Estimating the Microtubule GTP Cap Size In Vivo

• Published in: Current biology 2012-09, Vol.22 (18), p.1681-1687
• Main Authors: Seetapun, Dominique, Castle, Brian T., McIntyre, Alistair J., Tran, Phong T., Odde, David J.
• Format: Article
• Language: English
• Subjects: cortex; epithelial cells; Epithelial Cells - metabolism; Guanosine Triphosphate - metabolism; hydrolysis; Microtubule-Associated Proteins - metabolism; microtubules; Microtubules - metabolism; reaction kinetics; tubulin; Tubulin - metabolism
• ISSN: 0960-9822; 1879-0445
• DOI: 10.1016/j.cub.2012.06.068

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. [Display omitted] ► The microtubule (MT) GTP cap is ∼750 tubulin subunits in vivo and extends over 1 μm ► EGFP brightness can be calibrated in vivo using GFP-tubulin signals from MTs ► The presence of GTP-tubulin in the MT lattice influences MT shortening and rescue ► MT tip trackers, like EB1, can be highly concentrated at the MT plus end (∼100 μM)