Tuesday, November 10, 2015: 2:00 PM
151D/E (Salt Palace Convention Center)
Proper formation of the mitotic spindle is essential for chromosome separation and cell division. Single molecule studies have been essential in evaluating the mechanistic behavior of molecular motors, including conventional kinesin and myosin. 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 and stalk (N420); motor heads, stalk, and MT binding domain (N700); and the full length motor (FL) with its inhibitory tail (Coil-2). From these, characteristics such as step size, dwell times, and stall forces were measured. Binding assays were also performed on the MT binding domain (Coil-1) to determine the rupture force 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. Together, these results provide insight into the interaction between Kif15 and MTs and how motor sub-components contribute to spindle assembly in tetrameric-Eg5-independent cells.