Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from &lt;em&gt;S. cerevisiae&lt;/em&gt; Cells

Pandey, Himanshu; Zvagelsky, Tatiana; Popov, Mary; Sadan, Mayan; Yanir, Neta; Goldstein-Levitin, Alina; Siegler, Nurit; Hershfinkel, Shira; Abraham, Yahel; Abraham, Roy; Gheber, Levi A.; Gheber, Larisa

doi:10.3791/63425

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“…6 and 7 ) ( 17 , 18 ). To this end, we used a single-molecule motility assay, in which we characterized the activity of GFP-tagged full-length N-terminal variants, overexpressed and purified from S. cerevisiae cells, on fluorescently labeled GMPCPP-stabilized polarity-marked MTs ( 12 , 53 ). These experiments were conducted in the presence of 1 mM ATP and 70 mM KCl.…”

Section: Resultsmentioning

confidence: 99%

“…The sorting of Cin8 motors into single molecules and clusters was performed as previously described ( 13 , 29 , 53 , 54 ). Our examination of the fluorescence intensity of fluorescent Cin8 molecules as a function of time showed that the intensity was bleached in steps of ⁓50 a.u., with each step representing the photobleaching of 1 GFP.…”

Section: Methodsmentioning

confidence: 99%

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

Singh,

Siegler,

Pandey

et al. 2024

Sci. Adv.

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Several kinesin-5 motors (kinesin-5s) exhibit bidirectional motility. The mechanism of such motility remains unknown. Bidirectional kinesin-5s share a long N-terminal nonmotor domain (NTnmd), absent in exclusively plus-end–directed kinesins. Here, we combined in vivo, in vitro, and cryo–electron microscopy (cryo-EM) studies to examine the impact of NTnmd mutations on the motor functions of the bidirectional kinesin-5, Cin8. We found that NTnmd deletion mutants exhibited cell viability and spindle localization defects. Using cryo-EM, we examined the structure of a microtubule (MT)-bound motor domain of 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. Last, we showed that NTnmd mutants are defective in plus-end–directed motility in single-molecule and antiparallel MT sliding assays. These findings demonstrate that the NTnmd, common to bidirectional kinesin-5s, is critical for their bidirectional motility and intracellular functions.

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