Article

Millisecond spatiotemporal dynamics of FRET biosensors by the pair correlation function and the phasor approach to FLIM

Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, and Department of Development and Cell Biology, University of California, Irvine, Irvine, CA 92617.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 12/2012; 110(1). DOI: 10.1073/pnas.1211882110
Source: PubMed

ABSTRACT Here we present a fluctuation-based approach to biosensor Förster resonance energy transfer (FRET) detection that can measure the molecular flow and signaling activity of proteins in live cells. By simultaneous use of the phasor approach to fluorescence lifetime imaging microscopy (FLIM) and cross-pair correlation function (pCF) analysis along a line scanned in milliseconds, we detect the spatial localization of Rho GTPase activity (biosensor FRET signal) as well as the diffusive route adopted by this active population. In particular we find, for Rac1 and RhoA, distinct gradients of activation (FLIM-FRET) and a molecular flow pattern (pCF analysis) that explains the observed polarized GTPase activity. This multiplexed approach to biosensor FRET detection serves as a unique tool for dissection of the mechanism(s) by which key signaling proteins are spatially and temporally coordinated.

0 Followers
 · 
150 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Diffusion of a fluorescent protein within a cell has been measured using either fluctuation-based techniques (fluorescence correlation spectroscopy (FCS) or raster-scan image correlation spectroscopy) or particle tracking. However, none of these methods enables us to measure the diffusion of the fluorescent particle at each pixel of the image. Measurement using conventional single-point FCS at every individual pixel results in continuous long exposure of the cell to the laser and eventual bleaching of the sample. To overcome this limitation, we have developed what we believe to be a new method of scanning with simultaneous construction of a fluorescent image of the cell. In this believed new method of modified raster scanning, as it acquires the image, the laser scans each individual line multiple times before moving to the next line. This continues until the entire area is scanned. This is different from the original raster-scan image correlation spectroscopy approach, where data are acquired by scanning each frame once and then scanning the image multiple times. The total time of data acquisition needed for this method is much shorter than the time required for traditional FCS analysis at each pixel. However, at a single pixel, the acquired intensity time sequence is short; requiring nonconventional analysis of the correlation function to extract information about the diffusion. These correlation data have been analyzed using the phasor approach, a fit-free method that was originally developed for analysis of FLIM images. Analysis using this method results in an estimation of the average diffusion coefficient of the fluorescent species at each pixel of an image, and thus, a detailed diffusion map of the cell can be created. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.
    Biophysical Journal 12/2014; 107(12):2775-85. DOI:10.1016/j.bpj.2014.08.041 · 3.83 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Background Lipid rafts are cholesterol and saturated lipid-rich, nanometer sized membrane domains that are hypothesized to play an important role in compartmentalization and spatiotemporal regulation of cellular signaling. Lipid rafts contribute to the plasma membrane order and to its spatial asymmetry, as well. The raft nanodomains on the surface of CD4+ T lymphocytes coalesce during their interaction with antigen presenting cells (APCs). Sensing of foreign antigen by the antigen receptor on CD4+ T cells occurs during these cell-cell interactions. In response to foreign antigen the CD4+ T cells proliferate, allowing the expansion of few antigen-specific primary CD4+ T cell clones. Proliferating CD4+ T cells specialize in their function by undergoing differentiation into appropriate effectors tailored to mount an effective adaptive immune response against the invading pathogen. Results To investigate the role of lipid raft-based membrane order in the clonal expansion phase of primary CD4+ T cells, we have disrupted membrane order by incorporating an oxysterol, 7-ketocholesterol (7-KC), into the plasma membrane of primary CD4+ T cells expressing a T cell receptor specific to chicken ovalbumin323–339 peptide sequence and tested their antigen-specific response. We report that 7-KC, at concentrations that disrupt lipid rafts, significantly diminish the c-Ovalbumin323–339 peptide-specific clonal expansion of primary CD4+ T cells. Conclusions Our findings suggest that lipid raft-based membrane order is important for clonal expansion of CD4+ T cells in response to a model peptide. Electronic supplementary material The online version of this article (doi:10.1186/s12865-014-0058-8) contains supplementary material, which is available to authorized users.
    BMC Immunology 12/2014; 15(1):58. DOI:10.1186/s12865-014-0058-8 · 2.25 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Luminescence decay functions describe the time dependence of the intensity of radiation emitted by electronically excited species. Decay phasor plots (plots of the Fourier sine transform vs. the Fourier cosine transform, for one or several angular frequencies) are being increasingly used in fluorescence, namely in lifetime imaging microscopy (FLIM). In this work, a detailed study of the sum of two exponentials decay function is carried out revealing that sub-exponential, super-exponential and unimodal decays have different phasor signatures. A generalization of the lever rule is obtained, and the existence of an outermost phasor curve corresponding to intermediate-like decays is demonstrated. A study of the behavior of more complex decay functions (sum of three exponentials, stretched and compressed exponentials, phosphorescence with reabsorption and triplet-triplet annihilation, fluorescence with quantum beats) allows concluding that a rich diversity of phasor plot patterns exists. In particular, super-exponential decays can present complex shapes, spiraling at high frequencies. The concept of virtual phasor is also introduced.
    Chemical Physics 01/2015; 449:23. DOI:10.1016/j.chemphys.2015.01.007 · 2.03 Impact Factor