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- W2094090711 abstract "TIRF-based photoactivated localization microscopy (PALM) offers a powerful tool to interrogate the spatial distribution of proteins in the plasma membrane (PM) at scales inaccessible by conventional light microscopy. We employed PALM to map out the nanoscopic organization of PM and construct a mechanistic perspective of PM organization. Cluster analysis of PALM data is complicated by the multiple appearances of a single protein with associated stochastic uncertainty in position, and reversible blinking of individual fluorophores. We developed a statistical algorithm to rigorously analyze PALM-data by performing spatial pair auto- and cross-correlation on the entire ensemble of detected molecular peaks, and subsequently separating out the contribution resulting from multiple appearances of the same molecule from that arising due to the actual lateral organization of the protein molecules. Using this approach, we could describe protein organization in an accurate and quantitative way, and extract reliable physical parameters like cluster-radius and number of molecules in a cluster. We examined the distribution of a diverse set of PM-proteins carefully chosen based on their different membrane-anchors and differential partitioning in phase-separated PM vesicles. Correlation analysis of PALM data revealed distinct steady state organization of these proteins: Transferrin Receptor-PAGFP and VSVG-PAGFP were organized into discrete clusters of ∼150nm and 70nm radius, respectively. PAGFP-GL-GPI, Lyn-PAGFP and Lat-PAGFP, in contrast, exhibited random distribution. Interestingly, cross-correlation of 2-color PALM images showed dramatic reorganization of actin into ring-like structures around antibody-crosslinked PAGFP-GPI clusters (∼100-200nm); similar actin-structures can modulate protein-platforms during cell-signaling. The combination of PALM and correlation analysis provides a robust and quantitative approach to study PM reorganization during various physiological processes." @default.
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- W2094090711 date "2011-02-01" @default.
- W2094090711 modified "2023-09-28" @default.
- W2094090711 title "Nano-Scale Spatial Organization of Plasma Membrane Revealed by Pair-Correlation Analysis" @default.
- W2094090711 doi "https://doi.org/10.1016/j.bpj.2010.12.2108" @default.
- W2094090711 hasPublicationYear "2011" @default.
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