Quantifying subcellular distribution of fluorescent fusion proteins in cells migrating within tissues.
The movement of proteins within cells can provide dynamic indications of cell signaling and cell polarity, but methods are needed to track and quantify subcellular protein movement within tissue environments. Here we present a semiautomated approach to quantify subcellular protein location for hundreds of migrating cells within intact living tissue using retrovirally expressed fluorescent fusion proteins and time-lapse two-photon microscopy of intact thymic lobes. We have validated the method using GFP-PKCζ, a marker for cell polarity, and LAT-GFP, a marker for T-cell receptor signaling, and have related the asymmetric distribution of these proteins to the direction and speed of cell migration. These approaches could be readily adapted to other fluorescent fusion proteins, tissues and biological questions.
Melichar
Heather J
HJ
Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
Li
Ou
O
Herzmark
Paul
P
Padmanabhan
Raghav K
RK
Oliaro
Jane
J
Ludford-Menting
Mandy J
MJ
Bousso
Philippe
P
Russell
Sarah M
SM
Roysam
Badrinath
B
Robey
Ellen A
EA
D000818:Animals; D002465:Cell Movement / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013950:Thymus Gland / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins