Evidence that a single monolayer tubulin-GTP cap is both necessary and sufficient to stabilize microtubules.

Abstract

Evidence that 13 or 14 contiguous tubulin-GTP subunits are sufficient to cap and stabilize a microtubule end and that loss of only one of these subunits results in the transition to rapid disassembly (catastrophe) was obtained using the slowly hydrolyzable GTP analogue guanylyl-(a,b)-methylene-diphosphonate (GMPCPP). The minus end of microtubules assembled with GTP was transiently stabilized against dilution-induced disassembly by reaction with tubulin-GMPCPP subunits for a time sufficient to cap the end with an average of 40 subunits. The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was estimated from an observed 25-to 2000-s lifetime of the GMPCPP-stabilized microtubules following dilution with buffer and from the time required for loss of a single tubulin-GMPCPP subunit from the microtubule end (found to be 15 s). Rather than assuming that the 25-to 2000-s dispersion in cap lifetime results from an unlikely 80-fold range in the number of tubulin-GMPCPP subunits added in the 25-s incubation, it is proposed that this results because the minimum stable cap contains 13 or 14 tubulin-GMPCPP subunits. As a consequence, a microtubule capped with 13-14 tubulin-GMPCPP subunits switches to disassembly after only one dissociation event (in about 15 s), whereas the time required for catastrophe of a microtubule with only six times as many subunits (84 subunits) corresponds to 71 dissociation events (84-13). The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was also estimated with microtubules in which a GMPCPP-cap was formed by allowing chance to result in the accumulation of multiple contiguous tubulin-GMPCPP subunits at the end, during the disassembly of microtubules containing both GDP and GMPCPP. Our observation that the disassembly rate was inhibited in proportion to the 13-14th power of the fraction of subunits containing GMPCPP again suggests that a minimum cap contains 13-14 tubulin-GMPCPP subunits. A remeasurement of the rate constant for dissociation of a tubulin-GMPCPP subunit from the plus-end of GMPCPP microtubules, now found to be 0.118 s-1, has allowed a better estimate of the standard free energy for hydrolysis of GMPCPP in a microtubule and release of Pi: this is +0.7 kcal/mol, rather than -0.9 kcal/mol, as previously reported.

Estimates of lateral and longitudinal bond energies within the microtubule lattice

“…Prediction of strong longitudinal bonds and relatively weak lateral bonds is in agreement with structural observations (18). We also estimated that the potential mechanical energy of tubulin-GDP conformational stress in the MT lattice is 2.1-2.5 k B T, in approximate agreement with thermodynamic studies that suggest Ϸ2.8 k B T per tubulin-GDP is stored in the MT lattice (14,22,23).…”

“…Catastrophe frequency simulations began with an MT capped by 4 GTP-tubulin layers (52 dimers) and ran until the GTP-cap size (number of tubulin-GTP dimers) was zero. Experiments by Caplow and Shanks (14) suggested that Ͻ13 tubulin-GTP dimers at a MT tip would result in catastrophe. Here we used complete loss of the GTP cap as a convenient way to mark a catastrophe.…”

A driving and coupling “Pac-Man” mechanism for chromosome poleward translocation in anaphase A

“…For in vivo situation, the effective kinetochore diffusion coefficient includes the large chromosome effect and thus it is very small Ϸ400-800 nm 2 ͞s. Because the microtubule bending strain is usually low (Ϸ3 k B T) [this value is supported by experimental measurement (23) and theoretical calculations (21,22)], it alone could not drive the kinetochore to overcome local binding potential Ϸ12.5 k B T (5,14,19). Thus, according to Fig.…”

“…Upon microtubule depolymerization, the bending strain is released and provides a driving force for the kinetochore translocation (2,12,20). However, given the strong attachment of the kinetochore to microtubule of Ϸ12.5 k B T (5,14,19), as our calculations in this paper suggest, a bending strain of Ϸ3 k B T (2,3,(20)(21)(22)(23) is not sufficient. It has also been proposed that a ratchet-like biased one-dimensional diffusion model could account for the kinetochore translocation (17).…”

“…The bound GDP-tubulin subunit has a preferred angle with respect to its neighbor (0) ϭ 0.4 rad (24). The bending strain Ϸ3 k B T is thus stored in the subunits held in straight configuration (2,3,(20)(21)(22)(23), which is favored by lateral bonds Ϸ3 k B T between the neighboring subunits in the adjacent protofilaments (19,21,22). In this paper, the kinetochore is represented as the Dam1 ring-like structure from budding yeast (5-7), which has a radius of 16 nm and consists of 16 identical components (5)(6)(7), each carrying at least six positive charges q kt Ն 6 under physiological conditions (5,6).…”