Imaging molecular interactions in living cells - PubMed (original) (raw)
Review
Imaging molecular interactions in living cells
Richard N Day et al. Mol Endocrinol. 2005 Jul.
Abstract
Hormones integrate the activities of their target cells through receptor-modulated cascades of protein interactions that ultimately lead to changes in cellular function. Understanding how the cell assembles these signaling protein complexes is critically important to unraveling disease processes, and to the design of therapeutic strategies. Recent advances in live-cell imaging technologies, combined with the use of genetically encoded fluorescent proteins, now allow the assembly of these signaling protein complexes to be tracked within the organized microenvironment of the living cell. Here, we review some of the recent developments in the application of imaging techniques to measure the dynamic behavior, colocalization, and spatial relationships between proteins in living cells. Where possible, we discuss the application of these different approaches in the context of hormone regulation of nuclear receptor localization, mobility, and interactions in different subcellular compartments. We discuss measurements that define the spatial relationships and dynamics between proteins in living cells including fluorescence colocalization, fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy. These live-cell imaging tools provide an important complement to biochemical and structural biology studies, extending the analysis of protein-protein interactions, protein conformational changes, and the behavior of signaling molecules to their natural environment within the intact cell.
Figures
Fig. 1
Photobleaching Analysis Reveals the Mobility of FP-Labeled Proteins A, FRAP measures the recovery of fluorescence within a ROI after rapid photobleaching of the FP-labeled protein within that ROI. This monitors the movement of nonbleached FP-labeled protein (mobile fraction) from adjacent regions into the photobleached ROI over time. Tight binding of the bleached protein to cellular structures impedes complete recovery of fluorescence (immobile fraction). B, The FLIP assay measures the loss of fluorescence from a ROI after the continuous photobleaching of an adjacent region. The loss of fluorescence from the ROI over time defines kinetics of the mobile fraction of the FP-labeled protein. Incomplete loss of fluorescence defines the immobile fraction of FP-labeled protein that does not move into the continuously photobleached ROI. For both FRAP and FLIP, mathematical modeling of the kinetics of fluorescence recovery/loss should consider the possibility of one or more mobile and immobile fractions.
Fig. 2
Photoactivatable FP as Molecular Highlighters PA-GFP is activated 100-fold by a brief intense pulse of 400 nm light to a restricted ROI. This allows the FP-labeled protein to be selectively activated in the ROI. The measurement of fluorescence intensity in and away from the ROI with time after photoactivation defines, respectively, the immobile fraction of the FP-labeled protein and the kinetics of the mobile protein.
Fig. 3
FCS Measures Fluorescence Signals within a Very Small Optically Defined Observation Volume The amount of fluorescence detected from the observation volume over time will fluctuate with the concentration, retention, and rate of diffusion of the FP-labeled protein within the observation volume (X in the diagram). Correlation functions calculated from the fluctuation data are analyzed to determine the overall diffusion coefficients and concentrations of the proteins within the specified observation volume.
Fig. 4
FRET Microscopy Detects the Direct Transfer of Excitation Energy from a Donor Fluorophore to Acceptor Fluorophores that Is Limited to Distances of Less than about 80 Å A, When the donor is excited (blue arrow) and energy transfer occurs (red arrow), the donor fluorescence signal is quenched and there is sensitized emission from the acceptor (green arrow). FRET signals can be quantifying by measuring sensitized emission using SBT correction methods, or by measuring donor dequenching using pbFRET, as described in the text. B, FRET also can be detected by measuring the fluorescence lifetime of the donor fluorophore population. Comparing the fluorescence lifetime of a donor alone (red curve) with the donor in the presence of an acceptor (blue curve) indicates the quenching of the donor-protein population by the interactions with the acceptor-tagged proteins. C, For populations of cells that express varying levels of donor- and acceptor-labeled proteins, the FRET signals from individual cells will be proportional to the amount of acceptor-labeled protein available, reaching a maximum when all donor-labeled protein interacts with acceptor-labeled protein (closed circles). This relationship will not be true for coexpressed, but noninteracting labeled proteins (closed squares). This relationship aids the interpretation of the biochemical and structural contributors to FRET signals measured within a subcellular compartment.
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