Our results demonstrate that kinesin-5 motors, in particular Cin8p, promote net kMT and aMT disassembly in vivo. Since net kMT assembly is specifically suppressed for longer kMTs (i.e. whose plus ends are near the equator) during yeast metaphase, kinesin-5 motors must be mediating their effect, either alone or with binding partners, most strongly on longer kMTs. It is this length-dependent regulation of net kMT plus end assembly that establishes the congressed state of chromosomes that is characteristic of metaphase. These results are surprising to us, since the only established activity of kinesin-5 is its well known antiparallel MT sliding activity, and, to our knowledge, no effect on MT assembly has been reported previously. We also found that Cin8-GFP interacts frequently with MT plus ends in vivo, and exists in a spatial gradient on MTs. The close correspondence of the gradient in net kMT assembly and the gradient in motor distribution strongly suggests that Cin8p, either alone or with a binding partner, directly promotes kMT disassembly via its presence at the kMT plus-end. In addition, this conclusion is strengthened by the close similarity between the cin8Δ mutant and the cin8 motor-domain mutant (F467A) phenotypes.
It is interesting to consider the consequences of the disassembly promotion for spindle length regulation. It is well established that kinesin-5 promotes spindle pole separation by generating an outward extensional force, presumably via cross-linking of antiparallel MTs. The newly identified disassembly-promoting activity shortens kMTs, and thereby should generate an inward pulling force on the spindle poles via stretching of the intervening chromatin between the sister kMT plus ends. Thus, the two activities of Cin8p antagonize each other, and are expected to result in stable spindle pole separation during yeast metaphase.
As described above, the model for kinesin-5 motor dynamics in the metaphase budding yeast mitotic spindle that best agrees with experimental data is one in which motors bind randomly to kMTs (, top), and then walk toward kMT plus-ends where they act to promote net kMT disassembly (, middle). Here, because longer kMTs have a larger number of possible motor binding sites, the motor model predicts that the number of motor interactions at kMT plus-ends will increase with increasing kMT length, as shown recently for kinesin-8 molecular motors in vitro
(Mayr et al., 2007
; Varga et al., 2006
). Interestingly, the plus-ends of iMTs, which are the longest MTs in the yeast mitotic spindle, may attract fewer Cin8p motors than the plus-ends of kMTs, simply because iMT plus-ends are surrounded by potential anti-parallel motor attachments, frustrating the plus-end motility of crosslinking Cin8p motors. This explanation is supported by results for the non-crosslinking depolymerizing motor Kip3p, whose localization and deletion phenotypes are distinct from that of Cin8p. We found that Kip3-GFP behavior could be reproduced by taking the Cin8-GFP simulation and turning “off” the cross-linking. In this case, the non-crosslinking motor tends to accumulate more onto iMTs, and less so onto kMTs. The simple physical feature of MT crosslinking, enabled in the tetrameric Cin8p, and disabled in the dimeric Kip3p, is sufficient to explain all the experimental results.
The simplest molecular mechanism for length-dependent MT disassembly is that the kinesin-5 motor itself acts directly to promote MT plus-end disassembly. We speculate that mechanical stress between walking motor head domains would stress tubulin-tubulin bonds to destabilize the lattice and promote MT disassembly. Alternatively, kinesin-5 could carry a disassembly-promoting binding partner to promote net disassembly at MT plus-ends, although to our knowledge there are no known cargoes that are transported by kinesin-5 motors.
Either way, the role of kinesin-5 motors in regulating kMT assembly dynamics is a new property that we have now identified for a motor previously known only as a sliding motor that acts between antiparallel MTs. Because of the potent effect that CIN8
deletion has on kinetochore organization, it seems unlikely that a significant Cin8p-independent pathway will be found to also promote length-dependent disassembly. The effects of kinesin-5 on MT assembly will be important to consider as anticancer drugs directed toward inhibiting kinesin-5 sliding activity are presently in clinical trials (Sudakin and Yen, 2007