EB1-microtubule interactions in Xenopus egg extracts: role of EB1 in microtubule stabilization and mechanisms of targeting to microtubules

EB1 targets to polymerizing microtubule ends, where it is favorably positioned to regulate microtubule polymerization and confer molecular recognition of the microtubule end. In this study, we focus on two aspects of the EB1-microtubule interaction: regulation of microtubule dynamics by EB1 and the...

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Published in:Molecular biology of the cell 2002-10, Vol.13 (10), p.3614-3626
Main Authors: Tirnauer, Jennifer S, Grego, Sonia, Salmon, E D, Mitchison, Timothy J
Format: Article
Language:English
Subjects:
Animals
Cell Cycle - physiology
Cell Polarity - physiology
Centrosome - metabolism
Fluorescence Recovery After Photobleaching
Humans
Microtubule-Associated Proteins - genetics
Microtubule-Associated Proteins - isolation & purification
Microtubule-Associated Proteins - metabolism
Microtubules - metabolism
Microtubules - ultrastructure
Oocytes - cytology
Oocytes - physiology
Polymers - metabolism
Protein Binding
Tubulin - genetics
Tubulin - isolation & purification
Tubulin - metabolism
Xenopus laevis - physiology
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Summary:EB1 targets to polymerizing microtubule ends, where it is favorably positioned to regulate microtubule polymerization and confer molecular recognition of the microtubule end. In this study, we focus on two aspects of the EB1-microtubule interaction: regulation of microtubule dynamics by EB1 and the mechanism of EB1 association with microtubules. Immunodepletion of EB1 from cytostatic factor-arrested M-phase Xenopus egg extracts dramatically reduced microtubule length; this was complemented by readdition of EB1. By time-lapse microscopy, EB1 increased the frequency of microtubule rescues and decreased catastrophes, resulting in increased polymerization and decreased depolymerization and pausing. Imaging of EB1 fluorescence revealed a novel structure: filamentous extensions on microtubule plus ends that appeared during microtubule pauses; loss of these extensions correlated with the abrupt onset of polymerization. Fluorescent EB1 localized to comets at the polymerizing plus ends of microtubules in cytostatic factor extracts and uniformly along the lengths of microtubules in interphase extracts. The temporal decay of EB1 fluorescence from polymerizing microtubule plus ends predicted a dissociation half-life of seconds. Fluorescence recovery after photobleaching also revealed dissociation and rebinding of EB1 to the microtubule wall with a similar half-life. EB1 targeting to microtubules is thus described by a combination of higher affinity binding to polymerizing ends and lower affinity binding along the wall, with continuous dissociation. The latter is likely to be attenuated in interphase. The highly conserved effect of EB1 on microtubule dynamics suggests it belongs to a core set of regulatory factors conserved in higher organisms, and the complex pattern of EB1 targeting to microtubules could be exploited by the cell for coordinating microtubule behaviors.
ISSN:1059-1524
1939-4586
DOI:10.1091/mbc.02-04-0210