Combining atomic-resolution structure with high-resolution tracking in cells to dissect regulation and mechanism of the MKlp2 kinesin – MITOKINESIN

Mitosis involves the complex coordination of cytoskeleton rearrangements in which the action of nanomotors is temporally and spatially controlled to precisely divide chromosomes and cytoplasm of a cell. While basic features of molecular motor function have been understood, the most burning open questions concern the collective regulation of motors in complex cellular environments. Understanding the highly regulated series of events in mitosis is also central to medicine development. One of these motors, MKlp2, plays critical roles for the metaphase to anaphase transition and for cytokinesis. MKlp2 is overexpressed in mitotic cells and several human solid tumor cells. Its inhibition in pancreatic adenocarcinoma cells reduced cell growth and our collaborators have shown that MKlp2 inhibitors can kill tumor stem cells.

The MKlp2 motor domain is 60% larger than that of other kinesins, due to several unique inserts that are believed to be involved in regulation and possibly new functions for this kinesin. It belongs to the kinesin 6 class of molecular motors. Interestingly, two other class 6 kinesins with different insert sequences localize to different sites in the midbody of mammalian cells and play important, but distinct roles in cytokinesis. MKlp2 interacts with key kinases such as polo-kinase 1 (Plk1) and AuroraB, the enzymatic component of chromosome passenger complex (CPC), and contribute to the control of their spatial and temporal action during cell division. This microtubule motor protein also interacts with myosin II and this recognition is critical to bring kinases at the proper place to ensure furrow ingression. Whether the motor activity of Mklp2 is important at this location and whether myosin II activity is influenced by MKlp2 is unknown. While this motor has emerged as a crucial player/regulator in mitosis, its motor properties, precise cellular functions, as well as its regulation remain elusive.

MKlp2 is also interesting at the molecular level because it is a N-kinesin with unique features. These include an N-terminal extension, a short insertion in loop2 (important for MT binding), a long insertion in loop6 adjacent to the N-terminal extension in tertiary structure, and the neck linker that is about four times longer than that found in other kinesins. Because of the large insertions near the structural elements important for the force generation of N-kinesins, this motor is likely to have a different mechanism for generating its ‘powerstroke’. Whether the insertions contribute to and/or regulate its role in cytokinesis is unclear. Our preliminary data showing how the motor interacts with microtubule confirms its unusual property. Plk1 interaction, located close to the mechanical element of this kinesin, is likely to modulate how this motor produces force and how such force is used to allow transport of cargos or to organize microtubules during mitosis.

To understand how MKlp2 functions, high-resolution structures and a functional characterization are essential. We propose to undertake a thorough structural approach of this motor and its interaction with its partners. This study will be coupled with single-molecule studies in vitro and in vivo, pursuing for the latter a novel approach using single-walled carbon nanotubes (SWNTs) as precisely targetable and uniquely stable near-infrared fluorescent markers. We have further established multidisciplinary international collaborations to add transient kinetics and cryo-electron microscopy (cryoEM) experiments to study basic properties of this motor.

The project is both ambitious and innovative in focusing on MKlp2 and its regulators and in combining atomic structure determination with functional single-molecule studies in live cells. The ultimate goal is to define the properties of the motor by coupling structural, single-molecule, and cellular studies, and to break new ground by tracking the motor in cells using novel super-resolution live-cell imaging technique.