Biological & Soft Matter Seminar: Bidirectional Motility of Mitotic Kinesin-5 Motors: Old Dogma, New Insights

Abstarct:

One of the major factors that govern the mitotic spindle dynamics are the bipolar kinesin-5 motors (kinesin-5s). These motors were believed to move unidirectionally in the plus-end direction on the microtubules (MTs), thus performing essential functions in mitotic spindle assembly, maintenance of the bipolar structure and anaphase spindle elongation. Surprisingly, several reports from our and other laboratories have previously demonstrated that some kinesin-5 motors are bidirectional: they move in minus-end direction on the MTs as single-molecules and can switch directionality under a number of conditions. The mechanism of this bidirectional motility remains unknown. 

To address this unresolved problem, we apply an interdisciplinary approach combining live cell imaging, biophysical single molecule, and structural experiments to examine the activity of these motors in vivo and in vitro. We have previously shown that protein phosphorylation, motor clustering on the MTs and structural elements such as the neck-linker, loop 8 within the motor domain and the C-terminal tail, regulate the bidirectional motility of kinesin-5s. Recently, we examined the functions of the extended N-terminal non-motor domain (NTnmd), present in the bidirectional kinesin-5s, but absent in the exclusively plus-end directed kinesin motors (1). We found that NTnmd-deletion mutants exhibited cell viability and spindle localization defects. Using cryo-EM, we examined the structure of a MT-bound motor domain of bidirectional kinesin-5 Cin8, containing part of its NTnmd. Modeling and molecular dynamic simulations based on the cryo-EM map suggested that the NTnmd of Cin8 interacts with the C-terminal tail of β-tubulin. In vitro experiments on subtilisin-treated MTs confirmed this notion. We have also shown that NTnmd mutants are defective in plus-end–directed motility in single-molecule and antiparallel MT sliding assays. These findings demonstrate that the NTnmd-mediated interaction with MTs is critical for bidirectional motility and intracellular functions of bidirectional kinesin-5s.

1. S. K. Singh et al., Noncanonical interaction with microtubules via the N-terminal nonmotor domain is critical for the functions of a bidirectional kinesin. Science Advances 10, eadi1367 (2024).