Journal: bioRxiv

Article Title: The midbody interactome reveals new unexpected roles for PP1 phosphatases in cytokinesis

doi: 10.1101/569459

Figure Lengend Snippet: (a) Western blot analysis of protein extracts from HeLa cells stably expressing a CIT-K::AcGFP transgene synchronized at different stages of the cell cycle. The blots were probed with antibodies against the proteins indicated to the right. Numbers on the left indicate the size, in kDa, of the protein ladder. (b) Proportional Venn diagram showing the number of proteins identified at each cell cycle stage by AP-MS using CIT-K::AcGFP as bait. (c) Midbodies purified from HeLa S3 cells treated with siRNAs directed against either a random sequence (control) or CIT-K were fixed and stained to detect tubulin, CIT-K and MKLP1. Scale bars, 5 µm. (d) Logarithmic normalized protein ratios from two independent SILAC experiments were plotted against each other. Each point represents a single protein identified. Gray dots correspond to proteins that did not show any significant difference in abundance between control and CIT-K siRNA midbodies. Red and blue dots represent proteins that were either significantly enriched or less abundant after CIT-K depletion in both biological replicates ( p value < 0.01). (e) Western blot analysis of total protein extracts and midbodies purified from telophase HeLa S3 cells treated with siRNAs directed against either a random sequence (control) or CIT-K . The blots were probed with antibodies against the proteins indicated to the right. Numbers on the left indicate the size, in kDa, of the protein ladder.

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti α-tubulin (clone DM1A, Sigma, T9026), rabbit polyclonal anti-β-tubulin (Abcam, ab6046),), mouse monoclonal anti-cyclin B1 (clone GNS1, Santa Cruz, sc-245), mouse monoclonal anti-PP1α (clone G-4, Santa Cruz, sc-271762), mouse monoclonal anti-PP1β (clone A-6, Santa Cruz, sc-365678), mouse monoclonal anti-PP1γ (clone A-4, Santa Cruz, sc-515943), mouse monoclonal anti-CIT-K (BD Transduction Laboratories, 611377), mouse monoclonal anti-MYPT1 (clone C-6, Santa Cruz, sc-514261), (Abcam, ab2254), rabbit polyclonal anti-MKLP1 (clone N19, Santa Cruz Biotechnology, sc-867), rabbit polyclonal anti-phospho MKLP1 pS708 , rabbit polyclonal anti-tri-phospho CHMP4C pS210 pS214 pS215 , mouse monoclonal anti-Aurora B (clone AIM-1, BD Transduction Laboratories, 611082), mouse monoclonal anti-PRC1 (clone C-1, Santa Cruz, sc-376983), rabbit monoclonal anti-phospho PRC1 pT481 (Abcam, ab62366), rabbit polyclonal anti-phospho-histone H3 pS10 (Merck, 06-570), rabbit polyclonal anti-mono-phospho MRLC pS19 (Cell Signaling Technology, 3671), rabbit polyclonal anti-di-phospho MRLC pT18 pS19 (Cell Signaling Technology, 3674), goat polyclonal anti-RacGAP1 (Abcam, ab2270), mouse monoclonal anti-GST (Abcam, ab92), mouse monoclonal anti-MBP (NEB, E8032).

Techniques: Western Blot, Stable Transfection, Expressing, Purification, Sequencing, Staining

Journal: bioRxiv

Article Title: The midbody interactome reveals new unexpected roles for PP1 phosphatases in cytokinesis

doi: 10.1101/569459

Figure Lengend Snippet: (a) HeLa Kyoto cells were treated with siRNAs directed against either a random sequence (control) or MYPT1 siRNA and after 48 hours fixed and stained to detect the indicated epitopes and DNA (blue in the merged panels). DNA condensation and the shape and thickness of microtubule bundles at the intercellular bridge were used as criteria to stage telophase cells. Insets show a 3x magnification of the midbody. Bars, 10 µm. (b) Box plots showing the quantification of total and pS708 MKLP1 in control and MYPT1 siRNA late telophase cells. Fluorescence intensities of MKLP1 and MKLP1 pS708 were calculated as described in Methods. AU, arbitrary unit. The numbers of cells counted are indicated below each plot. p values were calculated using the student’s T-test. (c) HeLa Kyoto cells stably expressing GFP-MKLP1 were treated with either a random sequence (control) or MYPT1 siRNA and after 24 hours synchronized in telophase by thymidine/nocodazole block. Protein extracts were used in a GFP pull-down assay and then extracts and pull-downs were analyzed by Western blot to detect the proteins indicated to the right. The numbers on the left indicate the sizes of the molecular mass marker. (d) Schematic diagram of the MKLP1 protein. The Aurora B phosphorylation site at S708 and the VQF PP1 binding site are indicated. (e) MBP-tagged MKLP1, MKLP1 AQA or MBP alone were co-expressed in yeast along with either GST alone or GST-PP1β as indicated at the top. Protein extracts were used in a pull-down assay using amylose beads. Crude extracts and pull-downs were analyzed by Western blot to detect GST (top panels) and MBP (bottom panels). Numbers on the left indicate the size, in kDa, of the protein ladder. (f) GST-tagged wild type and AQA mutant MKLP1 fragments (aa 620-858) were expressed and purified in bacteria and then phosphorylated in vitro with recombinant Aurora B. After washing out Aurora B, the reactions were incubated with either MBP-tagged PP1β or a catalytically dead version (PP1β dead ) purified from yeast for the times indicated at the top. Proteins were then analyzed by Western blot using antibodies against MKLP1 pS708 and GST. (g) Graph showing the quantification of MKLP1 pS708 values normalized against the total amounts of GST-MKLP1 620-858 . (h) HeLa Kyoto cells stably expressing GFP-MKLP1, GFP-MKLP1 AQA or no transgene, were treated with either a random sequence (control) or an siRNA targeted against the MKLP1 3’UTR region, which is not present in the GFP-tagged MKLP1 transgenes, and after 48 hours were fixed and stained to detect the indicated epitopes and DNA (blue in the merged panels). DNA condensation and the shape and thickness of microtubule bundles at the intercellular bridge were used as criteria to stage telophase cells. Bars, 10 µm. (i) Quantification of multinucleate cells, a readout for cytokinesis failure, from the experiments shown in (h). More than 500 cells were counted in each experiment, n=3. Bars indicate standard errors.

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti α-tubulin (clone DM1A, Sigma, T9026), rabbit polyclonal anti-β-tubulin (Abcam, ab6046),), mouse monoclonal anti-cyclin B1 (clone GNS1, Santa Cruz, sc-245), mouse monoclonal anti-PP1α (clone G-4, Santa Cruz, sc-271762), mouse monoclonal anti-PP1β (clone A-6, Santa Cruz, sc-365678), mouse monoclonal anti-PP1γ (clone A-4, Santa Cruz, sc-515943), mouse monoclonal anti-CIT-K (BD Transduction Laboratories, 611377), mouse monoclonal anti-MYPT1 (clone C-6, Santa Cruz, sc-514261), (Abcam, ab2254), rabbit polyclonal anti-MKLP1 (clone N19, Santa Cruz Biotechnology, sc-867), rabbit polyclonal anti-phospho MKLP1 pS708 , rabbit polyclonal anti-tri-phospho CHMP4C pS210 pS214 pS215 , mouse monoclonal anti-Aurora B (clone AIM-1, BD Transduction Laboratories, 611082), mouse monoclonal anti-PRC1 (clone C-1, Santa Cruz, sc-376983), rabbit monoclonal anti-phospho PRC1 pT481 (Abcam, ab62366), rabbit polyclonal anti-phospho-histone H3 pS10 (Merck, 06-570), rabbit polyclonal anti-mono-phospho MRLC pS19 (Cell Signaling Technology, 3671), rabbit polyclonal anti-di-phospho MRLC pT18 pS19 (Cell Signaling Technology, 3674), goat polyclonal anti-RacGAP1 (Abcam, ab2270), mouse monoclonal anti-GST (Abcam, ab92), mouse monoclonal anti-MBP (NEB, E8032).

Techniques: Sequencing, Staining, Fluorescence, Stable Transfection, Expressing, Blocking Assay, Pull Down Assay, Western Blot, Marker, Binding Assay, Mutagenesis, Purification, In Vitro, Recombinant, Incubation

Journal: bioRxiv

Article Title: The midbody interactome reveals new unexpected roles for PP1 phosphatases in cytokinesis

doi: 10.1101/569459

Figure Lengend Snippet: During furrowing (top panel) MYPT1-PP1β accumulates at the cortex with no or very limited access to the centralspindlin pool that localizes to the central spindle midzone, which is highly phosphorylated by Aurora B and therefore can form clusters. After completion of furrow ingression, MYPT1/PP1β accumulates at the midbody ring whereas Aurora B localizes to the midbody arms (bottom panel). This allows PP1β to de-phosphorylate MKLP1 at S708, which could strengthen the association of centralspindlin with PRC1. See text for more details.

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti α-tubulin (clone DM1A, Sigma, T9026), rabbit polyclonal anti-β-tubulin (Abcam, ab6046),), mouse monoclonal anti-cyclin B1 (clone GNS1, Santa Cruz, sc-245), mouse monoclonal anti-PP1α (clone G-4, Santa Cruz, sc-271762), mouse monoclonal anti-PP1β (clone A-6, Santa Cruz, sc-365678), mouse monoclonal anti-PP1γ (clone A-4, Santa Cruz, sc-515943), mouse monoclonal anti-CIT-K (BD Transduction Laboratories, 611377), mouse monoclonal anti-MYPT1 (clone C-6, Santa Cruz, sc-514261), (Abcam, ab2254), rabbit polyclonal anti-MKLP1 (clone N19, Santa Cruz Biotechnology, sc-867), rabbit polyclonal anti-phospho MKLP1 pS708 , rabbit polyclonal anti-tri-phospho CHMP4C pS210 pS214 pS215 , mouse monoclonal anti-Aurora B (clone AIM-1, BD Transduction Laboratories, 611082), mouse monoclonal anti-PRC1 (clone C-1, Santa Cruz, sc-376983), rabbit monoclonal anti-phospho PRC1 pT481 (Abcam, ab62366), rabbit polyclonal anti-phospho-histone H3 pS10 (Merck, 06-570), rabbit polyclonal anti-mono-phospho MRLC pS19 (Cell Signaling Technology, 3671), rabbit polyclonal anti-di-phospho MRLC pT18 pS19 (Cell Signaling Technology, 3674), goat polyclonal anti-RacGAP1 (Abcam, ab2270), mouse monoclonal anti-GST (Abcam, ab92), mouse monoclonal anti-MBP (NEB, E8032).

Techniques:

Journal: Cerebral Cortex (New York, NY)

Article Title: SEPT7 Interacts with KIF20A and Regulates the Proliferative State of Neural Progenitor Cells During Cortical Development

doi: 10.1093/cercor/bhz292

Figure Lengend Snippet: Inducible knockout of Sept7 in NPCs causes early cell cycle exit and precocious neuronal differentiation. (A) Tamoxifen was administered to Sept7 inducible knockout mice at E9.5 and E10.5 consecutively. EdU was labeled at E14.5 and embryonic brains were collected 24 h afterwards. R1, R2, and R3 indicated the EdU-labeled cell ribbons located progressively from superficial regions to the apical surface. EdU+Ki67− cells were progenitor cells that had left the cell cycle. More EdU+Ki67− cells in the total population of EdU+ cells were present in the cortices of the homozygous mutant cortices. Scale bar represents 100 μm. **P < 0.01 (Student’s t-test). Error bars represent SD. (B) EdU+Ki67− cells that had migrated into the CP in the Nestin-CreERT2; Sept7fl/fl homozygous mutant cortices were positive for NeuN, indicating that these cells had become neurons. Arrows indicated examples of EdU+Ki67−NeuN+ cells. Scale bar represents 100 μm.

Article Snippet: Primary antibodies included Rabbit anti-Flag (Sigma F7425; 1:2000), Mouse anti-GFP (Roche; 1:200), Rabbit anti-SEPT7 (H-120) (Santa Cruz; 1:100), Goat anti-KIF20A(L-13) (Santa Cruz; 1:100), Rabbit anti-MKLP1(N-19) (Santa Cruz; 1:100), Mouse anti-BrdU (Sigma B2531; 1:100), Mouse anti-Ki67(BD Biosciences; 1:25), and Rabbit antiactivated caspase 3 (BD Biosciences; 1:200).

Techniques: Knock-Out, Labeling, Mutagenesis