Functional proteometrics for cell migration

Department of Pharmacology, University of North Carolina at Chapel Hill, 27599, USA.
Cytometry Part A (Impact Factor: 2.93). 07/2006; 69(7):563-72. DOI: 10.1002/cyto.a.20283
Source: PubMed


Advances in living cellular fluorescence biosensors and computerized microscopy enable a vision of fully automated high-resolution measurements of the detailed intracellular molecular dynamics directly linked to cellular behaviors. Given the heterogeneity of cell populations, a statistically relevant study of molecular-cellular dynamics is a key motivation for improved automation.
We explored automating computerized, microscope-based data extraction and analyses that monitor cell locomotion, rates of mitoses, and spatiotemporal activities of intracellular proteins via ratiometric fluorescent biosensors in mouse fibroblasts. Novel image processing methods included K-means clustering segmentation preprocessing followed by modified discrete, normalized cross-correlational alignment of two-color images; ratiometric processing for fluorescence resonance energy transfer (FRET) measurements; and intracellular spatial distribution measurements of RhoA GTPase activity.
The interdivision time was 19.4 h (mean) +/- 6.0 h (SD) (n = 7) for the GFP-histone cells in the two-by-two field that was scanned for 72 h. After registration and ratioing of the cells with the RhoA biosensor, increases in both cell protrusion and retraction were coincident with to increases in RhoA activity.
These advances lay the foundation for extracting and correlating measurements characterizing the functional relationships of spatial localization and protein activation with features of cell migration such as velocity, polarization, protrusion, retraction, and mitosis.

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Available from: Klaus M Hahn, Sep 05, 2014
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    • "To obtain an image's curvature perfectly even, it is necessary to implement a correction of the vignetting function . Typically, to solve this problem a single Empty Field (EF) image, or a sequence, is acquired to characterize the vignetting function and to correct it [4] [5] [7]. However , EF images can be acquired only in case the microscope setup keeps unchanged [6] and the instrumentation is (still) at one's disposal. "
    Proc. $7^th$ International Workshop on Biosignal Interpretation (BSI 2012); 01/2012
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    • "Nuclear localized fluorescent proteins are useful for cell tracking. Moreover, a label that shows a temporal change, such as correlation with DNA content, can be used in correction routines for during automated tracking [16], for instance to distinguish tracks of two cells that cross paths from two daughters arising from a single progenitor. To test utility of the nuclear fluorescent proteins in tracking applications, differentiating cells of the FACS-enriched hESCs were tracked automatically using a modified version of the Particle Tracking Plugin for ImageJ [17]. "
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    PLoS ONE 02/2009; 4(4):e5046. DOI:10.1371/journal.pone.0005046 · 3.23 Impact Factor
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    • "These include characteristic artifacts that warn of misalignment: ''edge artifacts'' where ratio values are high on one side of the cell and low on the opposite side, and bright shading on one side of objects within the cell such as the nucleus. Automated methods for image registration are available (Shen et al., 2004), but manual methods can be used successfully and remain more accurate in some cases. A Metamorph ''Color align'' plug‐in module can be used to determine the relative pixel shifts of up to three fluorescence channels. "
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