Abstract
Sub-diffraction imaging of plasma membrane localized proteins, such as the SNARE (Soluble NSF Attachment 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.
Highlights
SNARE proteins constitute a family of proteins which assemble a complex between vesicles and the plasma membrane to allow vesicle-to-membrane fusion and the release of vesicular content.[1,2] SNAREs are expressed at high levels in neurons and neurosecretory cells, the assembly of one to three complexes appears to be enough to drive fusion of a vesicle.[3]
Sub-diffraction imaging of plasma membrane localized proteins, such as the SNARE (Soluble NSF Attachment Protein Receptor) proteins involved in exocytosis, in fixed cells have resulted in images with high spatial resolution, at the expense of dynamical information
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
Summary
SNARE proteins constitute a family of proteins which assemble a complex between vesicles and the plasma membrane to allow vesicle-to-membrane fusion (exocytosis) and the release of vesicular content.[1,2] SNAREs are expressed at high levels in neurons and neurosecretory cells, the assembly of one to three complexes appears to be enough to drive fusion of a vesicle.[3]. Most investigations of the subdiffraction localization of SNAREs have utilized fixed cells combined with antibody-staining. This technique yields very high spatial resolution, because the fixation together with the use of bright chemical dyes allows for accurate localization of individual fluorophores. These investigations have resulted in the detailed description of sizes and numbers of SNARE-clusters in secretory cells. In PC12-cells, syntaxin-1 clusters are 50–100 nm in diameter, and have a density of 13–20 clusters μm−2,2,5,6 whereas SNAP-25 clusters appears to be a bit larger, ∼130 nm in diameter, and have a density of ∼10 clusters μm−2.2,6 The two cluster types only partially overlap.[7,8] This method has resulted in the identification of single SNAREs outside clusters, and numerical simulations have been used to suggest that clusters and individual molecules might be in a dynamic equilibrium.[5,6] in those experiments, which used fixed cells, these supposed dynamics could not be directly observed
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Schedule a callMore From: Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology
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