Figure 3. Quantification of protein association in the presomitic mesoderm.

(A) a schematic of cell transplantation and FCCS data collection. For FCCS, the confocal volume (drawn approximately to scale in blue in A and B) was positioned at a membrane interface between RFP and GFP expressing cells. (B) Confocal image of a mosaic embryo comprised of cells expressing mem-RFP and mem-GFP. Scale bar is 10μm. (C) Auto- and crosscorrelation measurements of Itgα5-RFP and Itgα5-GFP in a mosaic interface show low crosscorrelation. (D) Plots of crosscorrelation from individual measurements and mean values. (E) Table of crosscorrelation (Fcross) magnitude ±SD. Significance was examined using both t-test (two tailed, unequal variance) and Mann- Whitney U. There were no statistically significant differences in mem-GFP/mem-RFP compared to Itgα5-GFP/mem-RFP and Itgα5-GFP/Itgα5-RFP compared to Itgα5-GFP/Itgα5^FYLDD-RFP. Notably, either mem-GFP/mem-RFP or Itgα5-GFP/mem-RFP compared to either Itgα5-GFP/Itgα5-RFP or Itgα5-GFP/Itgα5^FYLDD-RFP differed with p<0.002. The Itgα5-RFP/Cdh2-GFP “trans” Fcross differs from the negative control (p<0.05) but not from the Itgα5-RFP/Cdh2-GFP “cis” Fcross. The Itgα5-GFP/Itgα5-RFP Fcross in the absence of Cdh2 is significantly lower (p<0.05) than the Itgα5-GFP/Itgα5-RFP Fcross in the presence of Cdh2. See Figure S1 for apparent Kd calculations.