Optical approaches to study microtubule depolymerisation

Lab rotation project description

This seven week mini-project will explore the use of optical trapping/laser tweezers to study the impact of MCAK (a kinesin crucial for cell division and chromosome separation) variants (Friel lab) on the rigidity of single cells. The technique involves using an optical trapping microscope to hold a single cell in a fixed position whilst subjecting it to a fluid flow and hence a shear force. Any change in shape of the cell when subjected to the shear force can be used to quantify the ability of the cell to deform. You will first become familiar with the optical trapping setup by holding and manipulating micron-sized beads, before moving on to looking at the control and/or knock down cells (Huett lab). The knock down cells will be produced by replacing the expression of wild-type MCAK in cells with the MCAK variants developed by the Friel lab. The optical trapping system will be used in conjunction with fluorescent microscope to identify and further characterise the cells of interest. You will develop skills in optical microscopy, optical trapping, fluorescence imaging, image processing and analysis and single cell studies, alongside the communication and transferable skills crucial to working on any multidisciplinary project.

Fact file

Research theme:
MCO

Location of lab rotation:
TBC

Rotation: TBC

Main supervisor:
Amanda Wright amanda.wright@nottingham.ac.uk

School:
Faculty of Engineering

2nd supervisor:
Claire Friel
Alan Huett

Triangle project:
Yes

 

Linked PhD Project Outline

Chromosome segregation errors lead to aneuploidy, a condition associated with cancer and developmental abnormalities. A critical question therefore is how, for the healthy population, potential chromosome segregation errors are corrected prior to cell division. In this project, you will dissect the role of microtubule depolymerisation in aneuploidy. The focus will be on the human microtubule depolymerising kinesin, MCAK. This kinesin is a major direct regulator of microtubule dynamics during cell division. Reduction in MCAK activity in cells results in hyperstabilised chromosome-microtubule attachments leading to defects in chromosome segregation. The converse, excessive MCAK activity, leads to loss of microtubule stability and defects in capture and alignment of chromosomes. The Friel lab have been characterising the kinesin features required for this end sensing activity and have created many mutants with altered end sensing and depolymerisation activities.

This project will look to measure and characterise the properties of individual microtubules with different variants of MCAK activity (Friel lab), as well as knock down cells, produced by replacing the expression of wild-type MCAK in cells with the MCAK variants (Huett lab).

Single molecule studies: A technique known as nano-plasmonic trapping will be used to hold, isolate and align individual microtubules. Nano-plasmonic trapping uses near-field optical effects generate by nano-sized metal structures on the surface of a cover slip to create electric-field hot-spots 10s of nanometers in size all three dimensions. The patterned coverslips will be designed such the microtubules stretch out along the surface of the coverslip and lay parallel to the imaging plane. Nano-sized optical interrogation sites will be capable of monitoring, for example, changing fluorescent or Raman signal with time along the length of the microtubule.

Single cell studies: Here the more common, single-beam, gradient-force, optical trap will be used to hold single control and knock down cells in a fluid flow thus exerting a shear force on the cell. The shape change of the cell in relation to the applied shear force will be used to as a measure of the rigidity of the cell and its ability to deform. Since the microtubules provide structure and shape to the cytoskeleton, the data generated here will inform on the impact of the MCAK variants on the mechanical properties of the cell as a whole.

 

 

Biotechnology and Biological Sciences Doctoral Training Programme

The University of Nottingham
University Park
Nottingham, NG7 2RD

Tel: +44 (0) 115 8466946
Email: bbdtp@nottingham.ac.uk