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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.

SUBMITTER: Patel K

PROVIDER: S-EPMC3315288 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundLinker histone H1 has been studied in vivo and using reconstituted chromatin, but there have been few systematic studies of the effects of the cellular environment on its function. Due to the presence of many other chromatin factors and specific chaperones such as RanBP7/importin beta that regulate histone H1, linker histones likely function differently in vivo than in purified systems.Methodology/principal findingsWe have directly compared H1 binding to sperm nuclei in buffer versus Xenopus egg extract cytoplasm, and monitored the effects of adding nuclear import chaperones. In buffer, RanBP7 decondenses sperm nuclei, while H1 binds tightly to the chromatin and rescues RanBP7-mediated decondensation. H1 binding is reduced in cytoplasm, and H1 exhibits rapid FRAP dynamics in cytoplasm but not in buffer. RanBP7 decreases H1 binding to chromatin in both buffer and extract but does not significantly affect H1 dynamics in either condition. Importin beta has a lesser effect than RanBP7 on sperm chromatin decondensation and H1 binding, while a combination of RanBP7/importin beta is no more effective than RanBP7 alone. In extracts supplemented with RanBP7, H1 localizes to chromosomal foci, which increase after DNA damage. Unlike somatic H1, the embryonic linker histone H1M binds equally well to chromatin in cytoplasm compared to buffer. Amino-globular and carboxyl terminal domains of H1M bind chromatin comparably to the full-length protein in buffer, but are inhibited ?10-fold in cytoplasm. High levels of H1 or its truncations distort mitotic chromosomes and block their segregation during anaphase.Conclusion/significanceRanBP7 can decondense sperm nuclei and decrease H1 binding, but the rapid dynamics of H1 on chromatin depend on other cytoplasmic factors. Cytoplasm greatly impairs the activity of individual H1 domains, and only the full-length protein can condense chromatin properly. Our findings begin to bridge the gap between purified and in vivo chromatin systems.

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

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