The mitotic functions of kinesin-related proteins are achieved by virtue of their motor activity that is binding to and moving along microtubules. Therefore, to understand how kinesin-related proteins perform their mitotic roles, we are characterizing their in vitro function by single molecule fluorescence and multi-motor MT-gliding assays.
 
     The majority of the members of the kinesin superfamily carry their catalytic domain at their N-terminus and are plus-end directed, i.e., they move towards the dynamic plus-ends of the MTs. Until recently, minus-end motion was seen only for the structurally distinct and non-processive kinesin-14 family members, which carry the catalytic domain at their C-terminus. Using a single molecule motility assay, we have recently shown that the S. cerevisiae kinesin-5 Cin8 and Kip1 (N-terminal motors) move processively towards the minus-end of MTs. Remarkably, Cin8 and Kip1 molecules switched from minus-end to plus-end directed motility as a function of ionic strength, in a multi-motor MT-gliding assay and between antiparallel MTs. These recent findings break the long-standing dogma that processive N-terminal kinesin motors move unidirectionally towards the plus-ends of MTs and thus challenge us with new fundamental questions: a – How general is minus-end directed motility among the kinesin-5 motors? b – What is the structural basis of the minus-end directionality of N-terminal motors? c – What is the impact of directionality switch on spindle dynamics and mitosis? d – Are motile properties and directionality of kinesin-5 regulated by phosphorylation? Our goal is to address these important questions in order to better understand kinesin-5 function and regulation during mitosis.
 
 
 

 
 

 

 
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The S. cerevisiae Kinesin-5 Cin8 is a minus-end directed motor 
 
The S. cerevisiae Kinesin-5 Cin8 is a minus-end directed motor.
 
Left: time-lapse images of single molecule of WT Cin8 (green) moving on a polarity-marked microtubule (red). The bright seed (yellow) marks the minus-end of the microtubule (s).
 
Right: kymographs on minus-end directed movement of Cin8 in whole cell yeast extracts and insect (sf9) cells.
 
 The S. cerevisiae Kinesin-5 Kip1 is a minus-end directed motor
 
The S. cerevisiae Kinesin-5 Kip1 is a minus-end directed motor.
 
Top: time-lapse images of single molecule of WT Kip1 (green) moving on a polarity-marked microtubule (red). The bright seed (red) marks the minus-end of the microtubule (s).
 
Bottom: a kymograph of the same minus-end directed motility event shown at the top panel.