Single-Molecule Investigations Into How Plant Kinesin-12 Motor Proteins Accurately Align The Cell Division Plane

Plant cytokinesis is the ultimate step in cell division that partitions the parent cell into two daughter cells by the physical insertion of a cell plate. The accurate placement of the cell plate aided by the microtubule-based phragmoplast is indispensable as it governs proper morphology and further plant development. However, how a plant cell correctly orients and inserts the maturing cell plate into the right location of the plasma membrane is poorly understood. Two kinesin-12 members, phragmoplast orienting kinesin 1 and 2 (POK1 and POK2), are involved in the process, but how these molecular machines work is not known. It has been hypothesized that these POK motors guide the phragmoplast by interacting with the peripheral microtubules emanating from it. In this thesis, single-molecule fluorescence and force spectroscopy of motor domains of POK1 and POK2 has been carried out to determine how these Arabidopsis thaliana motors function mechanically. It was found that POK1 and POK2 kinesin motors are moderately fast, plus-end-directed microtubule motors, which are capable of exerting forces. Interestingly, both motors switch between processive and diffusive modes that was quantifi ed by an exclusive-state mean-squared-displacement analysis. Furthermore, the motors are the weakest kinesins characterized to date attributed to the latter switching behaviour. After establishing the polarity of peripheral microtubules in the root meristem of Arabidopsis, jointly the data support a model that POK motors push against the peripheral microtubules of the expanding phragmoplast. These pushing forces contribute to the proper guidance of the phragmoplast to possibly re ne cell plate fusion site and orientation of the cell plate. Intriguingly, the single-molecule characterization of the tail domain of POK2 revealed a novel microtubule-associated motor distinct from kinesins, dyneins, and myosins. This motor exhibits slow, processive, plus-end motility. Moreover, this tail domain is also capable of membrane binding preferentially to the phosphatoinositides. Therefore, this thesis proposes potential roles of POK2 in guiding as well as stabilizing and connecting the expanding phragmoplast to the plasma membrane. In sum, this thesis provides mechanical insights into how active motors may assist in accurate positioning of cell walls in plants. Moreover, the finding of a new cytoskeletal motor may open avenues for detailed investigation and new discovery of related molecular machines