title: 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 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.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
authors.forename: Kieren
authors.initials: K
authors.lastname: Patel
authors.forename: Eva
authors.initials: E
authors.lastname: Nogales
authors.forename: Rebecca
authors.initials: R
authors.lastname: Heald
mesh_terms: D000818:Animals; D003599:Cytoskeleton / Q000378:metabolism*; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology*; D010063:Ovum / Q000166:cytology / Q000378:metabolism*; D058105:Polymerization; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D008941:Spindle Apparatus / Q000378:metabolism*; D014404:Tubulin; D014982:Xenopus laevis
chemical_list: C497850:CLASP1 protein, human; D008869:Microtubule-Associated Proteins; D014404:Tubulin