Simultaneous quantification of cell motility and protein-membrane-association using active contours.
We present a new method for the quantification of dynamic changes in fluorescence intensities at the cell membrane of moving cells. It is based on an active contour method for cell-edge detection, which allows tracking of changes in cell shape and position. Fluorescence intensities at specific cortical subregions can be followed in space and time and correlated with cell motility. The translocation of two GFP tagged proteins (CRAC and GRP1) from the cytosol to the membrane in response to stimulation with the chemoattractant cAMP during chemotaxis of Dictyostelium cells and studies of the spatio-temporal dynamics of this process exemplify the method: We show that the translocation can be correlated with motility parameters and that quantitative differences in the rate of association and dissociation from the membrane can be observed for the two PH domain containing proteins. The analysis of periodic CRAC translocation to the leading edge of a cell responding to natural cAMP waves in a mound demonstrates the power of this approach. It is not only capable of tracking the outline of cells within aggregates in front of a noisy background, but furthermore allows the construction of spatio-temporal polar plots, capturing the dynamics of the protein distribution at the cell membrane within the cells' moving co-ordinate system. Compilation of data by means of normalised polar plots is suggested as a future tool, which promises the so-far impossible practicability of extensive statistical studies and automated comparison of complex spatio-temporal protein distribution patterns.
School of Life Sciences, The Wellcome Trust Biocentre, University of Dundee, Dundee, Scotland.
Dirk
D
Dormann
Thorsten
T
Libotte
Cornelis J
CJ
Weijer
Till
T
Bretschneider
D000465:Algorithms; D000818:Animals; D002449:Cell Aggregation; D002462:Cell Membrane / Q000378:metabolism*; D048429:Cell Size; D002630:Chemotactic Factors / Q000378:metabolism; D002633:Chemotaxis; D003198:Computer Simulation; D000242:Cyclic AMP / Q000378:metabolism; D004023:Dictyostelium / Q000166:cytology / Q000502:physiology*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D021381:Protein Transport; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D002630:Chemotactic Factors; D008164:Luminescent Proteins; D015800:Protozoan Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; C089420:dagA protein, Dictyostelium; C040985:phosphatidylinositol receptors; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP