Phluorin Tagging
Phluorin Tagging - Related Videos
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.
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.
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.
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.
FAQs
The signal depends on the pH-sensitive behavior of pHluorin. Inside an acidic synaptic vesicle, the attached fluorophore is quenched and produces little fluorescence. When fusion exposes the vesicle protein to the neutral extracellular environment, fluorescence increases. Subsequent retrieval and reacidification reduce the signal, creating a time-resolved readout of vesicle cycling.
Reacidification provides the mechanism for the declining phase of the fluorescence response. After a vesicle is retrieved from the plasma membrane, its lumen becomes acidic again, which quenches the pHluorin signal. Consequently, the fall in fluorescence is linked to vesicle recovery and recycling rather than simply indicating that the fluorescent tag has disappeared.
The sequence of signal changes connects separate stages of synaptic vesicle behavior. A fluorescence increase marks exposure during exocytosis, while a later decline accompanies retrieval and reacidification. Monitoring these changes in living neurons allows investigators to examine the progression of neurotransmitter release, endocytosis, and vesicle recycling rather than observing only a single endpoint.
A basic experiment follows fluorescence from pHluorin molecules fused to synaptic vesicle proteins while vesicles move through their cycle. Researchers observe when the signal rises as vesicles encounter neutral extracellular pH and when it falls after reacidification. This live-cell approach links molecular vesicle events with measurable changes in neuronal synaptic activity.
The method can provide readouts for neurotransmitter release, vesicle endocytosis, and recycling. These processes appear as related phases of the fluorescence response: exposure during exocytosis, followed by signal reduction as vesicles are retrieved and reacidified. Examining the complete response helps characterize synaptic vesicle cycling instead of focusing only on release itself.
Phluorin tagging connects intracellular pH changes with the molecular events that support neuronal communication. Because the signal can be followed in living cells, researchers can examine how synaptic vesicles behave during release and recovery. This makes the approach useful for characterizing synaptic function and for investigating changes associated with health and disease.