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Single granule pH cycling in antigen-induced mast cell secretion

Rebecca M. Williams and Watt W. Webb

Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA

f2E.mov

Series showing a single granule fusing repeatedly while maintaining its shape and approximate location. The first release event, at ~150 sec, is indicated by the entry of extracellular fluorescein (red) and the plasma-membrane-residing PM (green) into the granule and granule membrane respectively. The fluorescein fluorescence in the granule subsequently quenches due to granule acidification, reappears suddenly during another fusion event (~230 sec) and then re-quenches upon re-acidification again. As is typical in Ag-indiced stimulation, this cycle repeats itself several times within the relatively stationary granule.

f2F.mov

Series showing permanent or "full" fusion. The release event (~140 sec) is indicated by the entry of the aqueous and membrane dyes (red and green) into the granule and granule membrane respectively. Over the next couple minutes the granule membrane collapses into the plasma membrane until the two become indistinguishable.

f3wAg (with Ag)

Series showing the slow blinking associated with LS-loaded granules undergoing recycling. Granule acidity is visually assessed by the red/green ratio. Granule release events, indicated by granule alkalization and dye loss, are often followed by re-acidification and dye re-accumulation in the same granules.

f3woAg (without Ag)

Representative series showing LS-loaded granules in unstimulated cells. Both the color and intensity of the fluorescence within the granules remains relatively constant.

f4wAg (with Ag)

Series showing flashing in AO-labeled cells undergoing exocytosis. When a granule experiences a pH jump, the majority of trapped AO molecules within the granule shed their protons (pKa ~4.9) and become permeable to the granule membrane. Flashes in the green channel result from the rapid redistribution of previously quenched and red-shifted AO from the granule into the cytosol.

f4woAg (without Ag)

Representative series showing AO-labeled cells without the addition of exogenous Ag. The AO intensity and distribution remains relatively constant.

f7B

Series of AO- and PM-labeled cells showing granule-granule coalescence and subsequent collapse. The event is indicated by an alkalization of one of the granules ~170 sec (AO flash, green), structural coalescence by 180 sec and a collapse of the granule complex from 180-210 sec.

f7C

Series of images in a PM-labeled cell showing a tubular lipid structure that develops coincident with the granule-granule fusion to collapse event.

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