Investigation of integrin expression on the surface of osteoblast-like cells by atomic force microscopy

The transforming growth factor beta1 (TGF-beta1) is a human cytokine which has been demonstrated to modulate cell surface integrin repertoire. In this work integrin expression in response to TGF-beta1 stimulation has been investigated on the surface of human osteoblast-like cells. We used atomic force microscopy (AFM) and confocal laser scanning microscopy to assess integrin expression and to evaluate their distribution over the dorsal side of the plasma membrane. AFM probes have been covalently functionalized with monoclonal antibodies specific to the beta1 integrin subunit. Force curves have been collected in order to obtain maps of the interaction between the immobilized antibody and the respective cell membrane receptors. Adhesion peaks have been automatically detected by means of an ad hoc developed data analysis software. The specificity of the detected interactions has been assessed by adding free antibody in the solution and monitoring the dramatic decrease in the recorded interactions. In addition, the effect of TGF-beta1 treatment on both the fluorescence signal and the adhesion events has been tested. The level of expression of the beta1 integrin subunit was enhanced by TGF-beta1. As a further analysis, the adhesion force of the single living cells to the substrate was measured by laterally pushing the cell with the AFM tip and measuring the force necessary to displace it. The treatment with TGF-beta1 resulted in a decrease of the cell/substrate adhesion force. Results obtained by AFM have been validated by confocal laser scanning microscopy thus demonstrating the high potential of the AFM technique for the investigation of cell surface receptors distribution and trafficking at the nanoscale.