Cellular adhesion in 3D matrices

Cells sense physical forces and the mechanical properties of the microenvironment via several distinct mechanisms and cellular components. The first step of cellular adhesion to the ECM occurs via transmembrane heterodimers of the integrin family. Once integrin molecules adhere to the ECM, they are activated and form clusters. As the number of bound molecules increases, some of the focal complexes evolve from small (0.5-1µm in diameter) transient ‘dot-like’ contacts to elongated structures (3-10µm) which couple with actin and associated proteins. The mechanical coupling between the ECM and the cell cytoskeleton is controlled by the dynamics of the focal adhesion complexes (assembly, disassembly and turnover).

Mechanical stimuli are converted into chemical signals to regulate cell behavior through mature integrin focal adhesions and/or contractile units.
Essentially, all types of adherent cells form adhesions with the ECM, but their morphology, size and subcellular distribution will depend on the local properties of the matrix and can be very heterogeneous. Most of our current understanding of these mechanical complexes is derived primarily from studies of cellular adhesions formed on 2D rigid substrates. Recently, 3D models composed of single or multiple natural occurring ECM proteins were used to understand of how cellular mechanosensing occurs within 3D microenvironments. However, tuning critical parameters of the natural ECM independently is not possible; for example, in a collagen gel it is impossible to modulate stiffness without also altering the density of the adhesive ligand, pore size and porosity. In the last decades, many synthetic biomaterials have been developed to study cellular interactions in 3D, for instance poly(ethylene glycol)(PEG) and poly(vinyl alcohol)(PVA) hydrogels. They possess reproducible and tunable biochemical and physical properties, and can be readily modified with biomimetic motifs. However these materials lack some of the properties of natural gels, namely the non-linear elastic behaviour. Recently, the groups of Prof. Paul Kouwer (Nijmegen University, The Netherlands) and Prof. Alan Rowan (University of Queensland, Australia) were able to develop a fully synthetic material that mimics in all aspects the gels prepared from cellular filaments. We aim to use a library of PIC-based hydrogels to investigate the influence of bio-chemical and mechanical properties of the ECM in cell-matrix interactions, independently and at a molecular scale, using cutting-edge fluorescence microscopy techniques available at the Hofkens' laboratory. As focal adhesions are organized at the nanometer scale, an accurate description of their structure and dynamics can only be achieved with super-resolution microscopy. In order to image the focal adhesions below the diffraction limit of the light (< 200nm) and with minimal loss of temporal resolution (seconds), we will use a home-built, highly sensitive multi-focal plane wide-field microscope.