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Biophysical determinants of the adhesion strength of gap junctions

Final Report Summary - GAPJUNCTION STRENGTH (Biophysical determinants of the adhesion strength of gap junctions)

Gap junctions are intercellular channels present in almost every tissue and provide an example of cell-cell attachment. These cell-cell junctions are fundamental in multicellular organisms, for they facilitate cellular communication and signalling being responsible of metabolite transport, waste evacuation, water homeostasis and ion exchange. Defects in the function and formation of gap junctions are involved in diseases such as hypertension, deafness or cataracts. Gap junctions are pairs of hexameric half-channels called connexons, which coaxially dock to connect two adjacent cells. Connexons are themselves formed by six subunits of one or more connexin (Cx) type. Even if the function of gap junctions as intercellular channels is well characterised, almost nothing is known about the forces and energies that these structures can support. Furthermore, while the forces supported by 'classical' adhesion proteins such as cadherins have been extensively studied using various biophysical nanotools, the adhesion strength of gap junctions remains completely unknown. The overall goal of this project was to determine the biophysical mechanisms of the adhesion strength of gap junctions.

To accomplish this aim, we applied advance nanotools in various and novel modes to purified connexin proteins reconstituted in supported lipid membranes. We have carried out the first quantitative characterisation of the two-dimensional (2D) kinetics and binding strength of the interaction of a short peptide mimicking extracellular loop 2 of Cx26 with membrane reconstituted Cx26 using atomic force microscopy (AFM). We have developed a novel approach to characterise the 2D kinetics and adhesion strength by combining the imaging and force spectroscopy capabilities of AFM. We found a relatively fast dissociation rate, which inferred a rather dynamic bond; while a slow association rate reflected the reduced flexibility and small dimensions of extracellular loops. The induction of channel closure by millimolar concentrations of Ca2+ induced a slight increase in the dissociation rates, but no important change in the association kinetics or binding strength. The adhesion strength we found suggests that the whole hexameric complex may be able to support important forces before dissociation, comparable to that supported by integrins in the high affinity state. This part of the project has led to a publication in the Journal of Molecular Biology. The implementation of the described approach has also allowed us to acquire expertise in a novel AFM imaging mode capable of mapping the mechanical properties of membrane proteins with unprecedented submolecular resolution. This developing part of the project has led to an additional publication (currently under review).

During the progress towards the completion of the aims of the project, we have taken the AFM capabilities a step further by mapping the mechanical properties of membrane proteins at submolecular resolution. We expect that the further use of this AFM imaging mode to other membrane proteins will provide a new perspective to the relationship between structure, function and flexibility of protein domains. The Marie Curie project allowed us to develop an approach to characterise the 2D kinetics and binding strength of adhesion complexes. Application of the approach on whole connexon-connexon pairs is currently being implemented and will prove gap junctions as adhesion complexes capable of bearing important forces. The straightforward continuation of the project is the application of the developed methodology to living cells. We believe that the obtained results will open a door to a so far ignored function of gap junction complexes, which may be crucial for the structural integrity of multicellular organisms.