QUANTITATIVE THREE-DIMENSIONAL (3D) MAPPING OF BIOMOLECULAR DYNAMICS AND ORGANIZATION IN VITRO AND IN VIVO BY SPIM-FCS

Abstract

The mobility and organization of biomolecules in biological systems serve as major driving forces for molecular level processes that regulate cellular functions. Such cellular functions coordinate organism level processes in a concerted manner. This prompted extensive research on the quantification of biomolecular dynamics in live systems to link mobility to functionality. In this thesis, an imaging modality of fluorescence correlation spectroscopy (FCS) which combines fast array detectors to the plane illumination of single plane illumination microscopy (SPIM-FCS) was established to determine spatial maps of diffusivity of biomolecules in vitro and in vivo. Sub-resolution membrane distribution of biomolecules was also investigated by applying FCS diffusion law analysis to SPIM-FCS data. These results demonstrate, for the first time, the association of Wnt3 protein to plasma membrane domains in live zebrafish and its dependence on lipid components and post-translational Wnt3 palmitoylation. Such association is tightly correlated to proper Wnt signaling and brain development.