Vamp2-phlourin
Vamp2-phlourin - Related Videos
14:28
Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis
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Cited by 8 •
2016
Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.
03:54
Total Internal Reflection Fluorescence Microscopy for Visualization of Exocytic Events
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2025
Source: Winkle, C. C., et al. Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis. J. Vis. Exp. (2016)This video demonstrates total internal reflection fluorescence (TIRF) microscopy for visualizing exocytic events in cortical neurons. A pH-sensitive green fluorescent protein (GFP) marker fluoresces upon vesicle fusion, with selective TIRF excitation capturing membrane-proximal exocytosis in time-lapse imaging.
10:02
Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast
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Cited by 18 •
2016
Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.
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Fluorescence rises when fusion exposes pHluorin to the neutral extracellular environment, because the reporter is no longer quenched by the vesicle’s acidic interior. After membrane retrieval, the vesicle is reacidified and the signal declines. This rise-and-fall pattern lets investigators separate exocytosis from subsequent endocytosis and reacidification in living neurons.
VAMP2 links the reporter to synaptic vesicle membrane traffic, so the measured fluorescence follows the behavior of vesicles that carry this protein. A stimulation-associated increase therefore reports exposure of labeled vesicles during fusion, while later signal loss reflects their return to an acidic compartment. The molecular tag provides a presynaptic readout rather than a direct measurement of neurotransmitter concentration.
Fluorescence dynamics can be used to quantify neurotransmitter release, release probability, and presynaptic activity. The magnitude and timing of responses provide information about how strongly stimulation engages vesicle fusion and how the signal resolves during recycling. Comparing these measurements across conditions can reveal effects of synaptic stimulation, altered molecular machinery, or disease-related changes in presynaptic communication.
Synaptic stimulation can produce a rapid fluorescence increase as vesicles fuse with the presynaptic membrane, followed by a decline as retrieved vesicles become acidic again. Examining both phases is important because the initial change reflects exocytosis, whereas signal recovery toward baseline reflects later recycling-related events. The complete trace therefore provides temporal information about presynaptic membrane traffic.
Researchers monitor fluorescence from labeled neurons while applying synaptic stimulation and recording the reporter response over time. They then evaluate the stimulation-associated signal increase and its subsequent decline during vesicle retrieval and reacidification. This workflow converts visible fluorescence changes into measurements of presynaptic activity and vesicle cycling, while preserving observation of membrane traffic in living cells.
The reporter is useful when a study needs to determine whether altered neuronal communication is associated with presynaptic vesicle traffic. Researchers can compare fluorescence responses under different stimulation conditions or after changes affecting molecular machinery. The resulting measurements may reveal differences in release probability, presynaptic activity, or recycling behavior linked to disease-related changes.
By tracking vesicle fusion and recycling in real time, VAMP2-pHluorin provides a functional readout for testing how synaptic molecular components influence membrane traffic. Changes in the fluorescence response can be evaluated during stimulation to determine whether a manipulation affects exocytosis, the later recycling sequence, or overall presynaptic activity. This connects molecular mechanisms with observable synaptic behavior.