Multiple domains of human CLASP contribute to microtubule dynamics and organization in vitro and in Xenopus egg extracts

Kieren Patel,Eva Nogales,Rebecca Heald

doi:10.1002/cm.21005

Abstract

Cytoplasmic linker associated proteins (CLASPs) comprise a class of microtubule (MT) plus end-binding proteins (+TIPs) that contribute to the dynamics and organization of MTs during many cellular processes, among them mitosis. Human CLASP proteins contain multiple MT-binding domains, including tumor over-expressed gene (TOG) domains, and a Ser-x-Ile-Pro (SxIP) motif known to target some +TIPs though interaction with end-binding protein 1 (EB1). However, how individual domains contribute to CLASP function is poorly understood. We generated full-length recombinant human CLASP1 and a series of truncation mutants and found that two N-terminal TOG domains make the strongest contribution to MT polymerization and bundling, but also identified a third TOG domain that further contributes to CLASP activity. Plus end tracking by CLASP requires the SxIP motif and interaction with EB1. The C-terminal coiled-coil domain mediates dimerization and association with many other factors, including the kinetochore motor centromere protein E (CENP-E), and the chromokinesin Xkid. Only the full-length protein was able to rescue spindle assembly in Xenopus egg extracts depleted of endogenous CLASP. Deletion of the C-terminal domain caused aberrant MT polymerization and dramatic spindle phenotypes, even with small amounts of added protein, indicating that proper localization of CLASP activity is essential to control MT polymerization during mitosis. © 2012 Wiley Periodicals, Inc

Highlights

Microtubule (MT) assembly and organization are highly regulated by many associated proteins, including a subset that selectively localize to growing plus ends [Galjart, 2010]
Full-length human CLASP1 protein and domain fragments were generated as C-terminal green fluorescence protein (GFP) fusions using baculovirus/insect cell expression
The molecule appeared highly flexible, making image processing and the generation of class averages impossible. Despite this limitation, based on the apparent length of the Cytoplasmic linker associated proteins (CLASPs) particles, there seemed to be a mixture of monomers and dimers, it was difficult to distinguish between actual dimerization and particles that were close together

Summary

Introduction

Microtubule (MT) assembly and organization are highly regulated by many associated proteins, including a subset that selectively localize to growing plus ends (þTIPs) [Galjart, 2010]. Recent studies of the Schizosaccharomyces pombe homolog Cls1p/Peg showed that CLASP can promote MT rescues and suppress catastrophes independent of other proteins [Al-Bassam et al, 2010], but molecular mechanisms behind CLASP function and targeting to MT plus ends and cellular structures remain unclear. We use a series of purified CLASP1 proteins to provide biochemical insight into the structure and activity of human CLASP, its effects on MT dynamics, MT plus end targeting, interaction with other cellular factors, and its function in the context of the meiotic spindle in Xenopus egg extracts

Experimental Procedures

Results

Discussion