Journal article
Quantifying subcellular distribution of fluorescent fusion proteins in cells migrating within tissues
HJ Melichar, O Li, P Herzmark, RK Padmanabhan, J Oliaro, MJ Ludford-Menting, P Bousso, SM Russell, B Roysam, EA Robey
Immunology and Cell Biology | NATURE PUBLISHING GROUP | Published : 2011
DOI: 10.1038/icb.2010.122
Abstract
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 migrati..
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Awarded by National Institute of Allergy and Infectious Diseases
Funding Acknowledgements
We thank Tanja Schwikert for generating the memCFP transgenic construct, Jenny Ross, Ying Fang Liao, Kevin Zhou and Jeff LeDue for technical assistance, Ena Ladi for contribution of a control two-photon data set and Yousef Al-Kofahi and Arunachalam Narayanaswamy for valuable input in cell segmentation and tracking. We acknowledge the CHPS Imaging Core for microscopy assistance. This work was supported by the NIH (AI064227, AI065537 and AI065831 to ER, and EB005157 to BR), the HFSP (to ER and SR), the NHMRC (to SR and JO) and the California Institute for Regenerative Medicine post-doctoral fellowship (Training Grant Number T1-00007) (to HJM).