Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length.

Xiangdong Zheng,Haiteng Deng,Michael Sattler,Guangshuo Ou,Wenjing Li,Komal Soni,Anand Ramani,Ina Poser,Shan Feng,Arpit Wason,Marco Gottardo,Haitao Li,Shuangping Zheng,Anthony A Hyman,Jay Gopalakrishnan,Li Ming Gooi,Aruljothi Mariappan,Callaini Giuliano,Andrei Pozniakovsky,Maria Giovanna Riparbelli ,Per O Widlund

doi:10.1038/ncomms11874

Abstract

Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets β-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule's α-β surface of β-tubulin. Through this, PN2-3 forms a high-affinity complex with GTP-tubulin, crucial for defining numbers and lengths of centriolar/ciliary-microtubules. Surprisingly, two distinct mutations in PN2-3 exhibit opposite effects on centriolar/ciliary-microtubule lengths. CPAPF375A, with strongly reduced tubulin interaction, causes shorter centrioles and cilia exhibiting doublet- instead of triplet-microtubules. CPAPEE343RR that unmasks the β-tubulin polymerization surface displays slightly reduced tubulin-binding affinity inducing over-elongation of newly forming centriolar/ciliary-microtubules by enhanced dynamic release of its bound tubulin. Thus CPAP regulates delivery of its bound-tubulin to define the size of microtubule-based cellular structures using a ‘clutch-like' mechanism.

Highlights

Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation
Since cilium templates from a centriole that resides within a centrosome, it is conceivable that a centrosomal protein that can directly interact with cytoplasmic tubulin could play a role in selective regulation of tubulin incorporation during centriolarand ciliary-microtubule construction
By adopting a DARPin strategy, in which DARPin molecule didn’t affect binding of PN2-3C to tubulin, we solved the 2.1 Å crystal structure of bovine tubulin bound to PN2-3C (Fig. 1d,e, Table 1 and Supplementary Fig. 1)

Summary

Introduction

Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. Intraflagellar transport (IFT) machineries mediate the transport of ciliary building blocks of tubulin from the cytoplasmic ciliary base to the tip[6,7,8] It remains poorly understood how a fraction of tubulin is selected from its large cytoplasmic pool and the mechanisms that operate to deliver these specialized tubulin to construct defined lengths of centriolar- and ciliary-microtubules. A CPAP/Sas-4 mutant that does not bind tubulin caused shortening of centrioles and primary cilia[9,10,11] These data suggest the possibility that CPAP could play a role in delivering its bound tubulin at the site of centriole assembly and/or building centriolar-microtubules. Understanding the molecular basis of PN2-3-tubulin interaction is crucial in dissecting how cytoplasmic tubulin is sequestered by CPAP for the controlled delivery to define centriolar- and ciliary-microtubule lengths in cells. Our functional studies in human cells and flies identified that, through the different facets of its PN2-3 domain, CPAP defines centriolarand ciliary-microtubule lengths, firstly, through the sufficient binding of cytoplasmic tubulin and secondly by regulated release of its bound tubulin to define centriolar- and ciliary-microtubule lengths

Methods

Results

Conclusion