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Shaping the actin cortex of cells by Curly, a newly found actin bending protein

Primary Supervisor: Dr Darius Koester, Warwick Medical School

Secondary supervisor: Mohan Balasubramanian

PhD project title: Shaping the actin cortex of cells by Curly, a newly found actin bending protein

University of Registration: University of Warwick

Project outline:

Background and rationale of the project:

We recently identified a domain in IQGAP proteins, baptised Curly, that binds to actin filaments and induces the bending of actin filaments into tight rings in vitro (see figures and [1]). This fascinating phenomenon was unexpected and opens new avenues to shape actin filaments in live cells. The aim of this project is to test how expression and targeting of curly in cells can be used to alter the cell cortex and cell function in a controlled way.

You will learn how to handle mammalian cell cultures, generate protein constructs for cell expression, and image live and fixed cells using confocal and lattice light sheet fluorescence microscopy. Combining curly with the knocksideways tool developed by the Royle group [2] will allow to direct curly to specific cell locations (e.g. mitochondria, plasma membrane) and to follow how this change in cellular actin organisation affects cell function, shape and function of the targeted location.

An additional interesting aspect will be to understand, how curly induces actin bending on the molecular scale. Cross-linking mass spectrometry (XL-MS) is a powerful and unbiased structural biology tool to probe native protein structure / topology and protein-protein interactions / proximities at ~ 1nm resolution. In XL-MS a chemical cross-linker generates a covalent bond between the side chains of two amino-acids that are in proximity. With this tool, that is well established in the Balasubramanian group, you will determine the interaction interface between actin and curly and solve the question, which interactions lead to actin bending.

Characterising and establishing curly as a molecular tool for cell cortex modulation, would be a major step forward and would be of interest for the community working on questions of cell biology, bioengineering and biophysics.

Aims of the project:

  • Characterise effect of curly expression in mammalian cells on actin dynamics and organisation.
  • Characterise the curly – actin interaction interface using cross-linking mass spectrometry.
  • Generate functional curly constructs for knocksideways assay.
  • Characterise knocksideways of curly on actin localisation and cell function using live cell imaging and biomolecular assays.

Techniques and methodologies the student will learn:

  • Live cell fluorescence microscopy
  • Quantitative image analysis using ImageJ and Matlab
  • Cell culture, cell transfection with plasmids
  • Cloning of protein constructs
  • Mass spectrometry analysis
  • Basic biochemistry/ molecular biology techniques

References:

[1] paper describing effect of curly on actin filament curvature:

Palani, S., Ghosh, S., Ivorra-Molla, E., Clarke, S., Suchenko, A., Balasubramanian, M. K., & Köster, D. V. (2021). Calponin-homology domain mediated bending of membrane associated actin filaments. ELife, 10. https://doi.org/10.7554/eLife.61078

[2] paper describing the knocksideways assay developed by the Royle lab:

Küey, C., Larocque, G., Clarke, N. I. & Royle, S. J. Unintended perturbation of protein function using GFP nanobodies in human cells. J. Cell Sci. 132, (2019).

https://jcs.biologists.org/content/132/21/jcs234955

[3] paper describing the power of cross-linking mass spectrometry:

O’Reilly FJ, Rappsilber J. 2018. Cross-linking mass spectrometry: methods and applications in structural, molecular and systems biology. Nat Struct Mol Biol 2018 2511 25:1000–1008. doi:10.1038/s41594-018-0147-0

BBSRC Strategic Research Priority: Understanding the Rules of Life: Structural Biology & Systems Biology

    Techniques that will be undertaken during the project:

    • Biochemistry and molecular biology techniques
    • Advanced microscopy (single-molecule, super-resolution)
    • Advanced image analysis
    • Protein purification and in vitro reconstitution of biological membranes

    Contact: Dr Darius Koester, University of Warwick