Final Report Summary - MITDYN (Studies on the role of Plk1 localization and activity in the regulation of microtubule dynamics during mitosis)
Microtubules are long polar polymers composed of a- and -tubulin. Microtubules switch stochastic ally between growth and shrinkage, a tightly-controlled process known as dynamic instability that is critical for microtubule function in cellular processes. At the interphase to mitosis transition, the mi crotubule cytoskeleton undergoes rapid remodeling with an increase in its dynamics parameters, which allows microtubules to rearrange and assemble into a spindle, while also capturing and align ing chromosomes. Regulators of microtubule dynamics increase the catastrophe rate to an estimated two to ten-fold and decrease the rescue rate four-fold. In particular, the Kinesin-l3 family members are major microtubule depolymerases that hydrolyze ATP to promote microtubule catastrophe. Im portantly, kinesin-l3 proteins can also depolymerize taxol-stabilized microtubules and are implicated in the resistance to taxol in cancer cell lines. However they are tightly regulated by intrinsic and ex trinsic factors to control chromosome segregation spatially and temporally. Our lab focuses on the kinesin-l3, MCAK, as a paradigm for the mechanism and regulation of microtubule depolymerases. MCAK is recruited to the plus tips of microtubules via End-Binding (EB) proteins and to kineto chores through an interaction with Sgo2. At the onset of mitosis, the kinesin-8 Kifl8b associates with MCAK and EBl to generate a powerful microtubule depolymerizing complex and dramatically re models the microtubule cytoskeleton to allow efficient chromosome capture, alignment and segrega tion. However, the molecular mechanisms by which these proteins associate and synergize to pro mote microtubule depolymerization and facilitate timely mitotic progression is unclear.
In this project, we aim to understand the function and role of the Kinesin-8 Kifl8b in regulating spe cifically the Kinesin-l3 MCAK function during mitosis and the implication for spindle assembly, po sitioning and for chromosome segregation. First we are conducting cell biology and biochemistry studies of the Kinesin-8 to understand the molecular basis for the properties of Kifl8b and its func tion in the cell. We showed using gene knockout that the Kinesin-8 Kifl8b controls microtubule length to center the mitotic spindle at metaphase. Using in vitro reconstitution, we revealed that
Kifl8b is a highly processive plus end-directed motor that uses a C-terminal non-motor microtubule-bind ing region to accumulate at growing microtubule plus ends. This region is regulated by phosphoryla
tion to spatially control Kifl8b accumulation at plus ends and is essential for Kifl8b-dependent spindle positioning and regulation of microtubule length. Finally we demonstrated that Kifl8b shortens microtubules by increasing the catastrophe rate of dynamic microtubules. Overall, our work revealed that Kifl8b utilizes its motile properties to reach microtubule ends where it regulates astral microtubule length to ensure spindle centering.
The combination of structural, biophysical and cellular studies with aim of reconstituting the biolo gical complexity found at microtubule tips is groundbreaking and will allow us in future studies to understand how the combinatorial associations of microtubule regulators cooperate spatially and tem porally to coordinate plus end dynamics.
In this project, we aim to understand the function and role of the Kinesin-8 Kifl8b in regulating spe cifically the Kinesin-l3 MCAK function during mitosis and the implication for spindle assembly, po sitioning and for chromosome segregation. First we are conducting cell biology and biochemistry studies of the Kinesin-8 to understand the molecular basis for the properties of Kifl8b and its func tion in the cell. We showed using gene knockout that the Kinesin-8 Kifl8b controls microtubule length to center the mitotic spindle at metaphase. Using in vitro reconstitution, we revealed that
Kifl8b is a highly processive plus end-directed motor that uses a C-terminal non-motor microtubule-bind ing region to accumulate at growing microtubule plus ends. This region is regulated by phosphoryla
tion to spatially control Kifl8b accumulation at plus ends and is essential for Kifl8b-dependent spindle positioning and regulation of microtubule length. Finally we demonstrated that Kifl8b shortens microtubules by increasing the catastrophe rate of dynamic microtubules. Overall, our work revealed that Kifl8b utilizes its motile properties to reach microtubule ends where it regulates astral microtubule length to ensure spindle centering.
The combination of structural, biophysical and cellular studies with aim of reconstituting the biolo gical complexity found at microtubule tips is groundbreaking and will allow us in future studies to understand how the combinatorial associations of microtubule regulators cooperate spatially and tem porally to coordinate plus end dynamics.