Computational Analysis of Spatiotemporal Molecular Hierarchy in Single
Live Cells
Shaoying (Kathy) Lu
UCSD
Abstract:
Genetically encoded biosensors based on fluorescence resonance energy
transfer (FRET) have been widely applied to visualize the molecular
activity in live cells with high spatiotemporal resolution. The
enormous amount of video images and the complex dynamics of signaling
events presented tremendous challenges for data analysis and demand the
development of intelligent and automated imaging analysis methods
specifically designed for the studies of live cell imaging. We have
developed advanced and automated computational imaging analysis methods
for quantifying and simulating the motion of biosensors, reconstructing
the de facto molecular activities, and tracking and analyzing the
spatiotemporal molecular interactions in a single live cell with
high-throughput power. Based on fluorescence recovery after
photobleaching (FRAP) experiments, we have developed a finite element
(FE) method to analyze, simulate, and subtract the diffusion effect of
mobile biosensors. The results indicate that the Src biosensor located
in the cytoplasm moves 4-8 folds faster than those anchored on
different compartments in plasma membrane. The mobility of biosensor at
lipid rafts is slower than that outside of lipid rafts and is dominated
by two-dimensional diffusion. Furthermore, we have developed a general
correlative FRET imaging method (CFIM) to quantify the subcellular
coupling between an enzymatic activity and a phenotypic response in
live cells, e.g. at focal adhesions (FAs). CFIM quantitatively
evaluated the cause-effect relationship between Src kinase activation
and FA dynamics monitored in single cells. CFIM showed that the growth
factor-stimulated FA disassembly at cell periphery was linearly
dependent on the local Src activation with a time delay. The FA
disassembly per unit of Src activation (coupling capacity), as well as
the time delay, was regulated by cell-matrix interaction via different
integrin receptors. The results revealed a tight enzyme-phenotype
coupling in FA populations mediated by integrin avb3 but not in those
by integrin a5b1. Therefore, our computational analysis methods can
allow the high-throughput quantification of molecular motions and
interactions at subcellular levels in single live cells. The results
should advance our systems understanding of the hierarchical
interactions of signaling molecular network at subcellular
microdomains.