CENP-32 is required to maintain centrosomal dominance in bipolar spindle assembly

Centrosomes nucleate spindle formation, direct spindle pole positioning, and are important for proper chromosome segregation during mitosis in most animal cells. We previously reported that centromere protein 32 (CENP-32) is required for centrosome association with spindle poles during metaphase. In this study, we show that CENP-32 depletion seems to release centrosomes from bipolar spindles whose assembly they had previously initiated. Remarkably, the resulting anastral spindles function normally, aligning the chromosomes to a metaphase plate and entering anaphase without detectable interference from the free centrosomes, which appear to behave as free asters in these cells. The free asters, which contain reduced but significant levels of CDK5RAP2, show weak interactions with spindle microtubules but do not seem to make productive attachments to kinetochores. Thus CENP-32 appears to be required for centrosomes to integrate into a fully functional spindle that not only nucleates astral microtubules, but also is able to nucleate and bind to kinetochore and central spindle microtubules. Additional data suggest that NuMA tethers microtubules at the anastral spindle poles and that augmin is required for centrosome detachment after CENP-32 depletion, possibly due to an imbalance of forces within the spindle

Polyglutamylated Tubulin Binding Protein C1orf96/CSAP Is Involved in Microtubule Stabilization in Mitotic Spindles.

The centrosome-associated C1orf96/Centriole, Cilia and Spindle-Associated Protein (CSAP) targets polyglutamylated tubulin in mitotic microtubules (MTs). Loss of CSAP causes critical defects in brain development; however, it is unclear how CSAP association with MTs affects mitosis progression. In this study, we explored the molecular mechanisms of the interaction of CSAP with mitotic spindles. Loss of CSAP caused MT instability in mitotic spindles and resulted in mislocalization of Nuclear protein that associates with the Mitotic Apparatus (NuMA), with defective MT dynamics. Thus, CSAP overload in the spindles caused extensive MT stabilization and recruitment of NuMA. Moreover, MT stabilization by CSAP led to high levels of polyglutamylation on MTs. MT depolymerization by cold or nocodazole treatment was inhibited by CSAP binding. Live-cell imaging analysis suggested that CSAP-dependent MT-stabilization led to centrosome-free MT aster formation immediately upon nuclear envelope breakdown without γ-tubulin. We therefore propose that CSAP associates with MTs around centrosomes to stabilize MTs during mitosis, ensuring proper bipolar spindle formation and maintenance

CSAP depletion causes NuMA mislocalization during mitosis.

<p><b>(A)</b> At 72 h after transfection with control or pooled siRNAs for CSAP, U2OS cells were lysed and immunoblotted with anti-CSAP antibody. <b>(B)</b> Proportion of control or CSAP siRNA-transfected U2OS cells with γ-tubulin foci on mitotic spindles. Data represent the mean ± SD of three experiments. <b>(C, D, E)</b> Control (a) or CSAP (b, c) siRNA-transfected U2OS cells were fixed and immunostained for α-tubulin (Red), DNA (Blue), and γ-tubulin (<b>C</b>; Green), Aurora A (<b>D</b>; Green), or NuMA (<b>E</b>; Green). <b>(F)</b> Control (a) and CSAP (b) siRNA-transfected U2OS cells were fixed and immunostained for polyglutamylation (Green), CDK5RAP2 (Red), α-tubulin (white), and DNA (Blue). Scale bar, 5 μm. <b>(G)</b> Comparison of α-tubulin, γ-tubulin, Aurora A, NuMA, and polyglutamylation staining intensity on mitotic spindles in control (blue) or CSAP-depleted (red) cells. <b>(H, I)</b> Comparison of α-tubulin vs. NuMA <b>(H)</b> or polyglutamylation <b>(I)</b> immunostaining on the spindles around the centrosomes in control (blue) or CSAP-depleted (red) cells. Data represent the mean ± SD relative intensity. ** and *** indicate p < 0.01 and p < 0.005, respectively. (n = ~30).</p

Overexpression of CSAP promotes the formation of centrosome-free MT asters on mitotic spindles.

<p><b>(A)</b> U2OS cells transiently expressing GFP-CSAP (a) or GFP-centrin (mitosis, b-d; interphase, e). Cells were stained for α-tubulin (Red) and DNA (Blue). GFP-CSAP localizes to centrosomes; cells overexpressing GFP-CSAP show multiple GFP foci on the mitotic spindles. Scale bar, 5 μm. Over-expression is indicated by the graph on the left side. <b>(B, C)</b> Proportion of U2OS <b>(B)</b> or HeLa cells <b>(C)</b> transiently expressing GFP-centrin or GFP-CSAP with the number of GFP foci on mitotic spindles. Data represent the mean ± SD of three experiments. <b>(D)</b> U2OS cells transiently expressing TrAP (a) or TrAP-CSAP (b, c). Cells were stained for α-tubulin (red), γ-tubulin (green), and DNA (blue). Microtubule asters with γ-tubulin (arrowheads) and without γ-tubulin (arrows) are indicated. <b>(E)</b> Proportion of U2OS cells transiently expressing TrAP or TrAP-CSAP with the number of γ-tubulin foci on mitotic spindles. Data represent the mean ± SD of three experiments. <b>(F)</b> U2OS cells transiently expressing GFP-CSAP. The cells were stained forα-tubulin (red), γ-tubulin (green), and DNA (blue). Microtubule asters with γ-tubulin (arrowheads) and without γ-tubulin (arrows) are indicated. <b>(G)</b> Proportion of U2OS cells transiently expressing GFP-CSAP with the number of GFP-CSAP foci vs. γ-tubulin foci on mitotic spindles. <b>(H, I)</b> Comparison of α-tubulin <b>(H)</b> and γ-tubulin <b>(I)</b> on mitotic spindles in cells transiently expressing control (blue) or TrAP-CSAP (red). In <b>(I)</b>, two types of γ-tubulin foci at centrosomes and centrosome-free MT asters are separately shown. Data represent the mean ± SD relative intensity. *** indicates p < 0.005 (n = ~40).</p

Pericentrosomal material composition of centrosome-free MT asters.

<p><b>(A, C)</b> Mitotic U2OS cells transiently expressing control (a) and TrAP-CSAP (b). Cells were stained for α-tubulin (Red), DNA (Blue), and CDK5RAP2 (<b>A</b>; Green) or Aurora A (<b>C</b>; Green). <b>(B, D, E)</b> Mitotic U2OS cells transiently expressing GFP-CSAP (Green). Cells were stained for α-tubulin (White), DNA (Blue), and CDK5RAP2 (<b>B</b>; Red), Aurora A (<b>D</b>; Red), or pericentrin (<b>E</b>; Red). <b>(F)</b> Mitotic U2OS cells transiently expressing control (a) and TrAP-CSAP (b). The cells were stained for γ-tubulin (Red), DNA (Blue), and Aurora A. Scale bar, 5 μm. <b>(G, H)</b> Comparison of CDK5RAP2 <b>(G)</b> and Aurora A <b>(H)</b> at mitotic spindle poles in cells transiently expressing control (blue) or TrAP-CSAP (red). In <b>(H)</b>, two types of Aurora A foci at centrosomes and centrosome-free MT asters are separately shown. Data represent the mean ± SD relative intensity. *** indicates p < 0.005 (n = ~30).</p

Time-lapse analysis of CSAP-overexpressing cells during mitosis.

<p>Panels summarize time-lapse recordings of control U2OS <b>(A, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142798#pone.0142798.s001" target="_blank">S1 Movie</a>)</b> and TrAP-CSAP-overexpressing cells <b>(B, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142798#pone.0142798.s002" target="_blank">S2 Movie</a>)</b> expressing GFP–α-tubulin (upper) and H2B-mRFP (lower). Times are minutes after NEBD (0’).</p

Increasing NuMA and polyglutamylation on mitotic spindles containing centrosome-free MT asters.

<p><b>(A, D)</b> Mitotic U2OS cells transiently expressing control (a) and TrAP-CSAP (b). Cells were stained for α-tubulin (Red), DNA (Blue), and NuMA (Green). <b>(B)</b> Mitotic U2OS cells transiently expressing GFP-CSAP (<b>A</b>; Green) or polyglutamylation (<b>D</b>). Cells were stained for NuMA (Red), α-tubulin (White), and DNA (Blue). <b>(C)</b> Mitotic U2OS cells transiently expressing GFP-CSAP (Green). Cells were stained for polyglutamylation (Red), CDK5RAP2 (White), and DNA (Blue). Scale bar, 5 μm. <b>(E, F)</b> Comparison of NuMA <b>(E)</b> and polyglutamylation <b>(F)</b> on mitotic spindles in cells transiently expressing control (blue) or TrAP-CSAP (red). <b>(G, H)</b> Comparison of α-tubulin vs. NuMA <b>(G)</b> or polyglutamylation <b>(H)</b> on the spindles around the centrosomes in transiently expressing control (blue) or on centrosome-free MT asters in cells transiently expressing TrAP-CSAP (red). Data represent the mean ± SD relative intensity. *** indicates p < 0.005 (n = ~30).</p

Identification of Mitosis-Specific Phosphorylation in Mitotic Chromosome-Associated Proteins

During mitosis, phosphorylation of chromosome-associated proteins is a key regulatory mechanism. Mass spectrometry has been successfully applied to determine the complete protein composition of mitotic chromosomes, but not to identify post-translational modifications. Here, we quantitatively compared the phosphoproteome of isolated mitotic chromosomes with that of chromosomes in nonsynchronized cells. We identified 4274 total phosphorylation sites and 350 mitosis-specific phosphorylation sites in mitotic chromosome-associated proteins. Significant mitosis-specific phosphorylation in centromere/kinetochore proteins was detected, although the chromosomal association of these proteins did not change throughout the cell cycle. This mitosis-specific phosphorylation might play a key role in regulation of mitosis. Further analysis revealed strong dependency of phosphorylation dynamics on kinase consensus patterns, thus linking the identified phosphorylation sites to known key mitotic kinases. Remarkably, chromosomal axial proteins such as non-SMC subunits of condensin, TopoIIα, and Kif4A, together with the chromosomal periphery protein Ki67 involved in the establishment of the mitotic chromosomal structure, demonstrated high phosphorylation during mitosis. These findings suggest a novel mechanism for regulation of chromosome restructuring in mitosis via protein phosphorylation. Our study generated a large quantitative database on protein phosphorylation in mitotic and nonmitotic chromosomes, thus providing insights into the dynamics of chromatin protein phosphorylation at mitosis onset

Proteomics analysis with a nano random forest approach reveals novel functional interactions regulated by smc complexes on mitotic chromosomes

Packaging of DNA into condensed chromosomes during mitosis is essential for the faithful segregation of the genome into daughter nuclei. Although the structure and composition of mitotic chromosomes have been studied for over 30 years, these aspects are yet to be fully elucidated. Here, we used stable isotope labeling with amino acids in cell culture to compare the proteomes of mitotic chromosomes isolated from cell lines harboring conditional knockouts of members of the condensin (SMC2, CAP-H, CAP-D3), cohesin (Scc1/Rad21), and SMC5/6 (SMC5) complexes. Our analysis revealed that these complexes associate with chromosomes independently of each other, with the SMC5/6 complex showing no significant dependence on any other chromosomal proteins during mitosis. To identify subtle relationships between chromosomal proteins, we employed a nano Random Forest (nanoRF) approach to detect protein complexes and the relationships between them. Our nanoRF results suggested that as few as 113 of 5058 detected chromosomal proteins are functionally linked to chromosome structure and segregation. Furthermore, nanoRF data revealed 23 proteins that were not previously suspected to have functional interactions with complexes playing important roles in mitosis. Subsequent small-interfering-RNA-based validation and localization tracking by green fluorescent protein-tagging highlighted novel candidates that might play significant roles in mitotic progression