Superecliptic Phluorin
Superecliptic Phluorin - Related Videos
09:41
Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets
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Cited by 12 •
2017
We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.
09:59
Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking
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Cited by 3 •
2017
Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.
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.
10:30
Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo
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Cited by 10 •
2014
We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface. The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.
12:58
Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons
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Cited by 8 •
2012
By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.
FAQs
The key variable is the reporter’s exposure to two pH environments. Inside an acidic synaptic vesicle lumen, fluorescence is quenched; when fusion places the reporter at the relatively neutral extracellular surface, the signal rises. This pH contrast converts a membrane-trafficking event into an optical change that can be followed in living neurons.
A rise in fluorescence marks exposure after fusion, whereas a later decline reflects endocytosis followed by reacidification. Reading these phases in sequence lets investigators distinguish the outward fusion event from the recovery phase of vesicle recycling. The resulting time course is therefore informative about how rapidly synaptic vesicles move through these trafficking stages.
pH sensitivity makes the optical signal reversible rather than a permanent label of vesicle presence. Neutralization during fusion increases fluorescence, while return to an acidic lumen suppresses it again. This reversible behavior helps researchers associate successive signal changes with repeated trafficking events and separate vesicle exposure from later recycling.
As a genetically encoded reporter, it can be present in the membrane-trafficking system of living neuronal cells while fluorescence is monitored. Researchers then relate signal increases and decreases to exocytosis, endocytosis, and reacidification. This approach provides an optical time course of synaptic vesicle behavior rather than reducing the measurement to a single endpoint.
It can report synaptic vesicle exocytosis, recycling, and release kinetics, giving researchers a way to examine the cellular events underlying neurotransmission. Because the signal changes as vesicles fuse and then recover their acidic lumen, measurements can connect membrane-trafficking dynamics with the timing of neuronal communication.
Superecliptic Phluorin connects vesicle-trafficking measurements with cellular mechanisms that shape neurotransmission. By tracking fluorescence-linked changes in exocytosis and recycling, researchers can examine how synaptic vesicle dynamics contribute to communication between neurons and investigate their relevance to synaptic plasticity in living neural cells over time.