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Deciphering the molecular dynamics of cell-cell adhesion formation using a novel reconstituted cell surface system

Principal Supervisor: Dr Darius Vasco Köster, Warwick Medical School

Co-supervisor: Michael Smutny

PhD project title: Deciphering the molecular dynamics of cell-cell adhesion formation using a novel reconstituted cell surface system

University of Registration: University of Warwick

Project outline:

Cell-cell adhesion formation is one of the most crucial processes in the development of organisms and malfunctions lead usually to death of the embryo. Cadherins are major factors for the establishment for cell-cell adhesions, called adherens junctions. Control of these adhesions is e.g. crucial for tissue formation and organisation during development. Depending on cell fate, the expression levels of cadherin isoforms change, and it is assumed that the molecular composition of adherens junctions sets their strength and stability. The strength of adherens junction engagement could also direct cell differentiation. However, the molecular and mechanochemical details of adherens junction formation remain poorly understood as well as the role of the substrate’s mechanical properties or mechanical stimuli.

A main reason for our poor understanding is the fact that it is difficult to control the environment of cells within a tissue and to follow single protein dynamics within adherens junctions between two cells. We propose to use a combination of quantitative live cell imaging in controlled mechanical and biochemical environments as well as biochemical experiments to study this process.

The project will focus on a newly developed assay in our lab that employs a cell surface mimic consisting of supported lipid bilayers decorated with adhesion proteins for attaching cells (Fig. 1). This allows to study the role of adhesion protein density, mobility and mixture in adherens junction formation in cells settling on this substrate. Forming supported lipid bilayers on elastomers will allow to further tune the mechanical properties of the substrate as well as to apply static or periodic uniaxial stresses during cell-cell adhesion formation (1). Live cell fluorescence imaging using confocal, total internal reflection fluorescence and super resolution microscopy combined with image analysis tools to segment, track and quantify imaging data will give deep insights into the dynamics of cell-cell adhesion formation.

Pic2

In collaboration with the Smutny lab, we will test how different embryonic progenitor cells (blastomers, endoderm, ectoderm, mesoderm) respond to mobile E-cadherin presented on lipid bilayers. Dynamics of cell-cell adhesions (cluster formation, turnover) will be quantitatively assessed by imaging zebrafish GFP-E-cadherin. Further actomyosin dynamics by imaging lifeact-GFP and myosin2-GFP will identify dynamic changes in mechanical properties of the cells in response to varying E-cadherin levels and substrate stiffness.

The results of this project will provide major advances in our understanding of the dynamic regulation of cell-cell adhesions and molecular details of processes involved in maintaining tissue integrity.

References:

  1. Sinha B, et al. (2011) Cells respond to mechanical stress by rapid disassembly of caveolae. Cell144(3):402–13. doi: 10.1016/j.cell.2010.12.031

BBSRC Strategic Research Priority: Molecules, Cells and Systems

Techniques that will be undertaken during the project:

  • Molecular biology: plasmid design and construction, protein expression and purification (in cells and bacteria)
  • Cell biology: cell culture, genetic manipulation using transfection, RNAi and evtl. CRISPR/Cas9
  • Microscopy: Fluorescence light microscopy (confocal, TIRF, super resolution)
  • Biomimetic systems: lipid handling, supported lipid bilayer formation, substrate modifications (patterning, coating, stiffness modulation)
  • Biophysics: mechanical manipulations on single cells
  • Data analysis: Quantitative image analysis, programming of analysis routines (e.g. in Matlab) (optional), Computational modelling (optional)

Contact: Dr Darius Vasco Köster, University of Warwick