title: Functional proteometrics for cell migration.
abstract: 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.
authors.affiliation: Department of Pharmacology, University of North Carolina at Chapel Hill, 27599, USA.
authors.forename: Feimo
authors.initials: F
authors.lastname: Shen
authors.forename: Louis
authors.initials: L
authors.lastname: Hodgson
authors.forename: Andrew
authors.initials: A
authors.lastname: Rabinovich
authors.forename: Olivier
authors.initials: O
authors.lastname: Pertz
authors.forename: Klaus
authors.initials: K
authors.lastname: Hahn
authors.forename: Jeffrey H
authors.initials: JH
authors.lastname: Price
mesh_terms: 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*