GAPJUNCTION STRENGTH

Biophysical determinants of the adhesion strength of gap junctions

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 Obiettivo del progetto (Objective)

'Gap junctions are intercellular channels present in almost all epithelia and in many other specialized tissues. The correct function and formation of gap junctions are involved in diseases such as hypertension, deafness or cataracts. Gap junctions are formed by pairs of hexameric half-channels called connexons, which coaxially dock to connect two adjacent cells, assuring communication, signalling and adhesion between cells. Junctional microdomains are known to be form by thousands of closely packed connexon pairs. Even if the function as intercellular channels of gap junctions is widely studied, the forces supported by connexon pairs and the biophysical mechanism of microdomain formation are still unknown. The overall goal of this project is to determine the biophysical mechanisms of the adhesion strength of gap junctions. The proposed research will make use of state of the art biochemistry methods to purify and reconstitute connexons from eye lens fiber cells into raw membranes extracts. These will be combined with high end biophysical tools, atomic force microscopy and biomembrane force probe, to directly measure the binding forces between connexon pairs and to determine the kinetics and biophysical mechanisms of the aggregation and assembly of gap junctions. The expected outcomes of the proposed research will provide the first direct measure, at single and multiple molecule levels, of the adhesion strength of gap junctions explaining its underlying biophysical mechanisms. The interest of the project covers disciplines such as biophysics, molecular biology and nanotechnology.'

Introduzione (Teaser)

Cell-cell junctions are important for communication, transport, signalling, waste evacuation and water homeostasis. An EU-funded project has investigated how biophysical forces can influence the fulfilment of this vast range of functions.

Descrizione progetto (Article)

Communication and signalling between cells in almost all tissues is achieved by gap junctions or intercellular channels. Gap junctions are pairs of half-channels or connexons each having an assembly of six proteins (connexins) that come together. Although the functions and chemical components of gap junctions are well documented, little is known about the forces and energies these structures can support.

The 'Biophysical determinants of the adhesion strength of gap junctions' (Gapjunction Strength) project aimed to use advanced nanotools and atomic force microscopy (AFM) to measure the kinetics and binding strength between the cell-cell junctions. Project scientists used purified connexin proteins in reconstructed lipid membranes to obtain biophysical data on the intercellular bonds.

The Gapjunction Strength scientists applied force to the interacting protein molecules and measured the dissociation characteristics. A rapid dissociation rate is indicative of a dynamic bond while a slow association rate reflected reduced flexibility and small size of the specially constructed structures mimicking extracellular loops. Adhesion strength was found to be significant, suggesting that the bond can support important forces prior to dissociation.

Results of the project have been published in journals. The Journal of Molecular Biology has presented the group's results on the nature of the bond. Another paper is currently under review on a novel AFM imaging mode that is capable of mapping mechanical properties of membrane proteins at submolecular resolution.

Characterisation of the protein-protein bonds from a biophysical angle at gap junctions promises to hold the key to an understanding of the nature of intercellular channels. Crucial to the structural and biochemical integrity of multicellular organisation, gap junction function will guide the direction of future molecular therapies for associated diseases.