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- W2087698082 abstract "The dynamic assembly of receptors into endocytic structures such as clathrin-coated pits underlies cellular response to many external signals. However, the biophysical mechanisms for the regulated uptake of select receptors are unresolved. While commonly used bulk measurements access only ensemble-averaged behaviors, single molecule measurements have largely been limited to looking at only a few molecules in a single cell, lacking true ensemble information. Thus, until recently, the study of receptor capture by clathrin-coated pits has been limited by a scarcity of experimental methods capable of accessing information on ensembles of individual molecules within an individual cell. We addressed this by combining two recent technological advances to image receptors and clathrin-coated pits in living cells: two-color imaging with the photoswitchable PA-mCherry fluorescent label, and single particle tracking photoactivated localization microscopy (sptPALM).As compared to other monomeric red photoactivatable proteins, PAmCherry1 has higher pH stability, faster maturation and photoactivation kinetics, better photostability, and similar number of photons in single-molecule imaging. Furthermore, lack of background green fluorescence makes PAmCherry1 an advanced probe for two-color diffraction-limited microscopy and super-resolution techniques such as PALM. Two-color PALM imaging of PAmCherry1 tagged to the transferrin receptor (TfR) and PAGFP fused with the clathrin light-chain (CLC) were performed. Pair correlation analysis suggests clusters of less than 200 nm in size with both distinct and overlapping distributions of the TfR and CLC chimeras at sub-diffraction 25 nm resolutions. In addition, sptPALM was used to create spatially resolved maps of the trajectories of single receptor motions in conjunction with SPT of diffraction-limited clathrin-coated pits." @default.
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- W2087698082 date "2009-02-01" @default.
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- W2087698082 title "Visualizing the Receptor Assembly Into Clathrin-coated Pits with Super-resolution Two-color Palm and sptPALM" @default.
- W2087698082 doi "https://doi.org/10.1016/j.bpj.2008.12.2882" @default.
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