The color of lactotroph secretory granules stained with FM1-43 depends upon dye concentration
Joseph M. Johnson 1 and William J. Betz 1*
1 Univ. of Colorado Medical School
* To whom correspondence should be addressed. E-mail: bill.betz{at}uchsc.edu.
Submitted on May 10, 2007
Revised on June 11, 2007
Accepted on 15 November 2007
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Abstract |
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When pituitary lactotroph granules undergo exocytosis in the presence of FM1-43, their cores absorb dye and fluoresce brightly. We report that different granules fluoresce with different colors, despite being stained with a single fluorescent dye; emission spectra from individual granules show up to a 25 nm difference between the greenest and reddest granules. We found a correlation between granule color and average fluorescence intensity, suggesting that granule color depends upon dye concentration. We confirmed this in two ways: by increasing FM dye concentration in granules, which red-shifted granule color, and by partially photobleaching the FM dye in granules, which green-shifted granule color. Increasing stimulation intensity (by increasing KCl concentration) increased the proportion of red granules, indicating that granules exocytosing during intense stimulation bound more dye. This, perhaps, reflects differences in granule core maturation and condensation, in which mature granules with condensed cores bind more FM dye, but require more intense stimulation to be released. Concentration dependent color shifts of FM dyes may be useful for monitoring aggregation processes occurring on a size scale smaller than the optical limit.
Key Words:
endocytosis, exocytosis, fluorescence, neuroendocrine, red-shift
