Principal Supervisor: Dr Kayoko Tanaka, Department of Molecular and Cell Biology
Co-supervisor: John Schwabe and Peter Moody
PhD project title: Unravelling microtubule anchoring mechanism
University of Registration: University of Leicester
Microtubules (MTs) have a hollow directional tubular structure and convey numerous fundamental cellular activities. With help of motor proteins, cellular MT architecture plays an essential role in organelle movement. During this process, MTs need to be anchored at an appropriate site, such as the centrosome, the major microtubule organising centre (MTOC), in order to effectively convey the force generated by the motor proteins. Nuclear movement during neural progenitor cell development is an example of such MT-driven movement. Physiological importance of the movement has been highlighted by human genetic mutations in components involved in the process to cause brain developmental disorder including microcephaly and lissencephaly.
As the biomolecular complexes mediating MT anchoring are subject to physical forces during MT-driven movement, the anchoring mechanism is expected to involve specialised stable protein-protein interaction. Furthermore, the biophysical nature of these anchoring complexes, such as viscoelasticity, are likely to play a critical role to secure anchoring by damping the force impact. In this project, these hypotheses will be tested by employing structural, mechanochemical, biochemical, cell biological and molecular genetic approaches.
In this work, we will exploit the highly tractable fission yeast to reveal conserved principals that facilitate a general understanding of MT anchoring complexes. Previously, we have found that fission yeast cells develop a transient structure during meiotic prophase. We termed this structure the radial MT organising centre (rMTOC) as it organises cytoplasmic radial MT (rMT) arrays (Curr Biol., 22, 562-574, 2012). The rMT arrays, with the help of dynein-dynactin motor complexes, induce vigorous nuclear movement. Strikingly, when we over-expressed the major structural component, Hrs1, a coiled coil protein, in mitotic cells, Hrs1 could generate rMTOC that organized radial MT arrays. The rMTOC provides a unique opportunity to investigate the role of MT anchoring and the attachment matrices in force mitigation. The relatively straightforward environment in fission yeast enables implementation of multidisciplinary approaches to reveal detailed mechanistic information.
The hypothesis is that, in order to retain bonds to MTs, the anchoring/attachment complexes must possess a highly stable protein-protein interaction surfaces and viscoelastic property which provides strain relief from, and sufficient damping of, MT motion. To explore these hypothesis, the objectives of the project are:
(1) To obtain structural insights of mode of protein-protein interactions of the major rMTOC componets Hrs1.
(2) To develop an assay system to characterise the mechanochemical properties of the rMTOC and relevant material properties such as viscoelasticity.
(3) To measure the mechanochemical properties (force-extension relationship) of the rMTOC complex prepared from yeast cells expressing (i) wild type and (ii) mutant proteins in which the properties of the rMTOC are altered.
(4) To link the mechanochemical properties to the in vivo function of the rMTOC.
This will be the first exploration of the mechanochemical properties, and quantitative measurement of material parameters, for a MT anchoring and attachment matrix.
The experiments exploit a unique setting, the fission yeast rMTOC, which will allow the use of integrated multidisciplinary approaches.
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
- Cryoelectron microscopy
- Single particle analysis by electron microscopy of negative staining