Receptor-ligand internalization

Abstract The interaction between biologically active compounds and target cells has been studied extensively by various quantitative and qualitative approaches as it is the crucial first step in the chain of events leading to the final effect. Additionally it is a suitable process to be studied for pharmacological purposes. The visualization of receptor–ligand binding and internalization has been studied by immunocytochemistry techniques for both light and electron microscopy on fixed, permeabilized tissues or cells. The information achievable by these methods is limited and static, as the localization observed in fixed samples might not correspond to the actual binding site in living cells and the time course of the interaction cannot be evaluated with satisfactory precision. A technique is now available that allows the visualization of the interaction between directly fluoresceinated ligands and living cells and enables one to follow their possible internalization. In living adherent cells, such a methodological approach was hindered previously by the relatively poor resolution of the conventional fluorescence microscope and the unfavorable signal-to-noise ratio. As aconsequence, the visual information has not been exhaustive and subcellular localization has been limited to main cell compartments. More recently, confocal imaging has provided new insights in the observation of fluorescent specimens. The virtual absence of out-of-focus blurring allows a much better definition of probe localization at the subcellular level together with the possibility of exploiting the three-dimensional reconstruction capability of most confocal systems. We have coupled a self-constructed flow chamber to an inverted confocal scanning laser microscope that allows long-term observation of adherent cells under controlled microenvironmental conditions. This method not only provides images of intact, nonfixed cells, but also allows one to change culture conditions and to observe living cell responses directly or to perform two-step staining to identify subcellular structures involved in the observed processes