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

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

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