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


The Silences of the Archives, the Reknown of the Story.
The Martin Guerre affair has been told many times since Jean de Coras and Guillaume Lesueur published their stories in 1561. It is in many ways a perfect intrigue with uncanny resemblance, persuasive deception and a surprizing end when the two Martin stood face to face, memory to memory, before captivated judges and a guilty feeling Bertrande de Rols. The historian wanted to go beyond the known story in order to discover the world of the heroes. This research led to disappointments and surprizes as documents were discovered concerning the environment of Artigat’s inhabitants and bearing directly on the main characters thanks to notarial contracts. Along the way, study of the works of Coras and Lesueur took a new direction. Coming back to the affair a quarter century later did not result in finding new documents (some are perhaps still buried in Spanish archives), but by going back over her tracks, the historian could only be struck by the silences of the archives that refuse to reveal their secrets and, at the same time, by the possible openings they suggest, by the intuition that almost invisible threads link here and there characters and events.

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Available from: Michelle A Digman, Dec 30, 2014
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    • "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. "
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    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.
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    • "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. "
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    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.
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    • "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. "
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    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.
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