meiosis I and II. Note that cells with multiple mitochondrial derivatives are a common artifact of squashing spermatocytes with a coverslip (Cenci et al., 1994). Bar, 20 µm. (B and C) Quantification of spermatids after successful cytokinesis (1 nucleus/mitochondrial derivative) versus cytokinesis failure (>1 nucleus/mitochondrial derivative) for fwd mutant and rescued flies. A minimum of 400 cells derived from at least 10 males was counted for each genotype. (B) Rescue of fwd/Df with GFP-Fwd or GFP-FwdKD (GFP-KD5, GFP-KD10). (C) Rescue of fwd/Df with bovine PI4Kβ (bPI4K) or with wild-type (hPI4K#1, hPI4K#2) or kinase-dead (hKD#1, hKD#4) human PI4Kβ. (D) Quantification of the probability of meiotic cytokinesis failure of fwd mutant and rescued flies. Error bars show the least sum of squares difference between the observed proportion of cells with 1, 2, or 4 nuclei/mitochondrial derivative and the proportion predicted from the model, based on the calculated probability (Dyer et al., 2007). Because the error bars are small, the model appears to accurately reflect the probability of cytokinesis failure. The probability of cytokinesis failure is negligible in control flies (+/+, fwd/+, Df/+) and nearly 1 (100%) in fwd mutant flies (fwd/Df), which are completely or partially rescued by wild-type or KD transgenes, respectively.
