Abstract
Sub-diffraction imaging of plasma membrane localized proteins, such as the SNARE (SolubleNSFAttachment Protein Receptor) proteins involved in exocytosis, in fixed cells have resulted in images with high spatial resolution, at the expense of dynamical information. Here, we have imaged localized fluorescence bursts of DRONPA-fused SNAP-25 molecules in live chromaffin cells by Total Internal Reflection Fluorescence (TIRF) imaging. We find that this method allows tracking protein cluster dynamics over relatively long times (∼20 min.), partly due to the diffusion into the TIRF field of fresh molecules, making possible the simultaneous identification of cluster size, location and temporal evolution. The results indicate that the DRONPA-fused SNAP-25 clusters display rich dynamics, going from staying constant to disappearing and reappearing in specific cluster domains within minutes.
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Electronic supplementary information (ESI) available: Rescue data for the DRONPA_SNAP-25 construct. An example of a 2D Gaussian fit of a single molecule and an example of the localization accuracy. Two examples of a SRIC images. Average intensity image and second order SOFI image. Additional L(r)-r functions of live and fixed Chromaffin cells. Data on the effect of 405 nm light on the number of detected single molecules. Additional traces from live and fixed Chromaffin cells showing cluster dynamics. See DOI: 10.1039/c4pp00423j
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Antoku, Y., Dedecker, P., Pinheiro, P.S. et al. Spatial distribution and temporal evolution of DRONPA-fused SNAP25 clusters in adrenal chromaffin cells. Photochem Photobiol Sci 14, 1005–1012 (2015). https://doi.org/10.1039/c4pp00423j
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DOI: https://doi.org/10.1039/c4pp00423j