Microtubule minus-end stability is dictated by the tubulin off-rate

Abstract

Dynamic organization of microtubule minus ends is vital for the formation and maintenance of acentrosomal microtubule arrays. In vitro, both microtubule ends switch between phases of assembly and disassembly, a behavior called dynamic instability. Although minus ends grow slower, their lifetimes are similar to those of plus ends. The mechanisms underlying these distinct dynamics remain unknown. Here, we use an in vitro reconstitution approach to investigate minus-end dynamics. We find that minus-end lifetimes are not defined by the mean size of the protective GTP-tubulin cap. Rather, we conclude that the distinct tubulin off-rate is the primary determinant of the difference between plus- and minus-end dynamics. Further, our results show that the minus-end–directed kinesin-14 HSET/KIFC1 suppresses tubulin off-rate to specifically suppress minus-end catastrophe. HSET maintains its protective minus-end activity even when challenged by a known microtubule depolymerase, kinesin-13 MCAK. Our results provide novel insight into the mechanisms of minus-end dynamics, essential for our understanding of microtubule minus-end regulation in cells.

Mitotic Poisons in Research and Medicine

“…Tubulin dimers can be disconnected from or attached to both ends of the fiber. Interestingly, much faster changes occur at the plus end of the fiber, i.e., on the opposite side to the nucleation center [ 7 ]. Microtubules are essential for maintaining the cell shape, polarity, migration and division [ 5 ].…”

GTP-tubulin loves microtubule plus ends but marries the minus ends

“…1 B, right). Yet, the similar scaling of EB1 comet size with growth rate suggests that the rate of GTP hydrolysis at the minus end is similar to the plus end (10). Perhaps β-tubulin is also sensitive to the lattice state of its intra-dimer α-tubulin, which must also be capped by another dimer before the minus-end β-tubulin can become competent to hydrolyze GTP.…”

“…Unfortunately, at tubulin concentrations required for MT assembly, this off-rate is so small as to be almost nonexistent and is accordingly difficult to quantify (8). Strothman et al (10) have heroically measured this parameter in highly stable GMPCPP MTs by imaging their slow disassembly over the course of 15 h. In doing so, they have made the important discovery that the loss of GTP-tubulin (as modeled by GMPCPP tubulin) is more than twice as rapid at the plus end versus the minus end of the MT (Fig. 1 B, left).…”

“…Once either end transitions to disassembly, both ends of the MT disassemble at the same rate, suggesting that the GDP-tubulin off-rate is similar for both ends. Thus, Strothman et al (10) demonstrate that it is the off-rate for GTP-tubulin that is distinct for the two ends.…”

“…Like any study that adds a key piece to the puzzle of MT end kinetics, Strothman et al (10) makes us eager for more puzzle pieces. For example, if the coupled-random model can explain both the plus and the minus end, are the GTP hydrolysis rules the same at each end?…”