AbstractClathrin-mediated endocytosis is the major pathway by which cells internalize materials from the external environment. Dynamin, a large multidomain GTPase, is a key regulator of clathrin-mediated endocytosis. It assembles at the necks of invaginated clathrin-coated pits and, through GTP hydrolysis, catalyzes scission and release of clathrin-coated vesicles from the plasma membrane. Several small molecule inhibitors of dynamin’s GTPase activity, such as Dynasore and Dyngo-4a, are currently available, although their specificity has been brought into question. Previous screens for these inhibitors measured dynamin’s stimulated GTPase activity due to lack of sufficient sensitivity, hence the mechanisms by which they inhibit dynamin are uncertain. We report a highly sensitive fluorescence-based assay capable of detecting dynamin’s basal GTPase activity under conditions compatible with high throughput screening. Utilizing this optimized assay, we conducted a pilot screen of 8000 compounds and identified several “hits” that inhibit the basal GTPase activity of dynamin-1. Subsequent dose-response curves were used to validate the activity of these compounds. Interestingly, we found neither Dynasore nor Dyngo-4a inhibited dynamin’s basal GTPase activity, although both inhibit assembly-stimulated GTPase activity. This assay provides the basis for a more extensive search for robust dynamin inhibitors.
A highly-sensitive high throughput assay for dynamin's basal GTPase activity
