Imaging Constitutive Exocytosis with Total Internal Reflection Fluorescence Microscopy

Total internal reflection fluorescence microscopy has been applied to image the final stage of constitutive exocytosis, which is the fusion of single post-Golgi carriers with the plasma membrane. The use of a membrane protein tagged with green fluorescent protein allowed the kinetics of fusion to be...

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Bibliographic Details
Published in:The Journal of cell biology 2000-04, Vol.149 (1), p.23-31
Main Authors: Schmoranzer, Jan, Goulian, Mark, Axelrod, Dan, Simon, Sanford M.
Format: Article
Language:English
Subjects:
Animals
Bulk carriers
Cell Membrane - metabolism
Cell membranes
Cellular biology
Chromaffin cells
COS Cells
Cytoplasmic Granules - metabolism
Diffusion
Exocytosis
Fluorescence
Golgi Apparatus - metabolism
Kinetics
Mast cells
Membrane Fusion
Membrane Glycoproteins
Membrane Proteins - chemistry
Membrane Proteins - genetics
Membrane Proteins - metabolism
Membranes
Microscopy
Microscopy, Fluorescence - methods
Mutation - genetics
Neurons
Original
P branes
Proteins
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Secretion
Temperature
Transfection
Viral Envelope Proteins - chemistry
Viral Envelope Proteins - genetics
Viral Envelope Proteins - metabolism
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Summary:Total internal reflection fluorescence microscopy has been applied to image the final stage of constitutive exocytosis, which is the fusion of single post-Golgi carriers with the plasma membrane. The use of a membrane protein tagged with green fluorescent protein allowed the kinetics of fusion to be followed with a time resolution of 30 frames/s. Quantitative analysis allowed carriers undergoing fusion to be easily distinguished from carriers moving perpendicularly to the plasma membrane. The flattening of the carriers into the plasma membrane is seen as a simultaneous rise in the total, peak, and width of the fluorescence intensity. The duration of this flattening process depends on the size of the carriers, distinguishing small spherical from large tubular carriers. The spread of the membrane protein into the plasma membrane upon fusion is diffusive. Mapping many fusion sites of a single cell reveals that there are no preferred sites for constitutive exocytosis in this system.
ISSN:0021-9525
1540-8140
DOI:10.1083/jcb.149.1.23