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Unraveling structural mechanism underlining microtubule nucleation

Principal Supervisor: Kayoko Tanaka - Department of Molecular and Cell Biology

Co-supervisor: John Schwabe and Peter Moody

PhD project title: Unraveling structural mechanism underlining microtubule nucleation

University of Registration: Leicester

Project outline:

The primary step to produce dynamic microtubule (MT) architecture is MT nucleation. Although the major MT nucleator, the gamma-tubulin complex (y-TuC), is known to act as a “template/seed” to facilitate MT nucleation, the exact structural mechanism is not fully understood. Our recent success to biochemically purify y-TuC from a highly tractable model system fission yeast has opened up a path to take multidisciplinary approaches to obtain a holistic mechanical view of y-TuC-driven MT nucleation.

The project consists of 5 parts:

  1. To biochemically purify the yeast endogenous y-TuC by affinity purification and density gradient centrifugation. Supervision by Dr Tanaka.
  2. To assess the MT nucleation activity of the y-TuC in vitro employing fission yeast tubulin in collaboration with Prof. Cross (Warwick).
  3. To study structure of the y-TuC by electron microscopy (EM), both negative staining and cryo-EM, under supervision of Prof. Schwabe.
  4. To develop a genetically encoded tag for EM by designing metal binding proteins under supervision of Prof. Moody and Dr Tanaka in collaboration with Prof. Schiebel (Heidelberg) and use it to map the components in the -TuC.
  5. To apply methodologies (1)-(4) to analyse y-TuCs prepared from various yeast shut-off mutant strains of y-TuC components. Supervision by Dr Tanaka

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

  • Yeast genetics
  • Yeast live cell imaging using a confocal microscopy
  • Biochemical protein purification using immunoprecipitation and sucrose gradient centrifugation
  • In vitro microtubule nucleation assays using a dark-field microscopy
  • Transmission electron microscopy
  • X-ray crystallography
  • NMR
  • Cryoelectron microscopy
  • Single particle analysis by electron microscopy of negative staining

Contact: Dr Kayoko Tanaka, University of Leicester