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

Shinya Ohta,Mayako Hamada,Iyo Toramoto,Nobuko Sato,Daniela Cimini

doi:10.1371/journal.pone.0142798

Abstract

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.

Highlights

C1orf96, termed as Centriole, Cilia and Spindle-Associated Protein (CSAP or CCSAP), is required for proper cilia beating and is targeted to polyglutamylated microtubules (MTs; [1])
We found more than three GFP foci on mitotic spindles in GFP-CSAP overexpressing cells (81%; Fig 1B); this phenotype was dependent on the extent of GFP-CSAP overexpression (Fig 1A-b, 1A-c and 1Ad)
We showed that spindle association with excess CSAP caused formation of centrosome-free MT asters, in which γ-tubulin and CDK5RAP2 were not detected, a phenotype not reported in a previous study on CSAP function [1]

Summary

Introduction

C1orf, termed as Centriole, Cilia and Spindle-Associated Protein (CSAP or CCSAP), is required for proper cilia beating and is targeted to polyglutamylated microtubules (MTs; [1]). Immunoelectron microscopy of CCSAP in centrosomes has shown primary localization to the MT cylinder walls and secondary localization in the centriole lumen. CSAP is required for proper zebrafish development and lateral asymmetry in the brain [1,2,3]. Polyglutamylation at the C-terminus of α- and γ-tubulin accumulates in neuronal cultures and in the brain during development [4,5,6,7]. Extensive polyglutamylation of tubulin leads to MT stability [14,15,16]. The molecular function of microtubule polyglutamylation and CSAP binding to polyglutamylated MTs during mitosis remains unclear

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