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

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 also 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 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.

Acetylcholine Receptor (AChR) Clustering Is Regulated Both by Glycogen Synthase Kinase 3β (GSK3β)-dependent Phosphorylation and the Level of CLIP-associated Protein 2 (CLASP2) Mediating the Capture of Microtubule Plus-ends

“…The function of this second TOG-like domain is not yet well defined. It may contribute to MT binding to some extent (19); however, we have shown previously that this is mostly mediated by a third TOG-like domain C-terminal to the disordered central part of the protein (15,19,33), which is present in the rescue construct. Overexpression of the rescue construct may compensate for a slightly reduced MT binding due to the absence of the second TOG-like domain.…”

“…Compared with a nonmodified form of CLASP2, these proteins were shown to bind MTs and MT ends with different characteristics in cultured keratinocytes. However, these CLASP2 proteins (CLASP2-9XS/A, CLASP2-8XS/D, and CLASP2-WT) lack part of the N terminus of CLASP2␥, including a TOG-like domain (28), which has been suggested to contribute to the binding of CLASP to MTs (19).…”

“…MT binding is mediated by multiple weaker affinity interactions, including the central arginine-and serine-rich intrinsically disordered region (18), as well as TOG-like MT-binding domains (19). Both direct and indirect CLASP MT bindings are inhibited by phosphorylation of an array of GSK3␤ sites in the middle of the protein (16,20).…”

GSK3-mediated CLASP2 phosphorylation modulates kinetochore dynamics

“…However, the fulllength isoforms CLASP2γ and CLASP2α, generated by alternative splicing, contain additional N-terminal TOG domains (Fig. 1A), but how TOG1 and TOG2 in mammalian CLASP2 contribute to MT binding is unclear (Patel et al, 2012). Therefore, to test whether MT association of the longest CLASP2α isoform is cell cycle regulated, we measured EGFP-CLASP2α on MT ends as a function of cell cycle phases ( Fig.…”

TOG domain MT polymerases accelerate MT plus end growth via electrostatically-steered diffusion-to-capture and electrostatic templating of GTP-tubulin

“…TOG-family polymerases can increase the MT net growth rate at plus ends by up to ~10-fold. Several lines of evidence suggest that electrostatic interactions play a key role in TOG-tubulin interactions [10,11], but the detailed role of electrostatic interactions in determining the dynamics of complex formation and / or the catalysis of tubulin exchange at MT plus ends is so far little explored.…”