FIGURE 9.
Characterization of the Spir-2 dynamics and interactions by FCCS. A, averaged normalized autocorrelation (AC) functions of the indicated fluorescent molecules reveal the progressively slower diffusion according to molecular size as well as a pronounced long tail correlation for full-length Spir-2 (arrow). B, oligomerization of cytoplasmic full-length GFP-Spir-2 probed by single color FCS in the cytoplasm of HeLa cells. The relative brightness was calibrated with multiple GFP tandem fusion constructs. Both GFP-Spir-2 in the presence of BFA and the LAFA mutant are monomeric (n = 5–13 cells; 1xGFP versus Spir-2, not significant, p > 0.1). C and D, FCCS measured with mStrawberry-Fmn-2-FH2-FSI co-expressed with either GFP-Spir-2 or GFP-Spir-2-KIND. Focal volume was positioned in the confocal cross-sections (white cross) where intensity traces and correlation curves were recorded for both color channels simultaneously. The resulting auto- and cross-correlation functions allow quantification of the cross-correlation ratio (CCmax) as a measure for the degree of binding. Error bars represent S.D.