Promoting tissue repair and regeneration by manipulating intracellular pathways is currently a hot topic in biomedicine research. In living cells, external mechanical stimuli are converted into biochemical signals by the mechanotransduction processes. Cadherins are crucial cell membrane proteins that promote cell-cell adhesion through their calcium-dependent homophilic interactions. In addition, cadherins are important mechanosensors which induce intracellular cascades in order to activate collective epithelial remodeling during tissue repair. Thus, cadherins are an attractive target to manipulate intracellular pathways implicated in these processes.

Our main objective is to selectively bind magnetic microparticles (MMPs) functionalized with oriented E-cadherin fragments to the membrane of E-cadherin-expressing cells, and under the application of an external magnetic field, the pulling force on the MMPs will be used to trigger mechanotransduction processes and intracellular signallings.

To do so, we have generated different engineered molecules of E-cadherin, composed of the first two extracellular domains, which are enough to establish stable homophilic interactions with the cadherins present on the cellular membrane. We have used the wild type E-cadherin recombinant fragment, and two E-cadherin mutants generated by site directed mutagenesis, in order to control the binding affinity. The cadherin fragments have been modified with a histidine tag (His-Tag) at the C-terminus to allow their oriented attachment to NTA-Cobalt-MMPs via metal-chelate affinity.

We have optimized the functionalization of the MMPs with the E-cadherin fragments, and the amount of attached molecules has been measured by flow cytometry using an anti-E-cadherin antibody that recognize specifically these proteins present in MMPs. To test if the cadherin fragments were functional and could recognize other cadherin fragments we incubated the MMPs in presence or absence of Ca2+. Only in the presence of Ca2+ homophilic interaction between cadherins occurs and, therefore, aggregates of MMPs are formed.

Finally, we have inmmobilized the E-cadherin-MMPs on membrane of living cells that express E-cadherin, visualizing this attachment using fluorescence microscopy and SEM. MMPs have been incubated with the cells at different concentrations and times, to establish the optimal conditions of membrane labelling without premature MMP internalization. With this fine-tuning of the MMPs-cell interaction assays, the next step will be the activation of mechanotransduction processes using magnetic actuators.