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Spatial and Temporal Aspects of Exocytosis Studied on the Isolated Plasma Membranes

  • Ira MilosevicEmail author
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Part of the Methods in Molecular Biology book series (MIMB, volume 2233)

Abstract

Exocytosis of large-dense core vesicles in neuroendocrine cells is a highly regulated, calcium-dependent process, mediated by networks of interrelated proteins and lipids. Here, I describe experimental procedures for studies of selective spatial and temporal aspects of exocytosis at the plasma membrane, or in its proximity, using adrenal chromaffin cells. The assay utilizes primary cells subjected to a brief ultrasonic pulse, resulting in the formation of thin, flat inside-out plasma membranes with attached secretory vesicles and elements of cell cytoskeleton. In this model, secretion of plasma membrane–attached secretory vesicles was found to be dependent on calcium and sensitive to clostridial neurotoxins. Depending on the probe selected for secretory vesicle cargo, protein, and/or lipid detection, this simple assay is versatile, fast and inexpensive, and offers excellent spatial resolution.

Key words

Cell-free assay Secretion assay LDCVs Neuropeptide Y Isolated membrane sheets Membrane patches SNAREs SNAP-25 Syntaxin 1 TIRF alternative 

Notes

Acknowledgments

I thank Dr. N. Raimundo for a discussion, and Dr. A. Milosevic for help with the figures. This work is supported by the Emmy Noether Young Investigator Award (1702/1) of the German Research Foundation (DFG) and the Schram Stiftung (T287/25457). No competing financial interests are declared.

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© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  1. 1.European Neuroscience Institute (ENI)A Joint Initiative of the University Medical Center Göttingen and the Max Planck SocietyGöttingenGermany
  2. 2.Wellcome Centre for Human Genetics, Nuffield Department of MedicineNIHR Oxford Biomedical Research Centre, University of OxfordOxfordUK

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