Imaging Ca2+-triggered exocytosis of single secretory granules on plasma membrane lawns from neuroendocrine cells.
This cell-free assay for exocytosis is particularly useful when spatial information about exocytotic sites and biochemical access to the plasma membrane within less than a minute is required. It is based on the study of plasma membrane lawns from secretory cells exhibiting secretory granules filled with neuropeptide Y-green fluorescent protein (NPY-GFP). The sample is prepared by subjecting NPY-GFP-expressing cells to a brief ultrasound pulse, leaving behind a basal, flat plasma membrane with fluorescent attached secretory organelles. These sheets can then be incubated in defined solutions with the benefit that complete solution changes can be achieved in less than 1 min. Individual secretory granules are monitored in the docked state and during exocytosis by video microscopy.
Lang
Thorsten
T
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
D000818:Animals; D020013:Calcium Signaling; D002462:Cell Membrane / Q000378:metabolism*; D002474:Cell-Free System; D005089:Exocytosis; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D018715:Microscopy, Video; D009474:Neurons / Q000378:metabolism*; D009478:Neuropeptide Y / Q000235:genetics / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism*; D014162:Transfection; D014465:Ultrasonics
D009478:Neuropeptide Y; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins