Intracellular functions and motile properties of bi-directional kinesin-5 Cin8 are regulated by neck linker docking

Abstract

In this study, we analyzed intracellular functions and motile properties of neck-linker (NL) variants of the bi-directional S. cerevisiae kinesin-5 motor, Cin8. We also examined – by modeling – the configuration of H-bonds during NL docking. Decreasing the number of stabilizing H-bonds resulted in partially functional variants, as long as a conserved backbone H-bond at the N-latch position (proposed to stabilize the docked conformation of the NL) remained intact. Elimination of this conserved H-bond resulted in production of a non-functional Cin8 variant. Surprisingly, additional H-bond stabilization of the N-latch position, generated by replacement of the NL of Cin8 by sequences of the plus-end directed kinesin-5 Eg5, also produced a nonfunctional variant. In that variant, a single replacement of N-latch asparagine with glycine, as present in Cin8, eliminated the additional H-bond stabilization and rescued the functional defects. We conclude that exact N-latch stabilization during NL docking is critical for the function of bi-directional kinesin-5 Cin8.

Noncanonical interaction with microtubules via the N-terminal nonmotor domain is critical for the functions of a bidirectional kinesin

“…S1). These findings indicate that these variants were either defective in minus-end–directed motility and/or exhibited reduced affinity to MTs ( 12 , 29 ). Consistently, cells expressing these two variants (Cin8Δ39–69 and Cin8Δ70–75) exhibited a considerably lower percentage of short bipolar spindles compared to wt Cin8, indicating that these variants were defective in bipolar spindle assembly ( Fig.…”

“…We next examined the binding of wt Cin8 and NTnmd deletion to the MTs by determining the number of Cin8 motors bound to subtilisin-treated MTs compared to untreated MTs in the presence of ATP ( Fig. 7 ) ( 13 , 29 ). First, we found that on untreated MTs, the number of NTnmd mutants (Cin8Δ1–38 and Cin8Δ1–69) bound to MTs was markedly reduced compared to the wt Cin8 ( Fig.…”

“…For this purpose, we labeled the plus-ends of the MT with a bright rhodamine seed ( 12 ) and polymerized two sets of MTs, in the presence and absence of biotin-labeled tubulin. The biotinylated MTs were attached to the surface, followed by the addition of biotin-lacking MTs in the presence of Cin8 ( 12 , 29 ). Consistent with previous reports ( 10 , 12 , 29 ), we observed that the majority of antiparallel MT sliding events (86%) mediated by the wt Cin8 were plus-end–directed ( Fig.…”

“…The biotinylated MTs were attached to the surface, followed by the addition of biotin-lacking MTs in the presence of Cin8 ( 12 , 29 ). Consistent with previous reports ( 10 , 12 , 29 ), we observed that the majority of antiparallel MT sliding events (86%) mediated by the wt Cin8 were plus-end–directed ( Fig. 9 and movie S1), as is to be expected from the physiological functions of Cin8 in spindle assembly.…”

“…We also demonstrated that the deletion of this large insert is essential for bidirectional motility on the single-molecule level and in surface MT gliding assays ( 13 ). In the second study, we demonstrated that the flexibility of NL anchoring to the motor domain of Cin8 is essential for Cin8 functionality ( 29 ). In light of the knowledge that flexibility of the NL is correlated with the two-headed MT bound state of plus-end–directed kinesin-motors ( 65 – 67 ), it is possible that the flexibility of the NL docking of Cin8 may also enable the two-headed bound state, enabling stepping in two directions.…”