In this study we showed that the SIN promotes spindle elongation and appropriate nuclear positioning through Sid2 phosphorylation of the kinesin Klp2. Sid2 phosphorylation keeps Klp2 off MTs by inhibiting its interaction with the EB1 homologue Mal3. Mal3 loads Klp2 on MT plus ends through binding to two SxIP motifs in a predicted unstructured region of the tail of Klp2. Sid2 phosphorylation of Klp2 at two sites proximal to the EB1/Mal3-binding motifs prevents this interaction. These results are reminiscent of several recent studies, which showed that phosphorylation near EB1/Mal3-binding motifs can disrupt association with EB1 by interfering with electrostatic interactions that contribute to binding (Honnappa et al., 2009
; Kumar et al., 2009
; Vacher et al., 2011
). However, the inhibitory phosphorylation sites on Klp2 are not immediately flanking the EB1 binding sites as in the other examples, but instead are between 80 and 110 residues away. It is possible that the predicted unstructured nature of the Klp2 tail allows the phosphoserines to act at a greater distance to inhibit electrostatic interactions that contribute to binding. Alternatively, Klp2 phosphorylation could result in a structural transition, perhaps from a disordered to a more ordered state (Kissinger et al., 1999
; Mendoza-Espinosa et al., 2009
), which prevents Klp2 binding to Mal3 by making the SxIP binding motifs less accessible. The N-terminal sequence of myosin regulatory light chain is one example where phosphorylation results in a more stable domain. More detailed structural analysis will be required to determine the precise mechanism of phosphoinhibition of the Klp2–Mal3 interaction.
Our results showed that in cells delayed in telophase (cps1-191
), loss of either SIN regulation of Klp2 (klp2-2A
cells) or PAA microtubules (myp2
cells) caused a small displacement of nuclei toward the cell center. However, elimination of both SIN regulation of Klp2 and PAA microtubules (klp2-2A myp2
cells) caused a strong nuclear clustering defect similar to SIN mutants. Of interest, cps1-191
cells slowly form septa, and klp2-2A myp2 cps1-191
cells often showed mispositioned nuclei being “cut” by the ingressing septa. Surprisingly, klp2-2A myp2
cells did not display “cut” phenotypes or obvious mispositioning of nuclei toward the cell center in normally dividing cells, suggesting that SIN-dependent nuclear positioning is most important for preventing “cut” nuclei when cytokinesis has been delayed. It is possible that in normally dividing cells, where the septum ingresses quickly, the nuclei are kept away from the cell center by the interphase nuclear positioning system (Tran et al., 2001
), which uses pushing forces from MTs associated with the nuclear envelope. In this scenario, ingression of the actomyosin ring and septum may provide a barrier for MTs associated with the nuclei to push against to keep the nuclei away from the division apparatus.
We presume that the SIN inhibits the MT localization of Klp2 to prevent its minus end–directed sliding activity from pulling telophase nuclei together. By this model, Klp2 would slide MTs emanating from opposite nuclei or the nucleus and PAA MTs to pull the nuclei toward the cell center. Our results also show that PAAs perform a Klp2-independent function in positioning the telophase nuclei. One possible function for PAA MTs could be that they interact with MTs associated with the nuclei via motor proteins to provide pushing forces to move the nuclei away from the cell center. One candidate motor could be the plus end–directed kinesin-6 Klp9, which transiently localizes at equatorial MTs in telophase before returning to the nucleoplasm (Fu et al., 2009
Just as inappropriate minus end–directed MT sliding activity in telophase may pull the nuclei together, this same type of MT sliding activity would also be predicted to interfere with spindle elongation. Consistent with this model, Klp2-2A (in contrast to wild-type Klp2) was observed not just on astral MTs but also faintly on anaphase spindles. In addition, both SIN inhibition and the klp2-2A
mutation slowed down spindle elongation in anaphase. Thus the SIN may inhibit Klp2 in anaphase to prevent its minus end–directed sliding activity from interfering with spindle elongation, although we cannot rule out other possibilities, such as subtle changes in MT organization or dynamics. It makes sense that the SIN might regulate anaphase spindle elongation, since the SIN becomes active in anaphase (Guertin and McCollum, 2001
; Feoktistova et al., 2012
). Furthermore, regulation of both spindle elongation and ingression of the actomyosin ring and septum by a single pathway may ensure proper coordination of these key events.
This work contributes to a broader understanding of kinesin-14 regulation and the dynamics involved in nuclear positioning. Klp2 plays a role in nuclear positioning during interphase and karyogamy but is inactivated during late mitosis to allow for rapid anaphase spindle elongation and telophase nuclear positioning. This study uncovered the mechanism governing EB1/Mal3-dependent loading of Klp2 onto MTs and SIN-mediated inhibition of the Klp2–Mal3 interaction. Furthermore, we also showed that phosphorylation at sites not immediately adjacent to the Mal3/EB1-binding motifs can regulate this interaction. This mode of regulation may be conserved in other kinesin-14 proteins because the Klp2 orthologue in Drosophila melanogaster
, called ncd, localizes to MT plus ends via EB1 and is inhibited during anaphase B (Sharp et al., 2000
; Goshima et al., 2005
). It will be interesting to determine whether other members of the kinesin-14 family are regulated by phosphorylation like Klp2.