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Quantitative Imaging of Lymphocyte Membrane Protein Reorganization and Signaling

Peter M. Kasson ^* , Johannes B. Huppa  , Michelle Krogsgaard  , Mark M. Davis   and Axel T. Brunger  ^¶ ||

^* Biophysics Program, Medical Scientist Training Program, Howard Hughes Medical Institute, Department of Microbiology and Immunology, ^¶ Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, and ^|| Stanford Synchrotron Radiation Laboratory, Stanford University School of Medicine, Stanford, California

Correspondence: Address reprint requests to Axel Brunger, Tel.: 650-736-1031; Fax: 650-736-1961; E-mail: brunger{at}stanford.edu.

Changes in membrane protein localization are critical to establishing cell polarity and regulating cell signaling. Fluorescence microscopy of labeled proteins allows visualization of these changes, but quantitative analysis is needed to study this aspect of cell signaling in full mechanistic detail. We have developed a novel approach for quantitative assessment of membrane protein redistribution based on four-dimensional video microscopy of fluorescently labeled proteins. Our analytic system provides robust automated methods for cell surface reconstruction, cell shape tracking, cell-surface distance measurement, and cluster formation analysis. These methods permit statistical analyses and testing of mechanistic hypotheses regarding cell signaling. We have used this approach to measure antigen-dependent clustering of signaling molecules in CD4⁺ T lymphocytes, obtaining clustering velocities consistent with single-particle tracking data. Our system captures quantitative differences in clustering between signaling proteins with distinct biological functions. Our methods can be generalized to a range of cell-signaling phenomena and enable novel applications not feasible with single-particle studies, such as analysis of subcellular protein localization in live organ culture.