Regulation of Clathrin-mediated Endocytosis by Hierarchical Allosteric Activation of AP2

The critical initiation phase of clathrin-mediated endocytosis (CME) determines where and when endocytosis occurs. Heterotetrameric adaptor protein 2 (AP2) complexes, which initiate clathrin-coated pit (CCP) assembly, are activated by conformational changes in response to phosphatidylinositol-4,5-bisphosphate (PIP2) and cargo binding at multiple sites. However, the functional hierarchy of interactions and how these conformational changes relate to distinct steps in CCP formation in living cells remains unknown. We used quantitative live-cell analyses to measure discrete early stages of CME and show how sequential, allosterically regulated conformational changes activate AP2 to drive both nucleation and subsequent stabilization of nascent CCPs. Our data establish that cargoes containing Yxxφ motif, but not dileucine motif, play a critical role in the earliest stages of AP2 activation and CCP nucleation. Interestingly, these cargo and PIP2 interactions are not conserved in yeast. Thus, we speculate that AP2 has evolved as a key regulatory node to coordinate CCP formation and cargo sorting and ensure high spatial and temporal regulation of CME

A highly-sensitive high throughput assay for dynamin's basal GTPase activity.

Clathrin-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 more potent and chemically desirable dynamin inhibitors

Assay validation and application for high-throughput screening of 8000 compounds.

<p><b>A)</b> The assay was validated in a preliminary mock screen that compared reactions containing no enzyme (positive control for inhibition) to uninhibited reactions containing DMSO (negative control for inhibition). <b>B)</b> A quantile-quantile (Q-Q) plot of the pilot screen of 8000 compounds. The compounds are ranked from 0 to 8000 according to their two-point robust Z score. Primary hits were chosen based on a RZ score of greater than 3.</p

Optimization of assay sensitivity to measure GDP production.

<p><b>A)</b> Cartoon depicting the Transcreener GDP fluorescence polarization reaction <b>B)</b> The GDP antibody was titrated to determine its optimal concentrations for 10, 100 and 500 μM GTP. Optimal antibody concentrations are represented by the highlighted points (n = 1, measured in triplicates). <b>C)</b> Standard curve representing the conversion of 0 to 100% GDP from 10 μM GTP. This curve was used to convert the fluorescence polarization data to GDP released (n = 1, measured in triplicates). Data are presented as mean ± SD.</p

Confirmation screen and dose-response studies.

<p><b>A</b>) Flowchart of high-throughput screening used to identify and validate the hits from the 8000-compound screen. The 42 preliminary hits were chosen based on their RZ score and lack of intrinsic fluorescence. These were further narrowed down to 4 compounds that were chosen based on their ability to inhibit the GTPase activity of Dyn1 in a dose-dependent manner and their commercial availability <b>B)</b> Chemical structures of the 4 compounds that were tested in dose-response studies. <b>C)</b> 11-point dose response curve of the 4 validated hits from the primary screen of 8000 compounds measured using 50 nM Dyn1 and 10 μM GTP (n = 1, measured in triplicates). Data are presented as mean ± SD.</p

Detection of basal GTP hydrolysis by Dyn1.

<p><b>A)</b> Dyn1 was titrated from 0.3 nM to 5000 nM in the presence of 10 μM, 100 μM or 500 μM GTP and GTP hydrolysis was measured as ΔmP after 60 min incubation at room temperature (n = 1, measured in triplicates). <b>B)</b> The GTP hydrolysis by Dyn1 was linear during 60-minute incubations at room temperature (n = 4, each measured in duplicates). <b>C)</b> The GTP binding mutant, Dyn1^S45N, shows no activity at any concentration of GTP (n = 1, measured in triplicates). <b>D)</b> The basal GTPase activity is proportional to Dyn1 concentration from 0 to 1000 nM, indicating no cooperativity under these concentrations (n = 3, each measured in triplicates). Data are presented as mean ± SD.</p

Comparison of commercially available inhibitors with compound 24.

<p><b>A)</b> Dynasore and Dyngo-4a were tested alongside compound 24 in the Transcreener assay (n = 1, measured in triplicates). <b>B)</b> Dose response curves for the inhibitory effects of Dynasore, Dyngo-4a and compound 24 on the lipid nanotube-stimulated GTPase activity of 100 nM Dyn1 assayed in the presence of 300 μM lipid nanotubes and 25 μM GTP and measured using the malachite green assay (n = 3, each measured in triplicates). Data are presented as mean ± SD.</p