A simple method for measurement of cell-substrate attachment forces: application to HIV-1 Tat.

In order to understand the importance of cell attachment to HIV-1 Tat, we quantified the strength of cell attachment to immobilized Tat in microtiter plate wells by the application of buoyant force. By replacing the attachment medium with dense medium, and subjecting the attached cells in the microtiter plates to centrifugal force in the conventional upright position, weakly binding and strongly binding cells could be discriminated (and separated) by varying the centrifugal speed. The strength of attachment of HT1080 cells to Tat was compared with that of the well-known extracellular matrix (ECM) proteins fibronectin and vitronectin. We observed that all three proteins mediated significant attachment of HT1080 cells both at 4 degrees C and 37 degrees C. However, unlike the ECM proteins, Tat was unable to engage in higher strength binding when the temperature was raised to 37 degrees C. The relatively weak binding of HT1080 cells to Tat (in the order of 3.0 mudynes/picomole of coated Tat) and lack of strengthening of binding to Tat at physiologic temperature suggests that this protein does not mimic adhesion molecule function. We anticipate that the methodology developed and described here will be useful in a wide variety of cell-matrix and cell-cell interaction studies.