Abstract
Microtubule assembly from purified tubulin preparations involves both microtubule nucleation and elongation. Whereas elongation is well documented, microtubule nucleation remains poorly understood because of difficulties in isolating molecular intermediates between tubulin dimers and microtubules. Based on kinetic studies, we have previously proposed that the basic building blocks of microtubule nuclei are persistent tubulin oligomers, present at the onset of tubulin assembly. Here we have tested this model directly by isolating nucleation-competent cross-linked tubulin oligomers. We show that such oligomers are composed of 10-15 laterally associated tubulin dimers. In the presence of added free tubulin dimers, several oligomers combine to form microtubule nuclei competent for elongation. We provide evidence that these nuclei have heterogeneous structures, indicating unexpected flexibility in nucleation pathways. Our results suggest that microtubule nucleation in purified tubulin solution is mechanistically similar to that templated by gamma-tubulin ring complexes with the exception that in the absence of gamma-tubulin complexes the production of productive microtubule seeds from tubulin oligomers involves trial and error and a selection process.
Highlights
Microtubules are fibrous elements in the cytoplasm of eukaryotic cells, where they perform a wide variety of functions
Our results suggest that microtubule nucleation in purified tubulin solution is mechanistically similar to that templated by ␥-tubulin ring complexes with the exception that in the absence of ␥-tubulin complexes the production of productive microtubule seeds from tubulin oligomers involves trial and error and a selection process
The ethylene glycol bis succinimidylsuccinate (EGS) suspension was centrifuged at 200,000 ϫ g for 30 min, and the pellet was recovered in 390 l of PEM buffer
Summary
Preparation of 1/1 GTP-Tubulin Complexes—Tubulin purification from bovine brain was performed as described [13]. Purified tubulin (100 –150 M) was incubated in PEM buffer (100 mM Pipes, pH 6.7, 1 mM EGTA, 1 mM MgCl2) for 10 min at 4 °C in the presence of 1 mM GTP. Free nucleotides were removed using Biogel P30 chromatography. The GTP-tubulin concentration was adjusted in PEM buffer, and the aliquots were stored in liquid nitrogen. Fluorescent Labeling of Tubulin—Tubulin was labeled with carboxytetramethylrhodamine succinimidyl ester as described [15], with one recycling step. Microtubule Assembly and Length Measurements—Microtubule assembly was carried out in PEM buffer with purified tubulin (12 M), GTP (1 mM), and various amounts of ethylene glycol bis succinimidylsuccinate (EGS) suspension. Was monitored at 350 nm, 35 °C, in a spectrophotometer
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