Estimating the microtubule GTP cap size in vivo

Dominique Seetapun, Brian T. Castle, Alistair J. McIntyre, Phong T. Tran, David J. Odde

Research output: Contribution to journalArticlepeer-review

64 Scopus citations

Abstract

Microtubules (MTs) polymerize via net addition of GTP-tubulin subunits to the MT plus end, which subsequently hydrolyze to GDP-tubulin in the MT lattice. Relatively stable GTP-tubulin subunits create a "GTP cap" at the growing MT plus end that suppresses catastrophe. To understand MT assembly regulation, we need to understand GTP hydrolysis reaction kinetics and the GTP cap size. In vitro, the GTP cap has been estimated to be as small as one layer [1-3] (13 subunits) or as large as 100-200 subunits [4]. GTP cap size estimates in vivo have not yet been reported. Using EB1-EGFP as a marker for GTP-tubulin in epithelial cells, we find on average (1) 270 EB1 dimers bound to growing MT plus ends, and (2) a GTP cap size of ∼750 tubulin subunits. Thus, in vivo, the GTP cap is far larger than previous estimates in vitro, and ∼60-fold larger than a single layer cap. We also find that the tail of a large GTP cap promotes MT rescue and suppresses shortening. We speculate that a large GTP cap provides a locally concentrated scaffold for tip-tracking proteins and confers persistence to assembly in the face of physical barriers such as the cell cortex.

Original languageEnglish (US)
Pages (from-to)1681-1687
Number of pages7
JournalCurrent Biology
Volume22
Issue number18
DOIs
StatePublished - Sep 25 2012

Bibliographical note

Funding Information:
We thank M.K. Gardner for technical assistance with the MT growth simulation, P. Wadsworth for the LLCPK1α cell line, L. Cassimeris for the EB1-EGFP LLCPK1 cell line, J. Mueller for the p2×EGFP construct, and R.Y. Tsien for the pmCherry-α-tubulin construct. Funding support was provided by National Institutes of Health grants GM-071522 and GM-076177 and National Science Foundation grant MCB-0615568.

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