Functional proteometrics for cell migration.
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.
Shen
Feimo
F
Department of Pharmacology, University of North Carolina at Chapel Hill, 27599, USA.
Hodgson
Louis
L
Rabinovich
Andrew
A
Pertz
Olivier
O
Hahn
Klaus
K
Price
Jeffrey H
JH
D000818:Animals; D002465:Cell Movement / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology* / Q000502:physiology*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D019044:Image Cytometry / Q000379:methods; D051379:Mice; D041681:NIH 3T3 Cells; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*