The pH cycling of individual granules in secreting (serotonin-loaded) mast cells is quantitatively examined using multicolor multiphoton fluorescence microscopy. A typical exocytosis event consists of maximal calcium rise at time zero, granule alkalization a few seconds later and, finally, complete contents release at a fraction of a second after alkalization. Membrane fusion is either transient, as indicated by subsequent granule reacidification, or ‘full’, as indicated by a granule disappearance with a collapse of its membrane into the plasma membrane. The relative frequency of these two coexisting behaviors (the ‘kiss-to-collapse’ ratio) is approximately 2:1. A typical transiently fusing granule experiences multiple alkalization/acidification cycles after addition of exogenous antigen. Between recycling granules, coalescence events are frequent, with 80% resulting in a collapse of the formed granule complex to the plasma membrane. The full dynamics of secretion encompass a complex combination of these granule activities.

Albillos
A.
,
Dernick
G.
,
Horstmann
H.
,
Almers
W.
,
Alvarez de Toledo
G.
,
Lindau
M.
(
1997
).
The exocytotic event in chromaffin cells revealed by patch amperometry.
Nature
389
,
509
512
Alvarez de Toledo
G.
,
Fernandez-Chacon
R.
,
Fernandez
J. M.
(
1993
).
Release of secretory products during transient vesicle fusion.
Nature
363
,
554
558
Artalejo
C. R.
,
Henley
J. R.
,
McNiven
M. A.
,
Palfrey
H. C.
(
1995
).
Rapid endocytosis coupled to exocytosis in adrenal chromaffin cells involves Ca2+, GTP, and dynamin but not clathrin.
Proc. Natl. Acad. Sci.USA
92
,
8328
8332
Aspinwall
C. A.
,
Brooks
S. A.
,
Kennedy
R. T.
,
Lakey
J. R. T.
(
1997
).
Effects of intravesicular H+and Zn2+on insulin secretion in pancreatic beta cells.
J. Biol. Chem
272
,
31308
31314
Barasch
J.
,
Gershon
M. D.
,
Nunez
E. A.
,
Tamir
H.
,
Al-Awqati
Q.
(
1988
).
Thyrotropin induces the acidification of the secretory granules of parafollicular cells by increasing the chloride conductance of the granular membrane.
J. Cell Biol
107
,
2137
2147
Barasch
J.
,
Kiss
B.
,
Prince
A.
,
Saiman
L.
,
Gruenert
D.
,
Al-Awqati
Q.
(
1991
).
Defective acidification of intracellular organelles in cystic fibrosis.
Nature
352
,
70
73
Betz
W. J.
,
Bewick
G. S.
(
1992
).
Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction.
Science
255
,
200
203
Betz
W. J.
,
Wu
L.-G.
) (
1995
).
Kinetics of synaptic-vesicle recycling.
Curr. Biol
5
,
1098
1101
Bonifacino
J. S.
,
Yuan
L.
,
Sandoval
I. V.
(
1989
).
Internalization and recycling to serotonin-containing granules of the 80K intergral membrane protein exposed on the surface of secreting rat basophilic leukaemia cells.
J. Cell Sci
92
,
701
712
Cao
Y.
,
Mager
S.
,
Lester
H. A.
(
1997
).
H+permeation and pH regulation at a mammalian serotonin transporter.
J. Neurosci
17
,
2257
2266
Ceccarelli
B.
,
Hurlbut
W. P.
,
Mauro
A.
(
1973
).
Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction.
J. Cell Biol
57
,
499
524
Chanturiya
A.
,
Chernomordik
L. V.
,
Zimmerberg
J.
(
1997
).
Flickering fusion pores comparable with initial exocytotic pores occur in protein-free phospholipid bilayers.
Proc. Natl. Acad. Sci.USA
94
,
14423
14428
Chow
R. H.
,
von Ruden
L.
,
Neher
E.
(
1992
).
Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells.
Nature
356
,
60
63
Curran
M. J.
,
Brodwick
M. S.
(
1991
).
Ionic control of the size of the vesicle matrix of beige mouse mast cells.
J. Gen. Physiol
98
,
771
790
Eiden
L. E.
,
Iacangelo
A.
,
Hsu
C.-M.
,
Hotchkiss
A. J.
,
Bader
M.-F.
,
Aunis
D.
(
1987
).
Chromogranin A synthesis and secretion in chromaffin cells.
J. Neurochem
49
,
65
74
Fernandez
J. M.
,
Villalon
M.
,
Verdugo
P.
(
1991
).
Reversible condensation of mast cell secretory products in vitro.
Biophys. J
59
,
1022
1027
Field
K. A.
,
Holowka
D.
,
Baird
B.
(
1997
).
Compartmentalized activation of the high affinity immunoglobin E receptor within membrane domains.
J. Biol. Chem
272
,
4276
4280
Gilon
P.
,
Shepherd
R. M.
,
Henquin
J.-C.
) (
1993
).
Oscillations of secretion driven by oscillations of cytoplasmic Ca2+as evidenced in single pancreatic islets.
J. Biol. Chem
268
,
22265
22268
Guo
Z.
,
Turner
C.
,
Castle
D.
(
1998
).
Relocation of the t-SNARE SNAP-23 from lamellipodia-like surface projections regulates compound exocytosis in mast cells.
Cell
94
,
537
548
Han
W.
,
Li
D.
,
Stout
A. K.
,
Takimoto
K.
,
Levitan
E. S.
(
1999
).
Ca2+-induced deprotonation of peptide hormones inside secretory vesicles in preparation for release.
J. Neurosci
19
,
900
905
Heuser
J. E.
,
Reese
T. S.
(
1973
).
Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction.
J. Cell Biol
57
,
315
344
Hide
M.
,
Beaven
M. A.
(
1991
).
Calcium influx in a rat mast cell (RBL-2H3) line.
J. Biol. Chem
266
,
15221
15229
Jahn
R.
,
Sudhof
T. C.
(
1999
).
Membrane fusion and exocytosis.
Annu. Rev. Biochem
68
,
863
911
Kaether
C.
,
Salm
T.
,
Glombik
M.
,
Almers
W.
,
Gerdes
H.-H.
) (
1997
).
Targeting of green fluorescent protein to neuroendocrine secretory granules: a new tool for real time studies of regulated protein secretion.
Eur. J. Cell Biol
74
,
133
142
Kim
T. D.
,
Eddlestone
G. T.
,
Mahmoud
S. F.
,
Kuchtey
J.
,
Fewtrell
C.
(
1997
).
Correlating Ca2+responses and secretion in individual RBL-2H3 mucosal mast cells.
J. Biol. Chem
272
,
31225
31229
Klingauf
J.
,
Kavali
E. T.
,
Tsien
R. W.
(
1998
).
Kinetics and regulation of fast endocytosis at hippocampal synapses.
Nature
394
,
581
585
Koenig
J. H.
,
Ikeda
K.
(
1989
).
Disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval.
J. Neurosci
9
,
3844
3860
Koenig
J. H.
,
Ikeda
K.
(
1996
).
Synaptic vesicles have two distinct recycling pathways.
J. Cell Biol
135
,
797
808
Kuchtey
J.
,
Fewtrell
C.
(
1996
).
Subcloning the RBL-2H3 mucosal mast cell line reduces Ca2+response heterogeneity at the single cell level.
J. Cell Physiol
166
,
643
652
Lee
C. J.
,
Dayanithi
G.
,
Nordmann
J. J.
,
Lemos
J. R.
(
1992
).
Possible role during exocytosis of a Ca2+-activated channel in neurohypophysial granules.
Neuron
8
,
335
342
Lollike
K.
,
Borregaard
N.
,
Lindau
M.
(
1998
).
Capacitance flickers and pseudoflickers of small granules, measured in the cell-attached configuration.
Biophys. J
75
,
53
59
Maiti
S.
,
Shear
J. B.
,
Williams
R. M.
,
Zipfel
W. R.
,
Webb
W. W.
(
1997
).
Measuring serotonin distribution in live cells with three-photon excitation.
Science
275
,
530
532
Marszalek
P. E.
,
Farrell
B.
,
Verdugo
P.
,
Fernandez
J. M.
(
1997
).
Kinetics of release of serotonin from isolated secretory granules. II. Ion exchange determines the diffusivity of serotonin.
Biophys. J
73
,
1169
1183
Miesenbock
G.
,
Angelis
D. A. D.
,
Rothman
J. A.
(
1998
).
Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.
Nature
394
,
192
195
Miesenbock
G.
,
Rothman
J. E.
(
1997
).
Patterns of synaptic activity in neural networks recorded by light emission from synaptolucins.
Proc. Natl. Acad. Sci.USA
94
,
3402
3407
Murthy
V. N.
,
Stevens
C. F.
(
1998
).
Synaptic vesicles retain their identity through the endocytotic cycle.
Nature
392
,
497
501
Nanavati
C.
,
Fernandez
J. M.
(
1993
).
The secretory granule matrix: a fast-acting smart polymer.
Science
259
,
963
965
Oliver
J. M.
,
Seagrave
J.
,
Stump
R. F.
,
Pfeiffer
J. R.
,
Deanin
G. G.
(
1988
).
Signal transduction and cellular response in RBL-2H3 mast cells.
Prog. Allergy
42
,
185
245
Palmgren
M. G.
(
1991
).
Acridine orange as a probe for measuring pH gradients across membranes: mechanism and limitations.
Anal. Biochem
192
,
316
321
Parsons
T. D.
,
Coorssen
J. R.
,
Horstmann
H.
,
Almers
W.
(
1995
).
Docked granules, the exocytotic burst, and the need for ATP hydrolysis in endocrine cells.
Neuron
15
,
1085
1096
Ryan
T. A.
,
Reuter
H.
,
Smith
S. J.
(
1997
).
Optical detection of a quantal presynaptic membrane turnover.
Nature
388
,
478
482
Ryan
T. A.
,
Reuter
H.
,
Wendland
B.
,
Schweizer
F.
,
Tsien
R. W.
,
Smith
S. J.
(
1993
).
The kinetics of synaptic vesicle recycling measured at single presynaptic boutons.
Neuron
11
,
713
724
Sabban
E. L.
,
Schwartz
J.
,
McMahon
A.
(
1990
).
Effect of compounds which disrupt proton gradients on secretion of neurosecretory proteins from PC12 pheochromocytoma cells.
Neurosci
38
,
561
570
Sheets
E. D.
,
Holowka
D.
,
Baird
B.
(
1999
).
Critical role for cholesterol in lyn-mediated tyrosine phosphorylation of FcRI and their association with detergent-resistant domains.
J. Cell Biol
145
,
877
887
Shupliakov
O.
,
Low
P.
,
Grabs
D.
,
Gad
H.
,
Chen
H.
,
David
C.
,
Takei
K.
,
de Camilli
P.
,
Brodin
L.
(
1997
).
Synaptic vesicle endocytosis impaired by disruption of dynamin-SH3 domain interactions.
Science
276
,
259
263
Smith
C.
,
Moser
T.
,
Xu
T.
,
Neher
E.
(
1998
).
Cytosolic Ca2+acts by two separate pathways to modulate the supply of release-competent vesicles in chromaffin cells.
Neuron
20
,
1243
1253
Smith
C.
,
Neher
E.
(
1997
).
Multiple forms of endocytosis in bovine adrenal chromaffin cells.
J. Cell Biol
139
,
885
894
Smith
J.
,
Thompson
N.
,
Thompson
J.
,
Armstrong
J.
,
Hayes
B.
,
Crofts
A.
,
Squire
J.
,
Teahan
C.
,
Upton
L.
,
Solari
R.
(
1997
).
Rat basophilic leukaemia (RBL) cells overexpressing Rab3a have a reversible block in antigen-stimulated exocytosis.
Biochem. J
323
,
321
328
Smith
P. A.
,
Duchen
M. R.
,
Ashcroft
F. M.
(
1995
).
A fluorimetric and amperometric study of calcium and secretion in isolated mouse pancreatic-cells.
Pflugers Arch
430
,
808
818
Spruce
A. E.
,
Breckenridge
L. J.
,
Lee
A. K.
(
1990
).
Properties of the fusion pore that forms during exocytosis of a mast cell secretory vesicle.
Neuron
4
,
643
654
Steyer
J. A.
,
Almers
W.
(
1999
).
Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy.
Biophys. J
76
,
2262
2271
Steyer
J. A.
,
Horstmann
H.
,
Almers
W.
(
1997
).
Transport, docking and exocytosis of single secretory granules in live chromaffin cells.
Nature
388
,
474
478
Takahashi
N.
,
Kadowaki
T.
,
Yazaki
Y.
,
Ellis-Davies
G. C. R.
,
Miyashita
Y.
,
Kasai
H.
(
1999
).
Post-priming actions of ATP on Ca2+-dependent exocytosis in pancreatic beta cells.
Proc. Natl. Acad. Sci. USA
96
,
760
765
Takahashi
N.
,
Kadowaki
T.
,
Yazaki
Y.
,
Miyashita
Y.
,
Kasai
H.
(
1997
).
Multiple exocytotic pathways in pancreaticcells.
J. Cell Biol
138
,
55
64
Torri-Tarelli
F.
,
Bossi
M.
,
Fesce
R.
,
Greengard
P.
,
Valtorta
F.
(
1992
).
Synapsin 1 partially dissociates from synaptic vesicles during exocytosis induced by electrical stimulation.
Neuron
9
,
1143
1153
Torri-Tarelli
F.
,
Villa
A.
,
Valtorta
F.
,
de Camilli
P.
,
Greengard
P.
,
Ceccarelli
B.
(
1990
).
Redistribution of synaptophysin and synapsin 1 during-latrotoxin-induced release of neurotransmitter at the neuromuscular junction.
J. Cell Biol
110
,
449
459
Valtorta
F.
,
Jahn
R.
,
Fesce
R.
,
Greengard
P.
,
Ceccarelli
B.
(
1988
).
Synaptophysin (p38) at the frog neuromuscular junction: its incorporation into the axolemma and recycling after intense quantal secretion.
J. Cell Biol
107
,
2717
2727
Weintraub
W. H.
,
Cleveland-Wolfe
P.
,
Fewtrell
C.
(
1994
).
Paracrine Ca2+ signaling in vitro: serotonin-mediated cell-cell communication in mast cell/smooth muscle cultures.
J. Cell. Physiol
160
,
389
399
Williams
R. M.
,
Shear
J. B.
,
Zipfel
W. R.
,
Maiti
S.
,
Webb
W. W.
(
1999
).
Mucosal mast cell secretion processes imaged using three-photon microscopy of 5-hydroxytryptamine autofluorescence.
Biophys. J
76
,
1835
1846
Xu
K.
,
Williams
R. M.
,
Holowka
D.
,
Baird
B.
(
1998
).
Stimulated release of fluorescently labeled IgE fragments that efficiently accumulate in secretory granules after endocytosis in RBL-2H3 mast cells.
J. Cell Sci
111
,
2385
2396
Zimmerberg
J.
,
Blumenthal
R.
,
Sarkar
D. P.
,
Curran
M.
,
Morris
S. J.
(
1994
).
Restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutin during cell fusion.
J. Cell Biol
127
,
1885
1894
This content is only available via PDF.

Supplementary information