Stumpff, Jason

The kinesin-5 motor protein KIF11 is crucial for mitotic spindle assembly, driving the separation of spindle poles through microtubule sliding. Src-family kinases phosphorylate KIF11 at multiple tyrosine residues within its motor domain, but the mechanistic consequences of these modifications remain unclear. Here, we dissect the role of phosphorylation at Y211 using phospho-mimetic (Y211E) and nonphosphorylatable mutants (Y211F) in biochemical, biophysical, and cellular assays. Optical trapping and Förster resonance energy transfer analyses reveal that Y211 phosphorylation slows neck-linker docking, reducing motor velocity and force generation under load. In human cells, Y211E expression impairs bipolar spindle formation and decreases spindle pole separation velocity, while Y211F shortens steady-state spindle length. Fluorescence recovery after photobleaching analyses show that Y211E accelerates motor turnover on spindle microtubules, consistent with the mutant motor’s heightened load sensitivity. Together, these findings support a model in which Src-mediated phosphorylation at Y211 acts as a rheostat to tune KIF11 mechanochemistry and spindle assembly dynamics, linking cancer-relevant kinase signaling to mitotic force generation.

Abstract The kinesin-5 motor protein KIF11 is crucial for mitotic spindle assembly, driving the separation of spindle poles through microtubule sliding. Src-family kinases phosphorylate KIF11 at multiple tyrosine residues within its motor domain, but the mechanistic consequences of these modifications remain unclear. Here, we dissect the role of phosphorylation at Y211 using phospho-mimetic (Y211E) and non-phosphorylatable mutants (Y211F) in biochemical, biophysical, and cellular assays. Optical trapping and Förster resonance energy transfer (FRET) analyses reveal that Y211 phosphorylation slows neck-linker docking, reducing motor velocity and force generation under load. In human cells, Y211E expression impairs bipolar spindle formation and decreases spindle pole separation velocity, while Y211F shortens steady-state spindle length. Fluorescence recovery after photobleaching (FRAP) analyses show that Y211E accelerates motor turnover on spindle microtubules, consistent with the mutant motor’s heightened load sensitivity. Together, these findings support a model in which Src-mediated phosphorylation at Y211 acts as a rheostat to tune KIF11 mechanochemistry and spindle assembly dynamics, linking cancer-relevant kinase signaling to mitotic force generation.

The chromokinesin KIF22 generates forces that contribute to mitotic chromosome congression and alignment. Mutations in the α2 helix of the motor domain of KIF22 have been identified in patients with abnormal skeletal development, and we report the identification of a patient with a novel mutation in the KIF22 tail. We demonstrate that pathogenic mutations do not result in a loss of KIF22’s functions in early mitosis. Instead, mutations disrupt chromosome segregation in anaphase, resulting in reduced proliferation, abnormal daughter cell nuclear morphology, and, in a subset of cells, cytokinesis failure. This phenotype could be explained by a failure of KIF22 to inactivate in anaphase. Consistent with this model, constitutive activation of the motor via a known site of phosphoregulation in the tail phenocopied the effects of pathogenic mutations. These results suggest that the motor domain α2 helix may be an important site for regulation of KIF22 activity at the metaphase to anaphase transition. In support of this conclusion, mimicking phosphorylation of α2 helix residue T158 also prevents inactivation of KIF22 in anaphase. These findings demonstrate the importance of both the head and tail of the motor in regulating the activity of KIF22 and offer insight into the cellular consequences of preventing KIF22 inactivation and disrupting force balance in anaphase.

Abstract Diffuse midline gliomas with the H3.3 K27M mutation are lethal brain tumors in children. H3 K27M causes global loss of Lys27 triple methylation (Lys27me3), inducing epigenetic reprograming. Here we show that H3.3 K27M also causes decreased H3.3 Ser31 phosphorylation on mitotic chromosomes. We show that H3.3 K27M DIPG cells have reduced pericentromeric phospho-Ser31 and increased rates of chromosome missegregation compared to normal, diploid human cells. CRISPR-editing K27M to M27K restored phospho-Ser31 to WT levels and dramatically decreased the rate of chromosome missegregation. We confirm that Chk1 is the H3.3 Ser31 kinase: K27M mutant H3.3 protein exhibits ~60% reduced Chk1 phosphorylation of Ser31 in vitro. Chk1 knockdown completely abolishes phospho-Ser31 in cells and these have increased rates of chromosome missegregation. In normal, diploid cells, expression of K27M or an S31A non-phosphorylatable mutant increased chromosome missegregation; this is suppressed by expressing a phosphomimetic double mutant (K27M/S31E) that restores phospho-Ser31. WT cells arrest following chromosome missegregation. However, cells expressing H3.3 K27M or S31A fail to arrest - despite having WT p53. Finally, we expressed H3F3AS31A and PDGFb in an RCAS/TVA mouse model of DIPG and ~80% developed diffuse high-grade brain tumors and show significantly decreased survival. Our results suggest that loss of phospho-Ser31 alone is oncogenic because H3.3 S31A-expressing cells are WT for K27me3. Our results demonstrate that H3.3 K27M inhibits Ser31 phosphorylation both in vitro and in vivo, leading to both chromosome missegregation and loss of subsequent G1 arrest – thus creating diffuse midline gliomas with both dynamic, complex karyotypes and epigenetic reprogramming.

Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding. Kinesin-5 tails decrease microtubule-stimulated ATP-hydrolysis by specifically engaging motor domains in the nucleotide-free or ADP states. Cryo-EM reveals that tail binding stabilizes an open motor domain ATP-active site. Full-length motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering. The tail is critical for motors to zipper together two microtubules by generating substantial sliding forces. The tail is essential for mitotic spindle localization, which becomes severely reduced in tail-deleted motors. Our studies suggest a revised microtubule-sliding model, in which kinesin-5 tails stabilize motor domains in the microtubule-bound state by slowing ATP-binding, resulting in high-force production at both homotetramer ends.

Significance Members of the kinesin superfamily serve a wide variety of functions, and a dominant narrative for these molecular motors has been that each member of the superfamily is uniquely specialized to serve a very limited set of functions. However, it is now appreciated that many members of this group serve several distinct physiological roles, and it has been unclear how these kinesins accomplish this functional flexibility. In this report, we describe a posttranslational modification of the kinesin 5 family member Eg5 that dramatically alters its chemomechanical behavior to make it function much more efficiently under load and in ensembles. This work provides the biophysical context required to mechanistically understand the effects of modified Eg5 in dividing cells.