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Abstract
Fluorescent proteins (FPs) have proven to be valuable tools for high-resolution imaging studies of vesicle transport processes, including exo- and endocytosis. Since the pH of the vesicle lumen changes between acidic and neutral during these events, pH-sensitive FPs with near neutral pKa, such as pHluorin, are particularly useful. FPs with pKa>6 are readily available in the green spectrum, while red-emitting pH-sensitive FPs are rare and often not well characterized as reporters of exo- or endocytosis. Here we tested a panel of ten orange/red and two green FPs in fusions with neuropeptide Y (NPY) for use as secreted vesicle marker and reporter of dense core granule exocytosis and release. We report relative brightness, bleaching rate, targeting accuracy, sensitivity to vesicle pH, and their performance in detecting exocytosis in live cells. Tandem dimer (td)-mOrange2 was identified as well-targeted, bright, slowly bleaching and pH-sensitive FP that performed similar to EGFP. Single exocytosis events were readily observed, which allowed measurements of fusion pore lifetime and the dynamics of the exocytosis protein syntaxin at the release site during membrane fusion and cargo release.
Highlights
Fluorescent proteins (FP) are extensively used as genetically encoded florescent tags [1,2] to address a multitude of biological questions
We targeted a panel of ten red FPs or EGFP/pHluorin to insulin granules with the goal of comparing their brightness and bleaching rates
The tandem dimers resulted in brighter granules (1.5-fold) than mEGFP or pHluorin (Fig 1C)
Summary
Fluorescent proteins (FP) are extensively used as genetically encoded florescent tags [1,2] to address a multitude of biological questions Their popularity is primarily due to the ease of labeling proteins of interest in living cells and organisms. Developed FPs are usually benchmarked for brightness, photochemistry, photostability, cytotoxicity, pH- and ion sensitivity, as well as subcellular targeting accuracy with a fairly standardized panel of subcellular localization sequences [3,4,5]. The latter does not include specialized organelles such as synaptic vesicles or dense core secretory granules, organelles that are involved in regulated exocytosis and release of neurotransmitters and hormones. Regulated exocytosis involves Ca2+- and SNARE-dependent fusion of secretory vesicles with the plasma membrane to empty its cargo [6,7], and FP-tagged markers are increasingly being used to study exocytosis in neuronal synapses [8] and even release of individual vesicles
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