Study of biomolecular interactions in vivo by multicolour fluorescence spectroscopy

Abstract

Fluorescence correlation spectroscopy (FCS) and its modality fluorescence cross-correlation spectroscopy (FCCS) are single molecule sensitive optical tools to study mobility, concentrations and interactions of molecules. Due to its non-invasive nature, it is gaining popularity in studying molecular processes in living cells and organisms. The presentation is divided into two sections. First, single-wavelength-FCCS (SW-FCCS) is applied to quantitate the binding between a small GTPases protein Cdc42 and one of its effectors IQGAP1. The dissociation constants (Kds) of their binding in 2D cell cultures are compared with that in zebrafish embryos, which is the first time a Kd is obtained from a live organism. The resulting one order of magnitude difference between the Kds suggests that a 2D cell model may not represent the biology of an organism. In the second section, the various factors affecting the quantitation of Kds in vivo with SW-FCCS are addressed, in particular, fluorescent proteins. Fluorescent proteins are the main choice of fluorescent labeling in cells, however they have a number of issues affecting the proper quantitation of Kds such as maturation issues, dark fractions and photobleached labels. Dark fractions of about 22 % and 40 % for the red fluorescent proteins mRFP and mCherry, respectively, are found. As these factors result in a fraction of undetected molecules, the Kds are overestimated. In addition, factors such as mis-match in detection volumes and the presence of endogenous proteins will also be addressed. The main goal is to be able to properly account for these factors so as to obtain a Kd which is independent of these factors. This is crucial for the development of FCCS and its application in vivo.