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Related Experiment Videos

Vesiculation and sorting from PC12-derived endosomes in vitro

Y Lichtenstein1, C Desnos, V Faúndez

  • 1Department of Biochemistry and Biophysics and Hormone Research Institute, University of California, San Francisco, CA 94143-0534, USA.

Proceedings of the National Academy of Sciences of the United States of America
|September 16, 1998
PubMed
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Researchers reconstituted synaptic vesicle formation in vitro using cytoplasmic proteins AP3 and ARF1. The study identified endosomes as the precursor organelle for synaptic vesicle biogenesis, distinct from plasma membranes.

Area of Science:

  • Cell Biology
  • Neuroscience
  • Membrane Trafficking

Background:

  • Synaptic vesicle formation is crucial for neurotransmission.
  • The specific organelle precursor for synaptic vesicle biogenesis in vitro was previously unclear.

Purpose of the Study:

  • To identify the organelle precursor involved in AP3 and ARF1-mediated synaptic vesicle formation.
  • To characterize the mechanism of synaptic vesicle biogenesis in vitro.

Main Methods:

  • Reconstitution of vesicle formation using PC12 cell homogenates, ATP, AP3, and ARF1.
  • Organelle characterization using temperature-dependent labeling (15°C vs. 4°C) and specific markers (transferrin, plasma membrane marker).

Main Results:

  • Budding activity was enriched in organelles labeling at 15°C but not 4°C.

Related Experiment Videos

  • The precursor organelle excluded plasma membrane markers.
  • The precursor contained internalized transferrin, identifying it as an endosome.
  • Vesicles formed in vitro from the endosomal precursor excluded transferrin.

    • Conclusions:

    • ARF1-mediated vesiculation generates synaptic vesicle-sized organelles from an endosomal precursor.
    • Synaptic vesicle protein sorting occurs concurrently with vesiculation from endosomes in vitro.