Detecting Protein Complexes in Living Cells from Laser Scanning Confocal Image Sequences by the Cross Correlation Raster Image Spectroscopy Method

Laboratory for Fluorescence Dynamics and Department of Biomedical Engineering, University of California, Irvine, California, USA.
Biophysical Journal (Impact Factor: 3.97). 02/2009; 96(2):707-16. DOI: 10.1016/j.bpj.2008.09.051
Source: PubMed

ABSTRACT We describe a general method for detecting molecular complexes based on the analysis of single molecule fluorescence fluctuations from laser scanning confocal images. The method detects and quantifies complexes of two different fluorescent proteins noninvasively in living cells. Because in a raster scanned image successive pixels are measured at different times, the spatial correlation of the image contains information about dynamic processes occurring over a large time range, from the microseconds to seconds. The correlation of intensity fluctuations measured simultaneously in two channels detects protein complexes that carry two molecules of different colors. This information is obtained from the entire image. A map of the spatial distribution of protein complexes in the cell and their diffusion and/or binding properties can be constructed. Using this cross correlation raster image spectroscopy method, specific locations in the cell can be visualized where dynamics of binding and unbinding of fluorescent protein complexes occur. This fluctuation imaging method can be applied to commercial laser scanning microscopes thereby making it accessible to a large community of scientists.

Download full-text


Available from: Michelle A Digman, Dec 30, 2014
  • Source
    • "In a recent study, Digman et al. used ccRICS to show the differences between cytoplasmic diffusion and binding of adhesion molecules. Moreover, the authors created maps of molecular interactions and their dynamics in the cell (Digman et al., 2009). RICS distinguishes between diffusion and binding which is the main advantage compared to other fluctuation methods. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The structure of cell membranes has been intensively investigated and many models and concepts have been proposed for the lateral organization of the plasma membrane. While proteomics and lipidomics have identified many if not all membrane components, how lipids and proteins interactions are coordinated in a specific cell function remains poorly understood. It is generally accepted that the organization of the plasma membrane is likely to play a critical role in the regulation of cell function such as receptor signalling by governing molecular interactions and dynamics. In this review we present different plasma membrane models and discuss microscopy approaches used for investigating protein behaviour, distribution and lipid organization.
    Molecular Membrane Biology 07/2014; 31(5). DOI:10.3109/09687688.2014.937469 · 1.73 Impact Factor
  • Source
    • "As we have previously demonstrated, correlation analysis can also be applied to detect molecular complexes (Choi et al., 2011). If two molecules tagged with different fluorophores (e.g., GFP and mCherry) reside within the same molecular complex, they will produce similar temporal fluorescence intensity fluctuation patterns, which will be revealed by calculating the crosscorrelation function (see Materials and Methods; Wiseman et al., 2004; Brown et al., 2006; Digman et al., 2009). In the absence of a complex, the intensity fluctuations are independent, and the cross-correlation function will be featureless and indistinguishable from the noise level. "
    [Show abstract] [Hide abstract]
    ABSTRACT: CD81 is a member of the tetraspanin family that has been described to have a key role in cell migration of tumor and immune cells. To unravel the mechanisms of CD81-regulated cell migration, we performed proteomic analyses that revealed an interaction of the tetraspanin C-terminal domain with the small GTPase Rac. Direct interaction was confirmed biochemically. Moreover, microscopy cross-correlation analysis demonstrated the in situ integration of both molecules into the same molecular complex. Pull-down experiments revealed that CD81-Rac interaction was direct and independent of Rac activation status. Knockdown of CD81 resulted in enhanced protrusion rate, altered focal adhesion formation and decreased cell migration, correlating with increased active Rac. Re-expression of wild type CD81, but not its truncated form lacking the C-terminal cytoplasmic domain, rescued these effects. The phenotype of CD81 knockdown cells was mimicked by treatment with a soluble peptide with the C-terminal sequence of the tetraspanin. Our data show that the interaction of Rac with the C-terminal cytoplasmic domain of CD81 is a novel regulatory mechanism of the GTPase activity turnover. Furthermore, they provide a novel mechanism for tetraspanin-dependent regulation of cell motility and open new avenues for tetraspanin-targeted reagents by the use of cell permeable peptides.
    Molecular biology of the cell 12/2012; 24(3). DOI:10.1091/mbc.E12-09-0642 · 5.98 Impact Factor
  • Source
    • "With the progresses in 3-D imaging techniques such as magnetic resonance imaging (MRI), computer tomography (CT) and microCT, the DVC approach has begun to be employed to analyze 3-D deformations of some typical materials within which there exist either specific textures or microarchitectural features, such as human cancellous bone [Zauel et al., 2006], wood [Forsberg et al., 2008, 2010], argillaceous rock [Lenoir et al., 2007] and solid foam material [Roux et al., 2008]. With the development of laser-scanning confocal microscopy (LSCM) in recent years, 3-D spatial information can now be captured via optical sectioning technique from live biological specimens, such as tissue explants [Roeder et al., 2002; Knight et al., 2006; Digman et al., 2009; Zanella et al., 2010], which is also very helpful for 3-D visualization of soft gels. Depending on tridimensional data acquired by LSCM, the DVC method can be employed to investigate 3-D mechanical deformations of soft gels. "
    [Show abstract] [Hide abstract]
    ABSTRACT: This paper develops a set of digital volume correlation (DVC) algorithms to address 3-D deformation measurements of soft gels with the aid of laser-scanning confocal microscopy. As an extension of the well-developed digital image correlation (DIC) method, the present DVC approach adopts a three-dimensional zero-normalized cross-correlation criterion (3-D ZNCC) to perform volume correlation calculations. Based on a 3-D sum-table scheme and the fast Fourier transform technique, a fast algorithm is first proposed to accelerate the integer-voxel correlation computations. Subsequently, two kinds of sub-voxel registration algorithms, i.e., 3-D gradient-based algorithm and 3-D Newton–Raphson algorithm, are presented to obtain the sub-voxel displacement and strain fields of volume images before and after deformation. Both a series of computer-simulated digital volume images and an actual agarose gel sample randomly embedded with fluorescent particles are employed to verify the 3-D deformation measurement capability of the proposed DVC algorithms, which indicates that they are competent to acquire 3-D displacement and strain fields of soft gels.
    International Journal of Applied Mechanics 06/2011; 03(02). DOI:10.1142/S1758825111001019 · 1.29 Impact Factor
Show more