CONTRIBUTION OF KINESIN-MEDIATED MICROTUBULE DYNAMICS TO THE REGULATION OF MITOTIC SPINDLE STABILITY: MOLECULAR MECHANISM OF KINESIN-MICROTUBULE INTERACTION
Open Access
- Graduate Program:
- Bioengineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 23, 2018
- Committee Members:
- William O Hancock, Dissertation Advisor
- William O Hancock, Committee Chair
- Peter J Butler, Committee Member
- Justin Lee Brown, Committee Member
- Melissa Rolls, Outside Member
- Keywords:
- Kinesin
- Microtubule
- Microtubule-associated protein
- Eg5 inhibitors
- Mitosis
- Size-scaling problem
- Single-molecule
- transient kinetics
- Tubulin dynamics
- Abstract:
- Faithful chromosome segregation relies on proper control of spindle architecture, which is controlled by dozens of regulatory proteins. Microtubule dynamic instability is central to defining spindle size and geometry, and kinesin motors are known to mediate tubulin dynamics during mitosis. The goal of this dissertation is to relate the chemomechanical characteristics of nanometer-scale molecular motors in the kinesin-5 family to the micron-scale microtubule dynamics that are essential for intracellular transport and cell division. In addition to sliding apart antiparallel microtubules, the kinesin-5, Eg5 has also been shown to promote microtubule polymerization, though the molecular mechanism remains elusive. This study includes three aspects of resolving the Eg5 microtubule polymerase mechanism. First, by comparing mechanochemical cycles of kinesin superfamily members, Eg5 motors were shown to primarily reside in a two-heads-bound state during its ATP hydrolysis cycle. This conformation has the potential to act as a “staple” that enhances tubulin-tubulin longitudinal bond stability in the microtubule lattice. Second, isolated Eg5 motor domains were shown to induce a curved-to-straight transition in tubulin, which promotes lateral tubulin-tubulin bonds. This curvature-modulating mechanism is shown to be conferred by family-specific sequences in Loop11, which is proximal to Switch-II in the motor domain and is located at the alpha-beta tubulin interface. Finally, small-molecule inhibitors that can mechanically regulate Eg5 motors were shown to also impact microtubule dynamics and spindle integrity both in vitro and in vivo. Inhibitors that induce a tight-binding rigor state of the motor have opposite effects from the better known Loop5 inhibitors that weaken microtubule binding. By linking the molecular mechanism of Eg5-microtubule interaction to cell-scale changes in microtubule architecture, this work should benefit drug discovery and chemical probe development for treating cytoskeleton-based neuropathies and cancer.