MIiSR: Molecular Interactions in Super-Resolution Imaging Enables the Analysis of Protein Interactions, Dynamics and Formation of Multi-protein Structures
Fig 7
Combining multiple analyses to characterize inter-protein interactions.
(A) Conventional microscopy images of dimerized Nef, detected by bimolecular fluorescence complementation (Nef, red) and the Golgi trafficking regulator TGN46 (green). ROI has a Mander’s colocalization ratio (Nef:TGN46) of 0.842. (B) GSDM image of the ROI indicated in panel A. (C-D) SAA plots and (E) quantification of Nef interactions with TGN46 (C, E) and the reverse quantification of TGN46 interaction with Nef (D,E). (F) Ripley’s H function analysis of Nef:TGN46 co-clustering. (G) TGN46 density in Nef-containing vesicles of increasing size. Nef-containing vesicles were identified using the OPTICS algorithm, and the density of TGN46 in each Nef-containing vesicle quantified. (H) Fraction of total detected TGN46 in small (<60 nm) and large (>60 nm) Nef vesicles. Images are representative of, and graphs quantify, 8 images collected in two experiments. Data are presented as mean ± SEM (E,H) or are representative plots from single images (C,D,F,G). Scale bars are 2.5 μm (A) or 0.5 μm (B), ROI (A) is 3.94 μm x 3.94 μm. SRP = Simulated Random Positions. * = p<0.05 compared to SRP, paired t-test (E) or vesicles <60nm, Students t-test (H).