title: Optical techniques for imaging membrane domains in live cells (live-cell palm of protein clustering).
abstract: It is now recognized that the plasma membrane is not homogeneous but instead contains a variety of membrane microdomains. These include lipid microdomains (lipid rafts) and protein clusters which exist on a range of size and time scales but are often small and short-lived. The small size and dynamic nature of membrane domains has made them difficult to study by conventional fluorescence microscopy approaches. Photoactivated localization microscopy (PALM) is a super-resolution technique capable of localizing the positions of individual molecules with tens of nanometers precision. Here, we describe a method for imaging membrane proteins using PALM, including live-cell PALM, to detect the molecular clustering of plasma membrane proteins using a statistical cluster-analysis method based on Ripley's K-function. While the method is applicable to a wide variety of proteins in various biological systems, to illustrate the technique, we will image and analyze the clustering behavior of the adaptor protein Linker for activation of T cells (LAT) at the T cell immunological synapse [Williamson, D. J., Owen, D. M., Rossy, J., Magenau, A., Wehrmann, M., Gooding, J. J. , and Gaus, K. (2011). Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events. Nat. Immunol.12, 655-662.].
authors.affiliation: Centre for Vascular Research, University of New South Wales, Sydney, Australia.
authors.forename: Dylan M
authors.initials: DM
authors.lastname: Owen
authors.forename: David
authors.initials: D
authors.lastname: Williamson
authors.forename: Astrid
authors.initials: A
authors.lastname: Magenau
authors.forename: Katharina
authors.initials: K
authors.lastname: Gaus
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis* / Q000378:metabolism*; D000465:Algorithms; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D058948:Cell Tracking / Q000379:methods*; D016000:Cluster Analysis; D049452:Green Fluorescent Proteins; D006801:Humans; D054992:Immunological Synapses / Q000378:metabolism; D019169:Jurkat Cells; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C110810:LAT protein, human; D008565:Membrane Proteins; D049452:Green Fluorescent Proteins