Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis

Fluorescence (auto)correlation spectroscopy (FCS) has developed into a widely used method for investigating molecular dynamics and mobility of molecules in vitro and in vivo. Dual-color cross-correlation, an extension of this technique, also assesses the concomitant movement of two spectrally distinguishable fluorescent molecules and has therefore proven superior to autocorrelation analysis to study interactions between different molecular species in solution. Here we explore the benefits of cross-correlation analysis when applied to live cells, by demonstrating its potential in analyzing endocytic processes. Bacterial cholera toxin (CTX) was labeled with Cy2 and Cy5 dyes on different subunits of the same holotoxin. Along the endocytic pathway, positive cross-correlation between the A and B subunits was first preserved, later followed by a loss in cross-correlation upon their separation in the Golgi. Furthermore, endocytosis of a mixture of only Cy2- and only Cy5-labeled holotoxins also gave rise to cross-correlation. Our results suggest that cross-correlation may be used to recognize whether different cargoes use the same endocytic pathway. Additionally, we show that cross-correlation is applicable to two-dimensional membrane diffusion. CTX bound to GM1-containing artificial giant unilamellar vesicles was diffusible, whereas CTX bound to the plasma membrane was immobile on the FCS time-scale, possibly because of raft-association of GM1.