When should endocytosis inhibitors be used in cell binding assays?

Question

I'm beginning to do some cell-binding assays and I would like for my proteins to not be endocytosed by my mammalian cells. Typical suggestions are for the cells to be kept on ice and that the binding experiment also occurs when the cells are chilled.However, to be sure it has been suggested that I use endocytosis inhbitors. So my questions are, when are the endocytosis inhibitors necessary? How would I know that endocytosis inhibitors are necessary? Which ones would I actually use?I recognize that there are both clathrin and non-clathrin mechanisms of internalization. Which mechanism would be more useful to study for an antibody-receptor interaction?

Answer 1

Endocytosis inhibitors will be essential in cell-binding assays in the case of ensuring that proteins or ligands remain on the cell surface without being internalized. These inhibitors are particularly necessary in surface binding studies, where the goal is to investigate the interaction between proteins or ligands and cell surface receptors without the complication of internalization processes. They are also crucial in quantitative binding assays to ensure that measurements reflect only surface interactions, as well as in studies of receptor occupancy and kinetics, where it is vital to confirm that all detected interactions occur at the cell surface.

To determine if endocytosis inhibitors are needed, preliminary experiments should be conducted without inhibitors to observe the extent of internalization, either through imaging or by comparing surface-bound versus internalized fractions. Control experiments using conditions known to facilitate rapid internalization can also help assess the necessity of inhibitors. The experimental conditions, including temperature and assay duration, should be considered since lower temperatures can reduce but not eliminate endocytosis, making inhibitors more crucial for longer assays.

Various endocytosis inhibitors target different pathways. Clathrin-mediated endocytosis inhibitors, such as chlorpromazine and Pitstop 2, disrupt clathrin-coated pit formation. Caveolae-mediated endocytosis can be inhibited by substances like filipin and nystatin, which disrupt cholesterol-rich lipid rafts. Macropinocytosis inhibitors, such as amiloride and its potent analog EIPA, reduce macropinocytosis by inhibiting Na+/H+ exchange. General endocytosis inhibitors like dynasore, which inhibits dynamin, and methyl-β-cyclodextrin, which depletes membrane cholesterol, affect multiple pathways.

In the context of antibody-receptor interactions, clathrin-mediated endocytosis is often the primary pathway, making inhibitors like chlorpromazine and Pitstop 2 commonly used. However, since some receptors may also utilize non-clathrin mechanisms like caveolae or macropinocytosis, it is important to investigate specific receptor pathways to tailor the use of inhibitors accordingly. In practical terms, starting with conditions where cells are kept on ice can reduce overall endocytic activity. Selecting inhibitors based on preliminary data or literature, and sometimes using a combination of inhibitors targeting multiple pathways, can ensure comprehensive inhibition. Validating the efficacy of inhibition by comparing internalization levels with and without inhibitors using methods such as fluorescence microscopy or biochemical assays is also crucial.

Overall, endocytosis inhibitors are vital in ensuring accurate surface binding measurements and preventing the interference of internalization in cell-binding assays, with the choice of inhibitors depending on the specific pathways involved in the receptor-ligand interactions being studied.