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Related Experiment Videos

Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins

G Miesenböck1, D A De Angelis, J E Rothman

  • 1Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.

Nature
|July 22, 1998
PubMed
Summary

Researchers developed novel pH-sensitive fluorescent protein sensors (pHluorins) to visualize neural activity. These DNA-encoded probes track neurotransmission and secretion in real-time within neural systems.

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Area of Science:

  • Neuroscience
  • Molecular Biology
  • Biochemistry

Background:

  • Neural information processing relies on distributed cellular activity, not just individual neurons.
  • Optical indicators, especially DNA-encodable protein-based probes, are crucial for visualizing this activity in situ.
  • Existing methods often require external cofactors or lack specificity for cellular compartments.

Purpose of the Study:

  • To develop novel, DNA-encodable optical sensors for monitoring neural secretion and neurotransmission.
  • To engineer pH-sensitive fluorescent proteins (pHluorins) for detecting vesicle dynamics.
  • To enable in situ, real-time visualization of synaptic activity and vesicle exocytosis.

Main Methods:

  • Structure-directed combinatorial mutagenesis was used to create pH-sensitive green fluorescent protein variants (pHluorins).

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  • pHluorins were genetically encoded and linked to vesicle membrane proteins for targeted localization.
  • The sensors were expressed in cells and tissues to monitor vesicle exocytosis, recycling, and synaptic transmission.

    • Main Results:

    • Engineered pHluorins were successfully targeted to secretory and synaptic vesicles.
    • The sensors accurately reported neurotransmission at individual synaptic boutons.
    • Single secretory granule fusion pore 'flicker' and exocytosis events were visualized.

    Conclusions:

    • Developed pHluorins are effective DNA-encodable probes for real-time monitoring of neural secretion and neurotransmission.
    • These sensors overcome limitations of previous optical indicators, enabling detailed study of vesicle dynamics.
    • The technology facilitates in situ investigation of fundamental neural processes at the cellular and subcellular levels.