Investigating the cellular functions of kinesin, microtubules and +TIPs using in vitro reconstitution, microscale engineering and novel statistical techniques

Open Access
Author:
Chen, Yalei
Graduate Program:
Cell and Developmental Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
October 09, 2014
Committee Members:
  • William O Hancock, Dissertation Advisor
  • William O Hancock, Committee Chair
  • Melissa Rolls, Committee Member
  • Christopher Martin Yengo, Committee Member
  • Richard Cyr, Committee Member
  • Thomas Nelson Jackson, Committee Member
Keywords:
  • microtubule
  • kinesin
  • cellulose synthase
  • spindle
  • micro-fabrication
  • in vitro reconstitution
  • statistics
Abstract:
Microtubules are cytoskeletal filaments that self-assemble from αβ tubulin dimers and are essential for cellular mechanics, cell division and intracellular cargo transport. Carrying out these functions requires numerous accessory proteins that bind to and interact with microtubules. The study of microtubules and microtubule associated proteins (MAPs) is relevant for understanding aging, developing cancer therapies, and providing approaches to treating neurodegenerative diseases. This thesis is devoted to using bioengineering tools and biophysical reconstitution methods to elucidate molecular mechanisms by which MAPs carry out their functional roles in mitosis, in organizing the neural cytoskeleton, and other processes. The mitotic motor kinesin-5 is best known as a homotetramer, however, motor properties independent of its homotetrameric configuration are not well understood. From in vitro assays, an engineered kinesin-5 dimer was found to stabilize microtubules by slowing GMPCPP microtubule depolymerization and promote dynamic microtubule growth by inhibiting catastrophe. Single-molecule experiments showed that kinesin-5 remains associated with microtubule plus-ends for a duration of 7 seconds. These results demonstrate that in addition to its role in sliding apart antiparallel microtubules, kinesin-5 is a microtubule stabilizer, polymerase and end-tracker. To maintain microtubule uniformity in dendrites of Drosophila neurons, microtubules growing into branches need to be guided towards the cell body. To investigate the mechanism of microtubule guidance, purified EB1 and kinesin-2 were dimerized to form an EB1-kinesin complex, and this complex was shown to be sufficient to steer the growth of one microtubule towards plus-end of another microtubule, providing a mechanism for maintaining uniform microtubule orientation, not only in neurons but also in other cells. Also, this work demonstrates that the dynamic EB1-microtubule interaction is sufficiently strong to carry out mechanical functions in cells. In vitro reconstitution can be used to investigate microtubules and MAPs in well-defined environments, but it is usually limited to single microtubules. To eliminate this constraint, micro-patterned electrodes were fabricated on elevated pedestals and high frequency AC fields were used to align microtubules on the opposite electrodes, thus forming a bipolar artificial mitosis spindle in 3D with microtubule plus-ends oriented towards the overlap zone. These aligned microtubules provide a platform for investigating MAPs in a spindle-like geometry. Cellulose synthase complex (CSC) is a membrane bound multi-subunit complex that synthesizes cellulose microfibrils and has great meaning in plant cell morphologies and commercial applications. To estimate the copy number and stoichiometry of CSC in a nondestructive way, GFP-CESA3 in Arabidopsis was photobleached under total internal reflection (TIRF) microscopy. A step detection algorithm was developed and the smooth bleaching traces were analyzed by to identify discrete bleaching steps. The resulting broad distribution of step sizes was analyzed by Gaussian Mixture Model to determine the unitary step size, thus allowing estimation of the copy number. These analyses can be applied both to other photobleaching studies and to molecular motor stepping.