GOLPH3 is required for cytokinesis in larval neuroblasts.

<p>(A) GOLPH3 accumulates at the cleavage furrow of larval dividing neuroblasts. Larval neuroblast stained for Tubulin (green), GOLPH3 (red) and DNA (blue). Arrow indicates the midzone. Scale Bar, 5 µm. (B) Western blotting from larval brain extracts. Polyclonal antibodies raised against GOLPH3 protein, recognized a band of the predicted molecular weight. The band is reduced in larval brain extracts from animals expressing <i>UAS::GOLPH3RNAi</i> under control of <i>Tub-GAL 4 Tub-GAL80^ts</i>. α-Tubulin was used as a loading control. Molecular masses are indicated in kilodaltons. (C) Normal male metaphase from wild type and tetraploid male metaphase from animals animals expressing <i>UAS::GOLPH3RNAi</i> under control of <i>Tub-GAL4 Tub-GAL80^ts</i>. Scale Bar, 5 µm. (D,E) Knockdown of <i>GOLPH3</i> in larval neuroblasts affects assembly of a stable, fully constricted contractile ring and impairs formation of a compact central spindle. Localization of Zipper (D), Sep2 (E) and RacGAP50C (F) in late telophase neuroblasts from wild type animals and in animals expressing <i>UAS::GOLPH3RNAi</i> under control of <i>Tub-GAL 4 Tub-GAL80^ts</i>, [<i>GOLPH3RNAi</i>]. Larval brains were stained for Tubulin (green), DNA (blue) and either Zipper, Sep 2 or RacGAP50C (red). Telophase stage is based on chromatin condensation. Arrows point to the midzone. Scale Bar, 5 µm.</p

Localization of PI(4)P is disrupted in telophase cells from <i>GOLPH3</i> mutants.

<p>(A,C) Live spermatocytes expressing either RFP-PH-FAPP (RFP-FAPP) or PLCδ-PH-GFP were imaged at the same exposure time during telophase. Note that the distribution of both RFP-PH-FAPP and PLCδ-PH-GFP is affected in <i>GOLPH3</i> mutant spermatocytes indicating a defective PI(4)P and PI(4,5)P2 subcellular localization and/or synthesis. Arrows indicate accumulation of RFP-PH-FAPP (A) and PLCδ-PH-GFP (C) at the cleavage furrows of wild type cells. (B) Fixed telophase spermatocyes expressing RFP-PH-FAPP (RFP-FAPP) stained for RFP (red), tubulin (green) and DNA (blue). Arrows indicate the cell midzone. Arrowheads indicate the enrichment of RFP signals at the poles of the wild type cell. Scale Bar; 10 µm.</p

GOLPH3 protein interacts with both cytokinetic proteins in testis extracts.

<p>(A,B) Co-IP of GOLPH3 with Zipper and Septins. (A) Equal fractions of protein extracts from testes expressing GFP-GOLPH3 were immunoprecipitated with either mouse anti-GFP (α-GFP) or non-specific mouse IgG (ctrl) and blotted for either Zipper or GFP. (B) Protein extracts from wild type testes were immunoprecipitated with antibodies against <i>Drosophila</i> GOLPH3 (α-GOLPH3) and blotted for either Sep2, Sep1 or GOLPH3. Preimmune serum was used as control (ctrl). (C) Bacterially expressed GST-GOLPH3 was purified by gluthatione-sepharose beads and incubated with testis lysate. GST bound to gluthatione-sepharose beads was used as a negative control. GST-GOLPH3 precipitated Zipper, Pav and Sep1 from testis protein extracts. Ponceau staining (Ponceau) is shown as a loading control. In all the experiments input is 4% of lysates. Molecular masses are indicated in kilodaltons.</p

Mutations in <i>GOLPH3</i> affect Golgi structure in primary spermatocytes.

<p>(A) G2 spermatocytes during the S5 stage, stained for Lva (red), Tubulin (green) and DNA (blue). Spermatocytes at this stage are characterized by three distinct chromatin clumps (corresponding to each major bivalents) and an interphase microtubule cytoskeleton (absence of asters). Left panels show enlargements of Golgi stacks in Lva images. Note that in wild type the size of Lva fluorescent units is approximately two fold when compared to <i>sau^z2217/Df(2L)Exel7010 (sau/Df)</i> mutants. Scale Bar; 10 µm (B) Average number of Golgi bodies per cell (± SEM), visualized by Lva staining in spermatocytes at S5 stage from wild type and <i>sau^z2217/Df(2L)Exel7010 (sau/Df)</i> mutant males. (C) Average size (± SEM), of Golgi stacks quantified by image J (Area in 2D images), in spermatocytes immunostained for Lva at S5 stage. Measures were normalized to control average size (set at 1). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004305#s4" target="_blank">Materials and Methods</a>.</p

<i>Drosophila</i> GOLPH3 is required for spermatocyte cytokinesis.

<p>(A) Multinucleate onion stage spermatids due to cytokinesis failure during meiosis in <i>GOLPH3</i> mutant males [<i>sau^z2217/Df(2L)Exel7010</i> and <i>sau^z2217/l(2)5379</i>] and in males expressing dsRNA against <i>CG7085</i> (<i>GOLPH3RNAi</i>), under control of the <i>Bam-GAL4</i> driver <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004305#pgen.1004305-Chen1" target="_blank">[74]</a>. (Nu) nuclei; (Nk) mitochondrial derivative; (4Nk) large mitochondrial derivative. Scale Bar; 10 µm. (B) Frequencies of spermatids containing 1, 2, 3, 4 or more than 4 nuclei per Nk, in <i>sau^z2217/Df(2L)Exel7010</i> and <i>sau^z2217/l(2)5379</i> mutant males and in males expressing dsRNA against <i>CG7085</i> (<i>GOLPH3RNAi</i>) under control of <i>Bam-GAL4</i>. Oregon R males and males carrying either the <i>Bam-GAL4</i> driver alone or the <i>UAS::GOLPH3RNAi</i> construct alone were used as control. (n); total number of spermatids (C) Alignment of <i>Drosophila</i> GOLPH3 (<i>Dm</i>) protein with human GOLPH3 (<i>Hs</i>). (Blue arrowhead) substitution of Glutamic acid by Lysine (E273K) in the <i>sau^z2217</i> male sterile allele; (red arrows) K167A and R170A mutations; (blue line) clathrin box sequence. (*) fully conserved residue; (<b>:</b>) conservation between groups of strongly similar properties; (<b>.</b>) conservation between groups of weakly similar properties.</p

<i>Drosophila</i> GOLPH3 localizes to the cleavage furrow in dividing spermatocytes.

<p>(A) Western blotting from adult testis extracts. Polyclonal antibodies raised against GOLPH3 protein, recognized a band of the predicted molecular weight. The band is reduced in testis extracts from <i>sau</i><b><i>^z2217</i></b>/<i>Df(2L)Exel7010</i> and <i>GOLPH3RNAi</i> males. α-Tubulin was used as a loading control. (B) Colocalization of GOLPH3 (anti-GOLPH3, red) with the Golgi protein Cog7-GFP (green) in interphase spermatocytes. (C) In dividing spermatocytes GOLPH3 protein (red) was enriched in Golgi derived vesicles and accumulated at the cleavage furrow (arrows) during telophase (green) Tubulin; (blue) DNA. Scale Bar; 10 µm.</p

Diagram illustrating how GOLPH3 function might be involved in cytokinesis.

<p>(A,C,E), wild type; (B, D, F) <i>sau^z2217/Df</i> mutants. (A) Golgi membranes are depicted in grey. Synthesis of PI(4)P at the Golgi requires function of the PI-4 kinase Fwd. Fwd also recruits Rab11 protein to Golgi where it becomes associated with secretory vesicles containing PI(4)P. GOLPH3 localization to the Golgi depends on the PI(4)P phosphoinositide. Wild type function of GOLPH3 is required for recruitment of PI(4)P-and Rab11-containing vesicles to the cleavage furrow. PI(4)P is the immediate precursor of PI(4,5)P2 that is generated in the cleavage furrow by PI(4)P 5-kinase. Binding to phosphatidic acid (PA), GOLPH3 might also contribute to PI(4)P 5-kinase activation and membrane curvature during cytokinesis. (B) In <i>sau^z2217/Df</i> mutant cells GOLPH3 protein fails to concentrate to Golgi membranes and to the cleavage furrow. PI(4)P- and Rab11-containing vesicles fail to accumulate at the cleavage furrow. PI(4)P- 5 kinase [PI(4)P 5K] activation might be also impaired in <i>sau^z2217/Df</i> mutants. (C) PI(4)P-GOLPH3 and PI(4,5)P2 regulate interaction of centralspindlin, septins and actomyosin with plasma membrane during cytokinesis. For the sake of simplicity, each cytokinesis protein is depicted separately. (D) In <i>sau^z2217/Df</i> mutant cells PI(4)P-GOLPH3 does not localize to the cleavage site. As a result, localization of centralspindlin at the equatorial cortex is not maintained, centralspindlin-associated microtubules fail to stably bundle at the midzone and F-actin ring assembly is impaired. <i>sau^z2217</i> mutations also affect stability of Myosin II and Septin filaments at the cleavage furrow. (E) In wild type telophase cells, rings containing F-actin, Myosin II and Septins constrict at the cleavage furrow during cytokinesis. (F) In <i>sau^z2217/Df</i> mutant cells F-actin ring assembly fails, Myosin II and Septin rings are patchy or thin and fail to constrict.</p

Rab11 forms a complex with GOLPH3 and depends on GOLPH3 for localization at the cleavage site.

<p>(A) Telophase spermatocytes from wild type and <i>sau^z2217/Df(2L)Exel7010 (GOLPH3)</i> stained for Tubulin (green), Rab11 (red) and DNA (blue). Arrows indicate accumulation of Rab11 at the cleavage furrow of wild type spermatocyte. Scale Bar; 10 µm. (B) GOLPH3 protein coprecipitated with Rab11 and Rab5 in testis extracts. Protein extracts from testes expressing either GFP-Rab5 or GFP-Rab11 were immunoprecipitated with GFP-trap beads (α-GFP) and blotted for either GFP or GOLPH3. Control binding beads (ctrl) were used in control experiments. Input is 4% of lysates. Molecular masses are indicated in kilodaltons. (C) Bacterially expressed GST-GOLPH3 was purified by gluthatione-sepharose beads and incubated with testis lysates expressing GFP-Rab11. GST bound to gluthatione-sepharose beads was used as a negative control. GST-GOLPH3 precipitated GFP-Rab11 from testis protein extracts. Ponceau staining (Ponceau) is shown as loading control. Input is 4% of lysates. Molecular masses are indicated in kilodaltons. (D) Interaction of GOLPH3 immobilized on a CM5 sensorchip with Rab11, measured via Surface Plasmon Resonance experiments. Sensorgrams were obtained using GOLPH3 as ligand and Rab11 as analyte. Rab11 concentrations were as follows: 50 nM (orange), 200 nM (red), 400 nM (magenta), 1.0 µM (green), 2.0 µM (cyan), 4.0 µM (blue), 10.0 µM (black). The interaction of immobilized GOLPH3 ligand with the analyte was detected through mass concentration-dependent changes in the refractive index on the sensor chip surface, expressed as resonance units (RU). The increase in RU relative to baseline (0–180 s) indicates complex formation, whereas the decrease in RU represents dissociation of Rab11 from immobilized GOLPH3 upon injection of buffer. K_D = 180 nM indicates that a high affinity complex is formed; the dissociation kinetics are very slow (k_d about 1×10⁻⁴ s⁻¹).</p

Binding to PI(4)P is essential for GOLPH3 function during cytokinesis.

<p>(A) Frequencies of spermatids (percentage) containing 1, 2, 3, 4 or more than 4 nuclei (nu) per Nk in testes from males of the indicated genotypes. (n); total number of spermatids counted per each genotype. Scale Bar; 10 µm. (B) Live spermatocytes of the indicated genotypes, expressing either GFP-GOLPH3 or a mutant version of GFP-GOLPH3 carrying K167A/R170L substitutions (GFP-GOLPH3^K167A) were imaged at the same exposure time. GFP-GOLPH3 failed to localize to Golgi stacks (fluorescent round bodies indicated by arrows) in <i>fwd³/Df(3L)7C [fwd³/Df]</i> mutant spermatocytes. GFP-GOLPH3^K167A failed to localize at the Golgi membranes in both wild type and <i>sau^z2217/Df(2L)Exel7010</i> (<i>sau^z2217/Df</i>) mutant spermatocytes. (C) Phase-contrast and corresponding fluorescence images of live telophase spermatocytes expressing either GFP-GOLPH3 (GOLPH3) or GFP-GOLPH3 carrying K167A/R170L substitutions (GOLPH3K^167A). Arrows indicate cleavage furrows. Scale Bar; 10 µm. (D) PIP strips (Echelon) were incubated with the indicated GST-tagged protein or GST, followed by anti-GST antibody for detection of lipid binding. PI(3)P ctrl and PI(4)P ctrl indicate GST-tagged control proteins that bind to either PI(3)P or PI(4)P. (E) Immunoblotting analysis of wild type testes expressing either GFP-GOLPH3 (wild type), GFP-GOLPH3^E273K (E273K) or GFP-GOLPH3^K167A/R170L (K167A). Giotto (Gio) was used as a loading control.</p

Figure S3 Validation of mouse anti-Rab1 and rabbit anti Rab1 antibodies by Western Blot and immunofluorescence assays. from Rab1 interacts with GOLPH3 and controls Golgi structure and contractile ring constriction during cytokinesis in <i>Drosophila melanogaster</i>

(a) Mouse anti-Rab1 S12085a was used in Western blotting to detect RFP-Rab1 and endogenous Rab1 proteins from either RFP-Rab1 males or wild type control Oregon-R males. (b) Western blot analyses of Rab1 depletion levels after RNAi knockdown using two different Rab1 antibodies respectively mouse anti-Rab1 S12085a and rabbit anti-Rab1 L12085a/169. Tubulin was used as loading control. (c) Quantification of band intensities in the Western blots using ImageJ software (version 1.42q; National Institute of Health, USA). The intensity of each band relative to the intensity of loading control (Tubulin), was normalized to the wild type control. The results are from three independent experiments. (d) Mouse anti-Rab1 S12085a was used to immunostain interphase-primary spermatocytes from wild type Oregon-R males and omt/Df mutant males. DNA was stained with DAPI. Note the absence of Rab1 signals in omt/Df. (e) Rab1 localization in DMel cells during cytokinesis. Cells were treated with Rab1 dsRNA (Rab1RNAi) or with Kanamycin dsRNA (control) for 72 hours and then fixed and stained with rabbit anti-Rab1 L12085a/169 to reveal endogenous Rab1, tubulin and DNA. Scale bars, 10 µm