Quantification of exocytosis kinetics by DIC image analysis of cortical lawns.
Cortical lawns prepared from sea urchin eggs have offered a robust in vitro system for study of regulated exocytosis and membrane fusion events since their introduction by Vacquier almost 40 years ago (Vacquier in Dev Biol 43:62-74, 1975). Lawns have been imaged by phase contrast, darkfield, differential interference contrast, and electron microscopy. Quantification of exocytosis kinetics has been achieved primarily by light scattering assays. We present simple differential interference contrast image analysis procedures for quantifying the kinetics and extent of exocytosis in cortical lawns using an open vessel that allows rapid solvent equilibration and modification. These preparations maintain the architecture of the original cortices, allow for cytological and immunocytochemical analyses, and permit quantification of variation within and between lawns. When combined, these methods can shed light on factors controlling the rate of secretion in a spatially relevant cellular context. We additionally provide a subroutine for IGOR Pro® that converts raw data from line scans of cortical lawns into kinetic profiles of exocytosis. Rapid image acquisition reveals spatial variations in time of initiation of individual granule fusion events with the plasma membrane not previously reported.
Program in Neuroscience, Amherst College, Amherst, MA 01002 USA.
James
J
Mooney
Program in Neuroscience, Amherst College, Amherst, MA 01002 USA.
Saumitra
S
Thakur
Department of Biology, Amherst College, Amherst, MA 01002 USA.
Peter
P
Kahng
Department of Biology, Amherst College, Amherst, MA 01002 USA ; Program in Neuroscience, Amherst College, Amherst, MA 01002 USA.
Josef G
JG
Trapani
Department of Biology, Amherst College, Amherst, MA 01002 USA ; Program in Neuroscience, Amherst College, Amherst, MA 01002 USA.
Dominic
D
Poccia
Cortical granules; DIC; Exocytosis; Membrane fusion; Sea urchin