The two coiled-coil domains of the fission yeast kinesin-6 Klp9 are required for motor tetramerization and spindle elongation.

The fission yeast kinesin-6 Klp9 localizes to the spindle midzone at anaphase to produce sliding forces to elongate the bipolar spindle. In the absence of Klp9 , anaphase spindle elongation is attenuated by half its normal rate. Klp9 functions as a microtubule plus end-directed tetrameric motor. Tetramerization is the key to its microtubule sliding function, as tetramerization allows Klp9 to bind antiparallel microtubules at the midzone. The amino acid sequence of Klp9 indicates two alpha-helical coiled-coils domains CC1 and CC2, important for protein-protein interactions. We seeked the potential oligomerization states of Klp9 via its coiled-coils using AlphaFold3. AlphaFold predicted that CC1 can form dimers and together with CC2 can form tetramers. The different oligomeric states enabled precise experimental verifications. We measured Klp9 motor GFP intensity and anaphase spindle elongation rate for the full-length Klp9 , Klp9-deletion (Klp9Δ), and truncated Klp9 containing no coiled-coils, or only CC1, or both CC1 and CC2. The results indicate that: 1) GFP intensity increases with increasing oligomeric state, and 2) attenuated anaphase spindle velocity is restored only in the Klp9 truncation containing both CC1 and CC2. The experimental data are consistent with prediction, indicating that CC1 contributes to Klp9 dimerization, and that CC1 and CC2 together contribute to Klp9 tetramerization.