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1.
Figure 1.

Figure 1. From: Dual roles for the Drosophila PI 4-kinase Four wheel drive in localizing Rab11 during cytokinesis.

PI4P-containing organelles localize at the midzone during cytokinesis. Phase–contrast (phase) and corresponding fluorescence time-lapse images of a dividing spermatocyte expressing RFP-PH-FAPP as a marker for PI4P and sGFP as a secretory marker. Fluorescence micrographs are shown as inverted images for clarity. Times are in min:sec. Arrowheads (t = 00:00) indicate parafusorial membranes. Arrows indicate the midzone. Colocalization of sGFP (green) and PI4P (magenta) appears white (overlay). Bar, 10 µm.

Gordon Polevoy, et al. J Cell Biol. 2009 December 14;187(6):847-858.
2.
Figure 3.

Figure 3. From: Dual roles for the Drosophila PI 4-kinase Four wheel drive in localizing Rab11 during cytokinesis.

PI4P and Fwd colocalize on Golgi membranes. (A and B) Phase–contrast (phase) and corresponding fluorescence micrographs of live squashed wild-type or fwd mutant (fwd/Df) spermatocytes expressing RFP-PH-FAPP (PI4P) with Fws-GFP (A) or with GFP-Fwd or GFP-FwdKD (B). Colocalization (arrows) of Fws-GFP, GFP-Fwd, or GFP-FwdKD (green) and RFP-PH-FAPP (magenta) appears white (overlay). Bar, 20 µm. Note the diffuse RFP-PH-FAPP signal in fwd/Df (A) and GFP-FwdKD; fwd/Df (B) spermatocytes (bottom panels). (C) Phase–contrast (phase) and corresponding fluorescence micrographs of a squashed dividing spermatocyte coexpressing GFP-Fwd and RFP-PH-FAPP (PI4P). Note the presence of PI4P and absence of GFP-Fwd at the midzone (arrowheads). Bar, 20 µm. (D) Phase–contrast (phase) and corresponding fluorescence micrographs showing time-lapse images of a dividing spermatocyte expressing GFP-Fwd. Fluorescence micrographs are inverted for clarity. Times are in min:sec. GFP-Fwd is primarily in puncta at the poles (t = 08:00–20:00) and fails to accumulate at the midzone (arrows). Note background mitochondrial autofluorescence (elongated dark structures) due to long exposure times. Bar, 10 µm.

Gordon Polevoy, et al. J Cell Biol. 2009 December 14;187(6):847-858.
3.
Figure 4.

Figure 4. From: Dual roles for the Drosophila PI 4-kinase Four wheel drive in localizing Rab11 during cytokinesis.

fwd is required for midzone accumulation of PI4P and sGFP. (A and B) Phase–contrast (phase) and corresponding fluorescence micrographs of live preparations of dividing wild-type and fwd mutant (fwd/Df) spermatocytes coexpressing sGFP and RFP-PH-FAPP (PI4P). Colocalization of sGFP (green) and RFP-PH-FAPP (magenta) appears white (overlay). (Arrows) Midzones of late telophase cells that have been squashed (B) or in which the cleavage furrow has regressed (fwd/Df) (A). Bars, 20 µm. (A) sGFP and PI4P colocalize at the midzone in wild type (wt; top panels), but not in fwd mutant (middle and bottom panels). (B) In squashed wild-type cells, sGFP and PI4P colocalize at the midzone (top panels). The few sGFP-positive puncta present at the midzone in fwd mutant cells (bottom panels) fail to colocalize with RFP-PH-FAPP. (C) Golgi proteins localize to the poles of dividing fwd mutant cells. Fluorescence micrographs of wild-type and fwd mutant cells expressing Fws-GFP (left panels) or stained for Lva (right panels). Distribution of Fws-GFP or Lva relative to nuclei (red) is shown (overlay). (Arrows) Midzones of representative dividing cells. Bar, 20 µm.

Gordon Polevoy, et al. J Cell Biol. 2009 December 14;187(6):847-858.
4.
Figure 6.

Figure 6. From: Dual roles for the Drosophila PI 4-kinase Four wheel drive in localizing Rab11 during cytokinesis.

Rab11 localizes to PI4P-containing organelles and its localization during cytokinesis requires fwd. (A and B) Phase–contrast (phase) and corresponding fluorescence micrographs of live squashed spermatocytes expressing GFP-Rab11 and RFP-PH-FAPP. Colocalization (arrows) of GFP-Rab11 (green) and RFP-PH-FAPP (magenta) appears white (overlay). Arrows indicate the midzone. Bars, 20 µm. (A) GFP-Rab11 and RFP-PH-FAPP (PI4P) colocalize in spermatocytes (top panels) and in dividing cells (bottom panels). Rab11 and RFP-PH-FAPP colocalize on organelles near the midzone of dividing cells (bottom panels). (B) In fwd mutant cells (fwd/Df), GFP-Rab11 and RFP-PH-FAPP colocalize to small puncta in spermatocytes (top panels) and show diffuse localization during cytokinesis (bottom panels). Note that pictures in A and B were taken on different days and the images in B were adjusted for brightness and contrast to reveal weak signals in fwd mutant. (C) Fluorescence micrographs of wild-type and fwd mutant (fwd/Df) male germ cells expressing GFP-Rab11. Arrowheads indicate elongating spermatids. GFP-Rab11 is uniformly distributed in wild type (left), but localizes to linear structures in fwd mutant (right). Bar, 20 µm.

Gordon Polevoy, et al. J Cell Biol. 2009 December 14;187(6):847-858.
5.
Figure 7.

Figure 7. From: Dual roles for the Drosophila PI 4-kinase Four wheel drive in localizing Rab11 during cytokinesis.

Rab11 acts downstream of fwd to promote completion of cytokinesis. (A) Phase–contrast (phase) and fluorescence images of dividing spermatocytes stained for Rab11 (green), Nuf (red), and DNA (blue). Arrows indicate the midzone. Colocalization of Rab11 and Nuf is yellow (overlay). Endogenous Rab11 and Nuf colocalize at the midzone in wild type (top panels). Neither Rab11 nor Nuf accumulate in the midzone in fwd mutant cells (middle panels). Localization of Rab11 and Nuf is restored upon expression of Rab11Q70L (bottom panels). Bar, 20 µm. (B) Quantification of the number of spermatids resulting from successful cytokinesis versus cytokinesis failure (as in Fig. 2, B and C). A minimum of 300 cells derived from at least 10 males was counted for each genotype. fwd mutant testes (fwd/Df) show a large proportion of cells with 2 or 4 nuclei. Overexpression of activated Rab11Q70L (Q70L#1, Q70L#3), but not wild-type Rab11 (Rab11#1, Rab11#3), partially rescues the fwd cytokinesis defect, producing increased numbers of cells with 1, 2, or 3 nuclei.

Gordon Polevoy, et al. J Cell Biol. 2009 December 14;187(6):847-858.
6.
Figure 5.

Figure 5. From: Dual roles for the Drosophila PI 4-kinase Four wheel drive in localizing Rab11 during cytokinesis.

Fwd binds and colocalizes with the recycling endosome regulator Rab11. (A) Yeast two-hybrid assays. (Left) Patches of yeast cells cotransformed with Fwd or FwdKD bait plasmids together with one of several prey plasmids (vector alone, Rab5, Rab7, Rab11, Rab11Q70L, and Rab11S25N). (Right) Filter X-gal assays performed on replicates of these patches. Blue color indicates a positive interaction. (B) Quantification of yeast two-hybrid results. LacZ reporter expression (β-galactosidase units) induced by various combinations of bait and prey plasmids (as in A). Statistically significant differences are: (single asterisk) P < 0.001, (double asterisk) P < 0.05, (triple asterisk) P < 0.01. (C) Co-immunoprecipitation (coIP) of Rab11 with Fwd and FwdKD expressed in COS-7 cells. Immunoblots were probed with anti-HA or anti-Flag, as indicated on the right. IP with anti-Flag antibody pulls down HA-Fwd and HA-FwdKD only when coexpressed with Flag-Rab11 (top panels). IP with anti-HA pulls down Flag-Rab11 only when coexpressed with HA-Fwd or HA-FwdKD (bottom panels). Note that a nonspecific protein of slightly lower mobility than Flag-Rab11 is present in all lanes of the anti-HA IP experiment. (D) Phase–contrast (phase) and corresponding fluorescence micrographs of spermatocytes coexpressing YFP-Rab11 and CFP-Fwd. Colocalization (arrows) of YFP-Rab11 (magenta) and CFP-Fwd (green) appears white (overlay). Bar, 20 µm.

Gordon Polevoy, et al. J Cell Biol. 2009 December 14;187(6):847-858.
7.
Figure 2.

Figure 2. From: Dual roles for the Drosophila PI 4-kinase Four wheel drive in localizing Rab11 during cytokinesis.

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.

Gordon Polevoy, et al. J Cell Biol. 2009 December 14;187(6):847-858.

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