Date of Award
Doctor of Philosophy (PhD)
Fluorescence fluctuation spectroscopy (FFS) is comprised of a set of statistical techniques used to learn information about fluorescence particles by analyzing intensity fluctuations in signals detected from a fluorescence spectrometer or microscope. In biological contexts, FFS is commonly used to quantify the concentrations, molecular brightnesses, and transport properties (e.g. diffusion coefficients) of labeled biomolecules. In particular, the molecular brightness parameter is valuable because it can be used to quantify oligomerization of biomolecules. Therefore, FFS has emerged as an attractive tool for measuring protein-protein interactions in live cells. This dissertation describes the development of image-based, multicolor FFS techniques. Image-based FFS is advantageous because it provides spatial resolution, generates more favorable statistics for slow or immobile molecules, distributes the excitation power over a wider part of the sample, and mitigates non-ideal photophysical effects such as photobleaching. Multicolor approaches allow the sensitive detection of heteromeric interactions by using spectrally distinct chromophores.Two novel implementations of image-based FFS are introduced in this work, two-color spatial intensity distribution analysis (2c-SpIDA) and two-color spatial cumulant analysis (2c-SpCA), and both allow measurements of protein-protein interactions from two-color laser scanning fluorescence microscopy images. We characterized these techniques in depth and determined that 2c-SpCA is more practical for live cell applications. Proof of principle experiments demonstrated this by measuring the interaction between two G proteins on the plasm membrane, Gβ1 and Gγ2. Activation of heterotrimeric G proteins through G protein coupled receptors (GPCRs) is an ubiquitous signaling mechanism in eukaryotic biology. The three principal molecular components of this mechanism are the GPCR, Gα subunit, and Gβγ subunit. To measure these three components simultaneously, we developed fluorescence covariance matrix analysis (FCMA) as an extension of 2c-SpCA. FCMA takes advantage of high spectral resolution images to permit FFS analysis of several chromophores simultaneously. Components were labeled with three spectrally distinct fluorescent proteins. As measured by FCMA, interactions between G protein subunits, Gαi1 and Gβ1γ2, were sensitive to stimulation of two GPCRs, the dopamine receptor D2 and the α-2A adrenergic receptor. Interactions between GPCRs and G proteins were not detectable above background, suggesting weak coupling that is more consistent with a collisional model of GPCR/G protein interactions. Altogether, the tools developed in this dissertation represent a significant contribution to our ability to quantify protein-protein interactions, especially in the field of GPCR/G protein biology.
David W. Piston
James A. Fitzpatrick, Maria S. Remedi, Jonathan R. Silva, Paul W. Wiseman,