Cytometry A. 2006 Jul;69(7):563-72.
Functional proteometrics for cell migration.
Shen F, Hodgson L, Rabinovich A, Pertz O, Hahn K, Price JH.
Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel
Hill, North Carolina 27599.
BACKGROUND:: 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. METHODS:: 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. RESULTS:: 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. CONCLUSIONS::
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. (c) 2006 International Society for
Analytical Cytology.
PMID: 16752422 [PubMed - as supplied by publisher]
