Imaging Constitutive Exocytosis with Total Internal Reflection Fluorescence Microscopy

doi:10.1083/jcb.149.1.23

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© The Rockefeller University Press, 0021-9525/2000/4/23/ $5.00
The Journal of Cell Biology, Volume 149, Number 1, April 3, 2000 23-32

Original Article

Imaging Constitutive Exocytosis with Total Internal Reflection Fluorescence Microscopy

Jan Schmoranzer^a, Mark Goulian^a, Dan Axelrod^b, and Sanford M. Simon^a
^a Laboratory of Cellular Biophysics, The Rockefeller University, New York, New York 10021
^b Department of Biophysics, University of Michigan, Ann Arbor, Michigan 48109-1055

Correspondence to: Sanford M. Simon, Laboratory of Cellular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10021. Tel:(212) 327-8130 Fax:(212) 327-8022 E-mail:simon{at}rockvax.rockefeller.edu.

Total internal reflection fluorescence microscopy has been appliedto image the final stage of constitutive exocytosis, which isthe fusion of single post-Golgi carriers with the plasma membrane.The use of a membrane protein tagged with green fluorescentprotein allowed the kinetics of fusion to be followed with atime resolution of 30 frames/s. Quantitative analysis allowedcarriers undergoing fusion to be easily distinguished from carriersmoving perpendicularly to the plasma membrane. The flatteningof the carriers into the plasma membrane is seen as a simultaneousrise in the total, peak, and width of the fluorescence intensity.The duration of this flattening process depends on the sizeof the carriers, distinguishing small spherical from large tubularcarriers. The spread of the membrane protein into the plasmamembrane upon fusion is diffusive. Mapping many fusion sitesof a single cell reveals that there are no preferred sites forconstitutive exocytosis in this system.

Key Words: membrane fusion, constitutive secretion, green fluorescent protein,vesicle, evanescent wave

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