Quantitative Analysis of Membrane Protein Localization and Signaling
Peter M. Kasson, Johannes B. Huppa, Michelle Krogsgaard, Mark M. Davis, and Axel T. Brunger
Stanford University

Fluorescence microscopy of labeled proteins yields a wealth of data on cell signaling processes. However, systems for quantitative analysis of such data have lagged behind the recent progress in data acquisition technology. As cellular protein redistribution plays a key role in proximal signaling and the establishment of cell polarity, this quantitative information is critical for the understanding and modeling of many signaling networks. We have developed a robust and automated system to analyze membrane protein redistribution based on four-dimensional datasets obtained via fluorescence video microscopy. Our system provides methods for cell surface segmentation and reconstruction, cell shape tracking, cell-surface parameterization, and cluster formation analysis. These methods enable statistical analyses and the testing of mechanistic hypotheses regarding cell signaling. Our system is novel both in its integration and in its surface-based approach, allowing a model-free analysis of protein redistribution across the entire cell. We validate our system by measuring receptor clustering in T lymphocytes undergoing activation, obtaining clustering velocities consistent with the previously reported single-particle tracking data that serve as our reference standard. We subsequently measure clustering of another lymphocyte signaling protein, observing a different clustering profile that corresponds to its distinct biological function. Our methods generalize to many different cell-signaling phenomena, allowing a quantitative measurement of these cell membrane processes and offering the ability to empirically derive parameters for spatial signaling network models.