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Curly, a novel IQGAP protein domain that bends actin filaments: characterisation and novel applications

Primary Supervisor: Dr Darius Köster, Warwick Medical School

Secondary supervisor: Mohan Balasubramanian

PhD project title: Curly, a novel IQGAP protein domain that bends actin filaments: characterisation and novel applications

University of Registration: University of Warwick

Project outline:

Actin filaments are central to a large number of biological processes in all domains of life. Dirven by the interplay with molecular motors, actin binding and actin modulating proteins, the actin cytoskeleton exhibits a variety of geometries. This includes structures with a curved geometry such as the contractile vacuoles and the cytokinetic contractile actomyosin ring. We recently identified a region from the IQGAP family of proteins, baptised “curly”, that stabilizes individual actin filaments in a curved geometry. This effect of curly is observed only when it is anchored to lipid membranes. Whereas F-actin is a semi-flexible with a persistence length of ten micro-meter, curly cooperatively stabilizes actin filaments into rings and arcs of high curvature. This novel and unprecedented observations provide a molecular mechanism to generate curved actin filaments which would align with highly curved membranes, e.g. along the short axis of dividing cells, to organize structures such as the cytokinetic actomyosin ring. This finding opens a new range of possibilities to control actin filament geometries on lipid membranes (see figures on the right). For example, it could be used to form contractile actin rings in lipid vesicles to generate synthetic cell division or expression of photo-activable curly in cells could be used to alter actin cortex architecture without external mechanical manipulation.

The aim of this project is to characterise the protein interactions and test how it can be used to modulate the cell actin cortex. The student will learn the key techniques to perform experiments with a focus on in vitro or cell assays depending on the student’s preferences. Given the novelty of the discovery of curly, the project can focus on various questions which all would provide novel insights for the cell biology and biophysics community.

Possible objectives:

  1. Use of curly as a tool to shape actin filaments in reconstituted networks and to understand the role of actin bending for the binding of associated proteins
  2. Reconstitution of a dividing lipid vesicle using curly, actin and myosin motors
  3. Role of curly in mammalian cells. Does it modulate the actin cortex architecture and dynamics as part of IQGAP proteins?
  4. Generation of theoretical models of how curly induces actin bending and comparison with experimental data

The PhD project can follow different trajectories depending on the student’s preference. For example, together with Nigel Burroughs (Maths department) theoretical modelling could be used to test different hypotheses of how curly induces actin ring formation. Another option is to work together with Till Brettschneider (Department of Computational Studies) to study cell cortex dynamics in mammalian cells and in dictyostelium using LLS microscopy and automated cell shape segmentation routines (developed in house). The idea would be to investigate possibilities to turn the protein domain curly into a versatile took for cell biologists. By taking advantage of the experience available in CMCB (e.g. genetic code expansion - Balasubramanian lab, or the FRB-FKBP protein targeting techniques -Royle lab) we envision to modify rng2 to act as a sensor/ modulator for actin filament curvature in live cells and to modulate local cell shape by targeting curly to particular areas and photo-activate it.

Another trajectory would be more focussed on in vitro experiments and to use curly as a tool to control membrane shape in reconstituted vesicles, with the possibility to generate smart vesicles that can be activated to undergo division of vesicle abscission upon light activation.

References:

  • An example of the potential of reconstituted acto-myosin systems on lipid bilayers:

Ditlev JA, Vega AR, Köster DV, et al (2019) A composition-dependent molecular clutch between T cell signaling condensates and actin. Elife 8:e42695: https://doi.org/10.7554/eLife.42695

  • paper describing the important role of IQGAP protein family in cell cortex regulation.

Briggs, M.W., and D.B. Sacks. 2003. IQGAP proteins are integral components of cytoskeletal regulation. EMBO Rep. 4: 571–574. https://doi.org/ 10.1038/sj.embor.embor867 

BBSRC Strategic Research Priority: Understanding the rules of life: Structural Biology

Techniques that will be undertaken during the project:

  • fluorescence microscopy: total internal reflection fluorescence (TIRF), confocal, lattice-light-sheet (LLS) and super-resolution (e.g. STORM)
  • Quantitative image analysis using ImageJ, Matlab, Python…
  • in vitro reconstitution of minimal, biomimetic systems: supported lipid membranes (SLBs), giant unilamellar vesicles (GUV), actin filament polymerization and handling of other purified proteins.
  • micro-manipulation of GUVs (e.g. micro-pipette aspiration)
  • Molecular Cell Biology techniques: cell culture, transfection with plasmids to express additional proteins
  • Immunofluorescence (IF) to visualise protein organisation in fixed cells.
  • biochemistry/ molecular biology techniques: western blots, protein purification

Typical pattern of working hours:

  • 37.5 hrs per week 9am-5pm on campus

Contact: Dr Darius Köster, University of Warwick