MIiSR: Molecular Interactions in Super-Resolution Imaging Enables the Analysis of Protein Interactions, Dynamics and Formation of Multi-protein Structures
Fig 3
Quantification of intermolecular interactions between Nef, PACS-1, AP-1 and the Golgi, using Spatial Association Analysis.
(A) Representative image of GFP-Nef, PACS-1-mCherry and Golgin97 staining in a CD4+ HeLa cell 48 hours post-transfection. (B) SAA histogram of panel A, plotting the distance between each Nef molecule and the nearest PACS-1 and nearest Golgin97. Horizontal and vertical dotted lines indicate the CDC below which molecules are potentially colocalized. (C) Quantification of Nef colocalization patterns with Golgin97 and PACS-1, with the fraction of Nef colocalized with both Golgin97 and PACS-1, associated with either Golgin97 or PACS-1, or not colocalized with either molecule indicated. (D) Images of and (E) quantification of AP-1 and Nef colocalization patterns during the endocytosis-targeting (16 hrs) and Golgi-targeting (48 hrs) phases of Nef activity. SRP = Simulated Random Positions. A,D are representative images of, and B,C,E quantify 3 experiments, 5 images per experiment. Data are presented as mean ± SEM, † = p < 0.05 compared to SRP, * p < 0.05 compared to NEF-Golgin97-PACS-1 (C) or 16 hrs (E). Scale bars 2.5 μm, inserts are 3.5 μm x 3.5 μm.