Single Molecule Characterization Of Mitotic Kif15 Reveals Capability To Generate Force In Anti-Parallel Microtubules
BIOPHYSICAL JOURNAL(2017)
Abstract
Proper formation of the mitotic spindle is essential for chromosome separation and cell division. Dimeric Kinesin-12, or Kif15, has been shown to serve as a backup mechanism for cell division, promoting spindle assembly in the absence of tetrameric Kinesin-5, or Eg5. The mechanism by which Kif15 aids in the assembly of the spindle is unknown. Here, single molecule motility assays were performed using optical tweezers to evaluate the mechanistic behavior of Kif15 on microtubules (MTs). Constructs containing various sub-components of the motor were utilized to determine the role of each domain, including the motor heads, stalk, MT binding domain (N700) and the full length motor (FL). Step size, dwell times, and stall forces were measured. Rupture assays were also performed on the MT binding domain (Coil-1) to determine the binding strength and the nature of the Coil-1/MT interaction. Motility and rupture assays were repeated on subtilisin-digested MTs (dMT) in which the negatively-charged carboxyl-terminal tail of MTs, called the E-hook, is removed. We found that the Coil-1 was stronger than the stall force of the motor due to its interaction with the E-hook, revealing that Kif15 has the ability to build up mechanical strain and slide MTs apart. In order to determine the importance of MT orientation, an in vitro optical trapping and fluorescence assay was developed to determine the force generating capabilities of Kif15 in different bundle environments. We determined that Kif15 slides anti-parallel MTs apart while parallel bundles remain stationary with a small amount of antagonizing force generation. Our studies provide insight regarding how Kif15 is able to rescue bipolar spindle assembly in the absence of Eg5.
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Key words
microtubules,mitotic kif15,generate force,single molecule characterization,anti-parallel
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