Super-resolution single molecule network analysis (SuperResNET) detects changes to clathrin structure by small molecule inhibitors

Specificity of small molecules for their target molecule in the cell is critical to determine their effective use as biologics and therapeutics. Small molecule inhibitors of clathrin endocytosis, Pitstop 2, and the dynamin inhibitor Dynasore, have off-target effects and their specificity has been challenged. Here, we used SuperResNET to apply network analysis to 20 nm resolution dSTORM single-molecule localization microscopy (SMLM) to test whether Pitstop 2 and Dynasore alter the morphology of clathrin coated pits in intact cells. SuperResNET analysis of dSTORM data from HeLa and Cos7 cells identifies three classes of clathrin structures: small oligomers (Class I); pits and vesicles (Class II); and larger clusters corresponding to fused clathrin pits and clathrin plaques (Class III). SuperResNET analysis of high resolution MinFlux imaging identifies Class 1 oligomers as well as Class 2 structures including morphologically identifiable clathrin pits and vesicles. SuperResNET feature analysis of dSTORM data shows that Pitstop 2 and Dynasore induce the formation of distinct homogenous populations of clathrin structures in HeLa cells. Pitstop 2 blobs are smaller and more elongated than those induced by Dynasore, indicating that these two clathrin inhibitors arrest clathrin endocytosis at distinct stages. Pitstop 2 and Dynasore are not impacting clathrin structure via actin depolymerization as the actin depolymerizing agent latrunculin A (LatA) induced larger heterogeneous clathrin structures. Ternary analysis of SuperResNET shape features presents a distinct profile for Pitstop 2 Class II structures. The most representative Pitstop blobs align with and resemble MinFlux clathrin pits while control structures resemble Minflux clathrin vesicles. SuperResNET analysis of SMLM data is therefore a highly sensitive approach to detect the effect of small molecules on target molecule structure in situ in the cell. ### Competing Interest Statement The authors have declared no competing interest.