In this study, we expand our earlier findings in fission yeast on a novel mitotic mechanism, that is kinesin-14 Klp regulation of γ-TuRC for spindle assembly. Here we identify two critical Tail elements in kinesin-14 Pkl1 that enable targeting to γ-TuRC and regulation by removal of γ-tubulin from γ-TuSC in vitro. This is the first report describing how γ-TuRC function is disabled and provides details of how this mechanism is accomplished by a kinesin-14. We refer to the critical Tail elements as a two-component TRIP domain, for targeting and regulatory interference at poles and use this domain to identify other kinesin-14 family members like human HSET that may share a similar mechanism as kinesin-14 Pkl1.
The integration of the TRIP domain with a Klp is logical in that it provides a means to link regulation of γ-TuRC to spindle functions controlled by other motor proteins. In eukaryotes, kinesin-14 and kinesin-5 families are ubiquitous and provide opposing activities in spindle assembly. In S. pombe
, kinesin-5 also localizes to spindle poles.22,23
We propose a new model that may apply, in which kinesin-5 association at or near γ-TuRC prevents Pkl1 from binding. This novel competitive relationship is distinct from that used by other microtubule-based kinesin-14 and kinesin-5 members.24
In most model eukaryotes, two to three kinesin-14 family members are present. An alignment of kinesin-14 sequences from fission yeast and mammals identified those subfamily members predicted to operate at MTOCs (). In human, mouse and rat, the mixed-charge cluster shown follows the sequence WDLK immediately upstream, similar to the TRIP domain. This domain is absent from Drosophila Ncd, and our cross-species studies previously revealed that human HSET but not Ncd rescues Pkl1 function in fission yeast.18
The TRIP domain and its mechanism to regulate γ-TuRC may therefore be of broad biological significance and useful in distinguishing kinesin-14 roles in vivo.
We propose the first model for regulation of γ-TuRC by kinesin-14 that includes Motor binding to γ-tubulin and γ-TuSC disruption by the TRIP domain through γ-tubulin removal. We previously demonstrated that the Pkl1 Motor domain binds to γ-tubulin helix 11.17
Despite the ability of the TRIP domain to act independently on γ-TuRC, it is clear that in vivo the Motor domain enhances this mechanism. This docking site of the Pkl1 Motor on γ-tubulin may partially overlap with sites used by γ-TuSC. The C-terminal domains of the GCP2/Alp4/Spc97 and GCP3/Alp6/Spc98 families of γ-TuSC proteins are proximate to γ-tubulin helix 11.10,25
One model is that the Pkl1 Motor domain, when bound to γ-tubulin, interferes with the C terminus of γ-TuSC subunits of γ-TuRC, thus priming the system for more efficient γ-tubulin removal by TRIP ().
Figure 4. Model for the mechanism of S. pombe kinesin-14 Pkl1 regulation of γ-TuRC. Pkl1 associates with the γ-TuRC in vivo to regulate bipolar spindle assembly through Tail and Motor domain interactions. The Pkl1 Motor domain (more ...)
How the γ-TuSC self-assembles and integrates with additional components to form the γ-TuRC is unknown. Also unclear is how the γ-TuRC can be regulated to control microtubule growth, dynamics and organization. In our peptide assays, Pkl1 removes a subset of γ-tubulin from the γ-TuRC (). A recent study of the budding yeast γ-TuRC indicates that additional γ-tubulin molecules are present along with the seven γ-TuSC subunits and are required for activating the complex26
(). Kinesin-14 Pkl1 might sever such additional γ-tubulins from the structure, preventing an active γ-TuRC. Alternatively, Pkl1 may sever γ-tubulin from γ-TuSC subunits, perturbing or entirely disrupting the γ-TuRC structure (). We favor a mechanism in which γ-tubulin remains attached to microtubule minus ends, consistent with a cap27
and with in vitro studies that indicate free minus ends depolymerize more slowly than plus ends28
shown by TEM to be structurally distinct.29
As well, the γ-tubulin internal structural domain may not allow rotation to accommodate protofilament bending, as occurs in β-tubulin for depolymerization.30,31
How γ-tubulin removal by Pkl1 is regulated and the rate at which γ-TuSC and γ-TuRC can recover remains unclear.
The novel mechanism of γ-TuRC regulation by kinesin-14 in S. pombe determined by parallel in vivo and in vitro analysis provides the first model for understanding how γ-TuRC can be perturbed to influence microtubules required for spindle assembly. For future experiments, we are developing a high-resolution, dynamic in vitro system for capture and analysis of a single γ-TuRC to determine which γ-tubulins are accessible, where removal is initiated, the critical number of γ-tubulins removed to inactivate the γ-TuRC, the rate of γ-TuRC recovery and TRIP domain recycling, and to monitor changes to protofilament and microtubule attachment at γ-TuSC of γ-TuRC following TRIP action. This coupled with in vivo studies to determine if mutants can be isolated that are resistant to the action of Pkl1 will provide added details. We expect the mechanistic information defined here to have a fundamental impact on the microtubule cytoskeleton field, revealing new roles for conserved Klp families in spindle assembly while also uncovering details of how structural changes in the γ-TuRC can be generated by motor proteins.