articles
Sequential-replenishment mechanism
of exocytosis in pancreatic acini
Tomomi Nemoto*†‡, Ryoichi Kimura*†, Koichi Ito†, Akira Tachikawa†‡, Yasushi Miyashita†, Masamitsu Iino†‡
and Haruo Kasai*‡§
*Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan
†University of Tokyo School of Medicine, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
‡CREST, Japan Science and Technology Corporation, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
§e-mail: [email protected]
Here we report exocytosis of zymogen granules, as examined by multiphoton excitation imaging in intact pancreatic
acini. Cholecystokinin induces Ca2+ oscillations that trigger exocytosis when the cytosolic Ca2+ concentration
exceeds 1 µM. Zymogen granules fused with the plasma membrane maintain their Ω-shaped profile for an average
of 220 s and serve as targets for sequential fusion of granules that are located within deeper layers of the cell. This
secondary exocytosis occurrs as rapidly as the primary exocytosis and accounts for most exocytotic events.
Granule–granule fusion does not seem to precede primary exocytosis, indicating that secondary fusion events may
require a plasma-membrane factor. This sequential-replenishment mechanism of exocytosis allows the cell to take
advantage of a large supply of fusion-ready granules without needing to transport them to the plasma membrane.
he mechanisms of intracellular membrane transport are conserved from yeast to mammals1. Because of their highly polarized subcellular organization and large, distinctive secretory
vesicles, known as zymogen granules, mammalian exocrine acinar
cells have been studied extensively as a model system with which to
characterize the biogenesis, transport and regulated exocytosis of
secretory vesicles2. Early electron-microscopic observation of these
T
a
b
e
SRB
[Ca2+] i (µM)
Fura-2
cells established that the release of the contents of secretory vesicles
is mediated by fusion of the granule membrane with the luminal
plasma membrane2, a process termed exocytosis. In the pancreas,
exocytotic secretion is activated by cholecystokinin (CCK) or acetylcholine as a result of an increase in the cytosolic concentration of
Ca2+ ([Ca2+]i; ref. 3). Ca2+-dependent exocytosis of zymogen granules is mediated by SNARE (soluble N-ethylmaleimide-sensitive
10 µm
100 s
0.2
0.1
Fluorescence intensity
(arbitrary units)
f
CCK 5 pM
200
0
Lifetime
c
[Ca2+]i
(µM)
[Ca2+]i
0.1
0.3
d
1
SRB
3
10
10 µm
0
4
8
Figure 1 Exocytosis and [Ca2+]i at low CCK concentration. a, b, Fluorescence
images of an acinus stained with fura-2 (a) and SRB (b). The preparation was preloaded with fura-2-AM and then immersed for 20 min in a solution containing 0.5
mM SRB. Scale bar represents 10 µm. c, d, Images of [Ca2+]i (c) and SRB (d) fluorescence during stimulation of acini with 5 pM CCK. Images were obtained at the
indicated times from the area marked with a dashed green line in a. [Ca2+]i is represented by the pseudocolour scale on the right. Arrows indicate the Ω-shaped profile
20
254 Time (s)
caused by exocytosis. e, f, Time courses of average [Ca2+]i within the red square in
a and average SRB fluorescence intensity within the blue circle in b during CCK
stimulation. Vertical lines indicate the times at which the images in c and d were
acquired. The dotted line marked ‘lifetime’ represents the lifetime of the Ω-shaped
profiles, measured as the time between the onset of their appearance and the time
of their complete flattening.
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articles
d
Fura-2FF
[Ca2+]i (µM)
Basal
Apical
10 µm
b Texas Red–dextran
10 µm
c
100 pM CCK
15
e
10
Fluorescence intensity
(arbitrary units × 102)
a
5
A
B
1
0
9
100 s
5
10 µm
10 µm
–100
0
42
106
192
282
500
700
Time (s)
Figure 2 Exocytosis and [Ca2+]i at a high CCK concentration. a, b,
Fluorescence images of an acinus stained with fura-2FF (a) and Texas Red–dextran
(b). The preparation was preloaded with fura-2FF-AM and then immersed for 20 min
in a solution containing 0.1 mM Texas Red–dextran. c, Images of Texas Red fluorescence obtained at the indicated times during stimulation with 100 pM CCK. d, Time
courses of average [Ca2+]i in the apical red square (A) and in the basal blue square
(B) in a during CCK stimulation. Vertical lines indicate the times at which the images
shown in c were acquired. e, Time course of average Texas Red fluorescence in
the apical area surrounded by the blue line in b. Vertical lines indicate the onset of
each Ca2+ spike.
factor (NSF) attachment-protein receptor) proteins in the plasma
membrane and granule membrane4, as is also the case for exocytosis of synaptic vesicles in neurons1. A specific pool of vesicles,
known as fusion-ready vesicles, is thought to be especially susceptible to Ca2+-regulated exocytosis5,6. However, the morphological status of such fusion-ready vesicles and the mechanism responsible
for their replenishment have remained unclear.
We have visualized the exocytosis of individual zymogen granules, using multiphoton excitation imaging in intact pancreatic
acini. In the cells of this tissue, the luminal plasma membrane is
adjacent to several layers of zymogen granules, and sequential exocytosis of granules in deeper layers has been proposed on the basis
of electron-microscopic analysis2,7,8. Here we demonstrate fusion of
zymogen granules closest to the plasma membrane during CCK
stimulation that increased [Ca2+]i to >1 µM, a result that is consistent with those of previous studies9,10. We also observed that the Ωshaped profile of each fused granule was relatively stable, and that
zymogen granules present within deeper layers of the cell became
fusion-ready as soon as those in the adjacent peripheral layer had
fused with the plasma membrane. This secondary exocytosis constitutes the predominant form of exocytosis in these exocrine cells,
and it may also have a role in secretion in other cell types.
granules were readily apparent in 3D reconstructed images of acini
immersed in a solution containing the polar fluid-phase tracer
sulphorhodamine-B (SRB; see Supplementary Information, Fig.
S1c). Thus, the enhanced depth penetration of two-photon excitation imaging enabled us, for the first time, to visualize sequential
processes of zymogen-granule exocytosis in exocrine glands.
Visualization of Ca2+-dependent exocytosis. We carried out simultaneous two-photon excitation imaging of [Ca2+]i and exocytosis
(Figs 1, 2), taking advantage of the fact that both fura-2 (or fura2FF) and SRB (or Texas Red–dextran) are excited at a wavelength of
830 nm. We separated the fluorescence from the two dyes with the
use of dichroic mirrors. The formation of Ω-shaped exocytotic
profiles was rarely induced at a low concentration (5 pM) of CCK
(Fig. 1), which triggers the generation of global submicromolar
Ca2+ spikes12,13; [Ca2+]i did not reach 1 µM in any region of the cell
(Fig. 1c). In Fig. 1d, for example, exocytosis of only one granule can
be detected. Exocytosis was frequently induced, however, at a higher concentration of CCK (100 pM; Fig. 2), which also induced
micromolar Ca2+ oscillations (Fig. 2d). Exocytosis was induced
selectively in the luminal region of the cell (Fig. 2c), and occurred
when CCK-induced Ca2+ oscillations increased [Ca2+]i to >1 µM
(Fig. 2d, e).
Exocytotic profiles exhibited the same bell-shaped dependence
on CCK concentration as that revealed by measurement of amylase
secretion14. Thus, the extent of exocytosis at high concentrations
(>1 nM) of CCK was less than that induced by lower CCK concentrations (Fig. 3a). At high concentrations of CCK, the frequency
with which Ca2+ spikes occurred was also less than the maximal
value (Fig. 3b), although the peak amplitude of [Ca2+]i was similar
to that at lower CCK concentrations (Fig. 3c).
Sizes and lifetimes of Ω-shaped profiles. We investigated whether
the Ω-shaped filling of fluid-phase tracers represents exocytosis of
individual zymogen granules, as well as whether granule–granule
fusion occurs before exocytosis. For these experiments, we relied on
the fact that zymogen granules contain materials that emit ultraviolet fluorescence when excited with a femtosecond-pulse laser at
720 nm (Fig. 4a). The locations and sizes of the spots of ultraviolet
fluorescence corresponded exactly to those of zymogen granules, as
Results
Two-photon excitation imaging compared with confocal imaging.
Two-photon excitation imaging exhibited greater depth penetration
than did confocal imaging in intact pancreatic acini. As shown by the
representative images scanned along the x- and z-axes of an acinar
preparation loaded with the acetoxymethyl ester of Oregon green
488 BAPTA-1, the lumen of acini surrounded by acinar cells was
apparent in two-photon images (see Supplementary Information,
Fig. S1a), whereas such luminal structures were not usually
detectable by confocal microscopy because of intense light scattering
by zymogen granules adjacent to the lumen11 (see Supplementary
Information, Fig. S1b). In addition, photobleaching of Oregon green
was more pronounced in confocal images than in two-photon excitation images (data not shown). Moreover, the Ω-profiles of fused
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b
Secondary
1
Primary
0.5
0
a
c Lucifer yellow
0.3
0.2
CCK
0.1
0
102
104
102
1
CCK (pM)
104
CCK (pM)
c
No. of observations
d
10
1
0.1
Difference
40
10 µm
30
d
20
10
0
1
102
CCK (pM)
104
0
120
240
360
480
Lifetime (s)
5 µm
Ultraviolet autofluorescence
2
1 µm
1
0
0
2
4
6
e
SRB
Fluorescence intensity
(arbitrary units × 103)
1
Peak [Ca2+]i (µM)
b Ultraviolet
Fluorescence intensity
(arbitrary units × 102)
No. of Ω-shaped profiles
(events per cell per min)
Total
No. of Ca2+ transients
(events per cell per min)
a
3
2
1 µm
1
0
0
2
Distance (µm)
Ultraviolet autofluorescence
15
10
5
0
0
0.4
1
Diameter (µm)
revealed in transmission images (data not shown). Ultraviolet autofluorescence was induced by three-photon excitation of the granular contents, as the fluorescence intensity increased by a factor of
eight when the power of laser excitation was doubled, as has previously been shown to be the case in mast cells 15. The molecular
entity responsible for the ultraviolet autofluorescence of the granule contents of acinar cells remains unknown.
The release of the ultraviolet-autofluorescent contents of individual zymogen granules induced by CCK (Fig. 4b) was always
accompanied by Ω-shaped filling of the fused granule with a fluidphase marker (Fig. 4c). The diameter of zymogen granules was estimated as 0.5–1.5 µm on the basis of the three-photon excitation
images (Fig. 4d, f), which was consistent with the size of the structures that were rapidly filled with fluid-phase markers (Fig. 4e, g).
Thus, each Ω-shaped profile represents exocytosis of an individual
zymogen granule.
These data also indicate that granule–granule fusion does not
precede exocytosis. If fusion between zymogen granules had taken
place before exocytosis, we should have detected filling with fluidphase tracers of structures larger than those that were actually
observed. An apparent granule–granule fusion seemed to precede
sequential exocytosis only once (Fig. 5b) out of 406 fusion events (1
out of 72 fusion events at 37 °C), but such an exceptional observation can be explained by the occurrence of 2 sequential exocytoses
within a sampling interval of 2 s. Zymogen granules therefore fused
with the membranes of other granules only after the latter had
become continuous with the luminal plasma membrane. It is still
possible that exocytotic granules might fuse each other under certain experimental conditions, as this occurs in vitro4,16. Our observations, however, indicate that such fusion is rarely induced by
stimulation with physiological concentrations of CCK in intact
mice pancreatic acinar cells, possibly because it requires larger
g
No. of granules
f
No. of granules
Figure 3 CCK-dependence and lifetime of Ω-shaped profiles. a, Dependence
of primary and secondary exocytotic events on the concentration of CCK. The number of Ω-shaped profiles revealed by filling with SRB was counted during a single
episode of Ca2+ spiking. b, c, Dependence of the frequency of Ca2+ spikes (b) and
of peak [Ca2+]i, (c) on the concentration of CCK. Data in a–c are means ± s.e.m.
from 21–33 cells. d, Lifetime histogram for Ω-shaped profiles formed by zymogen
granules. Only Ω-shaped profiles that did not give rise to subsequent secondary
exocytosis were counted.
4
6
Distance (µm)
1.6
SRB
50
0
0
0.4
1
1.6
Diameter (µm)
Figure 4 Diameters of zymogen granules and SRB-filled structures. a,
Ultraviolet autofluorescence of acini (orange) and Lucifer yellow fluorescence (blue)
excited by a femtosecond-pulse laser at 720 nm. The preparation was immersed in
a solution containing 1 mM Lucifer yellow. Intense spots of ultraviolet fluorescence
correspond well with the positions of zymogen granules adjacent to the acinar
lumen (blue). b, c, Disappearance of ultraviolet autofluorescence (b) and appearance of Lucifer yellow fluorescence (c) for the same zymogen granule in response
to stimulation with 100 pM CCK. Middle images were obtained 30 s after the upper
images; lower panels show the difference between each pair. d, e, Measurement of
the diameters of a zymogen granule (as revealed by ultraviolet autofluorescence)
(d) and of a structure that rapidly filled with SRB during CCK stimulation of acini (e).
Diameters were determined as the peak width at half-maximal fluorescence
(arrows). Intensity profiles of granules (insets) were obtained along the green lines
crossing the centres. f, g, Distributions of the diameters of zymogen granules at
rest (f; mean ± s.d. is 0.93 ± 0.18 µm (n = 35)) and of structures that rapidly filled
with SRB during CCK stimulation (g; mean ± s.d. is 1.04 ± 0.18 µm (n = 131)).
increases in [Ca2+]i than those that are induced physiologically4,16.
The Ω-profiles of fused granules were relatively stable, with the
associated fluorescence of fluid-phase markers decaying only gradually. The manner in which the fluorescence decayed indicates that
the granule membrane may flatten out as it becomes part of the
luminal membrane (Fig. 1d); this was confirmed by the observed
widening of the lumen after massive exocytosis (Fig. 2c). We measured the lifetime of Ω-shaped profiles as the time between the onset
of their appearance and the time of their complete flattening (Fig.
1f). A lifetime histogram constructed from 247 events indicated
that the mean lifetime of the Ω-shaped profiles was 220 ± 178 s
(mean ± s.d.) at room temperature (Fig. 3d), and 172 ± 169 s at
37 °C (n = 72). As an alternative to complete incorporation into the
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a
2'
1 2
1 2
1 2
1
3
3
2 µm
0s
6s
1
8s
2 1
2 1
20 s
22 s
2'
2 µm
10 s
14 s
58 s
b
60 s
c
0s
2s
5 µm
2 µm
2 µm
d
e
20
First
First
Second
Second
Third
Third
No. of events
No. of events
20
10
0
10
0
0
30
Onset of exocytosis (s)
60
0
30
Latency (s)
60
Figure 5 Sequential exocytosis of zymogen granules. a, Examples of
sequential exocytosis induced by stimulation with 100 pM CCK. Four granules (1,
2, 3 and 2′) and three granules (1, 2 and 2′) are shown fusing sequentially at the
indicated times in the upper and lower sets of images, respectively. b, A rare
occurrence of the formation of a structure consisting of two granules that
appeared within 2 s of CCK stimulation. c, Left panel, image showing sequential
exocytoses. Right panel, 3D reconstruction image obtained by x–y–z scanning of
the area indicated by a green square in the left panel. SRB was used as a fluidphase tracer in a–c. d, Histograms of the times between the onset of Ca2+ spiking
and exocytosis of the first, second and third granules in sequential exocytoses.
e, Latency histograms for exocytosis of the first, second and third granules in
sequential exocytoses.
luminal membrane, a small proportion of multiple Ω-shaped profiles appeared to undergo endocytosis en bloc, as indicated by the
observation that such multiple profiles were retained even after
washout of fluid-phase markers (data not shown). This is consistent with the large stepwise reductions in capacitance that have
been observed during the endocytotic phase10, as well as with the
detection by electron microscopy of large endocytotic vesicles in
salivary acinar cells during muscarinic stimulation17.
Sequential exocytosis of zymogen granules. During single episodes
of CCK-induced Ca2+ spiking (>30 s), granules that were present
within deeper layers of the cell frequently underwent exocytosis by
fusing with the Ω-shaped profiles of granules that had already
fused with the luminal membrane (Figs 2c and 5a, c). Moreover,
both primary and secondary exocytosis occurred with similar
delays. The latency of primary exocytosis was measured as the time
between the onset of Ca2+ spiking (>5 µM) and the formation of a
primary Ω-shaped profile. The latency histogram for primary exocytotic events revealed that the appearance of Ω-shaped profiles
peaked at 9 s and declined thereafter with a time constant of
13.7 ± 0.8 s (mean ± s.e.m., n = 129; Fig. 5d). We propose that this
latency predominantly represents the time required for exocytosis
after Ca2+ binding, given that [Ca2+]i increased rapidly (Fig. 2d) and
that Ca2+ binding should occur within 2 s at a [Ca2+]i of 5 µM,
assuming a rate constant of 105 M–1 s–1 for binding by the putative
Ca2+ sensor of zymogen granules10. Because the onset of exocytosis
for the second and third granules in a sequential series was delayed
by the latency of the preceding exocytotic events (Fig. 5d), we
defined the latency of secondary exocytosis as the time between the
exocytosis of a granule and that of a secondary granule that fuses to
its Ω-shaped profile. This latency therefore represents the time
required for secondary exocytosis after Ca2+ binding. The latencies
for exocytosis of the second and third granules in a series also
peaked at 9 s, and then declined with a time constant of 12.9 ± 1.5 s
(n = 51) and 13.7 ± 1.9 s (n = 26), respectively (Fig. 5e). No significant difference was apparent either among the mean latencies of
exocytosis for the first, second and third granules, or among the
latency distributions (Smirnov-test, P > 0.15), indicating that the
fusion competence of peripheral granules is similar to that of granules located within the deeper layers of the cell. It is possible that at
least some of the granules present in these deeper layers are already
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a
Sequential
exocytosis
Multigranular
exocytosis
b
v-SNARE
13 s
t-SNARE
13 s
Plasma membrane
Figure 6 Sequential-replenishment mechanism of exocytosis. a,
Granule–granule fusion and multigranular exocytosis do not occur during CCKinduced secretion in pancreatic acinar cells. b, Hypothetical molecular status of
granules in an acinar cell. At least some granules in the deeper layers of the cell
have an identical molecular status to that of the most peripheral granules. Granules
in the most peripheral layer constitute the readily releasable pool of granules by
virtue of their proximity to the luminal plasma membrane. Granules in the deeper
layers of the cell constitute the reserve pool, as their exocytosis requires the preceding exocytosis of more peripheral granules. A candidate factor derived from the
luminal plasma membrane and required for granule fusion is the t-SNARE protein
syntaxin-2. v- and t-SNARE, vesicle and target, respectively. Both primary and secondary exocytosis occur with a time constant of 13 s.
primed with ATP, given that a substantial proportion of zymogengranule exocytosis does not depend on cytosolic ATP10,18. A similar
conclusion has been reached for oocyte granules19, the yeast vacuole20, and synaptic-like microvesicles of PC12 cells21.
Sequential exocytosis involved up to six adjacent zymogen granules and often affected the deepest (4th to 6th) layer of mature
granules (Fig. 1b, c). Although the responsiveness of zymogen
granules varied greatly among acini, an average of 70% of total exocytosis was secondary in nature (Fig. 3a) and 85% of exocytosis
occurred in a sequential manner. The dominance of secondary exocytosis can readily be explained by the large number of granules
present within the deeper layers of the cell. Stimulation of acini
with CCK (100 pM) for 10 min induced exocytosis of 35% of
zymogen granules and involved an average of 3.5 layers of such
granules (data not shown).
Discussion
We have studied exocytosis in intact pancreatic acini by using multiphoton excitation imaging22. This approach allowed the visualization of the key aspects of CCK-induced exocytosis of zymogen
granules described in previous studies. Thus, exocytosis occurs
selectively at the luminal membrane, exhibits a bell-shaped
dependence on CCK concentration14, requires micromolar [Ca2+]i
(refs 9, 10), is synchronous with Ca2+ oscillations23,24, and proceeds
in a sequential manner, as shown by electron microscopy of acini 2,7,8
and of other cells25,26 (as well as by measurement of capacitance in
mast cells27). Our results have clarified the spatio-temporal organization of sequential exocytosis, and thereby give rise to a previously
unidentified mechanism for replenishment of secretory granules.
Granule–granule fusion frequently precedes exocytosis (multigranular exocytosis, Fig. 6a) in eosinophils28–30 and lactotrophs31. In
pancreatic acinar cells, however, we failed to observe multigranular
exocytosis (Fig. 6a), which is consistent with the results of electron
microscopy2,7,8 and capacitance measurement10,24. Although our
results do not rule out the possible occurrence of multigranular
fusion events in acinar cells, they indicate that zymogen granules
preferentially fuse with those granules that have been supplied with
a factor from the luminal plasma membrane (Fig. 6b).
Our observation that granules located within deeper layers of
the cell are able to undergo exocytosis in a sequential manner indicates that CCK-induced secretion requires neither prior contact of
granules with the luminal membrane per se nor complete preassembly of the fusion machinery that connects the luminal and
granule membranes. Furthermore, exocytosis of granules in the
deeper layers of the cell occurred with a delay that was similar to that
for exocytosis of peripheral granules, indicating that deeper granules may be silmilarly fusion-competent to peripheral granules. The
incomplete pre-assembly of the fusion machinery in acinar cells
probably underlies the slower rate of exocytosis of zymogen granules (Fig. 6b) relative to that of exocytosis of synaptic vesicles or
endocrine granules32. The plasma-membrane factor may be
required for complete assembly of the fusion machinery; a candidate for such a plasma-membrane factor is the t-SNARE protein
syntaxin-2, which is localized in the luminal plasma membrane4
and is required for fusion of zymogen granules to the plasma membrane4. Assuming a diffusion coefficient for an unrestrained membrane protein of 2 × 10–9 cm2 s–1 33, a mean square displacement of 1
µm would be achieved within 5 s. Such a high rate of diffusion may
account for the lack of further delay in the latency distribution of
secondary exocytosis. Other candidates for the plasma-membrane
factor include changes in granule content (pH, [Ca2+], osmolarity,
etc.) and in granule membrane potential that may activate the resident fusion machinery in the granule membrane.
The Ω-shaped profiles of fused granules persisted for 30–480 s
in acinar cells, which was sufficient to account for the occurrence
of sequential exocytosis with a mean latency of 13 s. The structural stability of Ω-shaped profiles is probably due to some extent
to cytoskeletal organization of the apical area 34. In addition, agonist-induced micromolar Ca2+ spikes persisted for >30 s and
encompassed multiple layers of granules as a result of release of
Ca2+ from intracellular stores, as has been observed previously10.
These properties of pancreatic acinar cells thus seem to favour
sequential exocytosis, which indeed accounts for most exocytotic
events in these cells. The sequential mechanism of exocytosis
allows efficient secretion of multiple layers of secretory vesicles
from a small surface area with minimal structural alterations, and
without the need for transport of granules to the plasma membrane or for unnecessary fusion among granules. Although the lifetime of Ω-shaped profiles seems to be shorter in endocrine cells35,
a sequential mechanism of exocytosis may still operate in these cells
if the time required for Ca2+-dependent exocytotic fusion is shorter than the lifetime of the Ω-shaped profiles31,36,37. Indeed, one of
the consequences of sequential exocytosis, vacuolation, has frequently been detected in endocrine cells, both by electron
microscopy37,38 and by capacitance measurement27,39,40. In presynaptic terminals, the Ω-shaped profiles of fused vesicles persist for >20
ms (ref. 41). Synaptic vesicles are densely packed, and vesicles in the
deeper layers of the terminal ‘dock’ with peripheral vesicles at the
same time that peripheral vesicles dock with the plasma membrane41.
In addition, the small size (50 nm) of synaptic vesicles allows rapid
(<12 ms) diffusion of a plasma-membrane factor into the membrane of a fused vesicle. Sequential exocytosis may therefore occur
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during repetitive or artificial stimulation32 of neurons when
increases in [Ca2+]i encompass the deeper layers of the presynaptic
terminal.
The sequential mechanism of exocytosis may operate generally
in cells in which secretory vesicles are densely packed in multiple
layers and in which efficient secretion is necessary. Multiphoton
excitation imaging has thus revealed a new aspect of regulated exocytosis as a result of its deep tissue penetration, and this technique
holds the potential for providing further insights into the mechanism of Ca2+-dependent exocytosis.
Methods
Preparation of mouse pancreatic acini.
Clusters of acini were obtained from the pancreases of 5–7-week-old mice by brief (1–2 min) digestion
with collagenase (1 mg ml–1) followed by gentle trituration. Acini were dispersed in a small chamber
with a solution (Sol A) containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM
HEPES–NaOH, pH 7.3, and 10 mM glucose. To visualize exocytosis, acini were immersed in an extracellular solution that contained a fluid-phase polar tracer and either 0.5–1.0 mM sulphorhodamine-B
(SRB; Molecular Probes), 0.1 mM Texas Red–dextran (Mr 3K) or 0.5–1.0 mM Lucifer yellow.
Exocytosis of zymogen granules was similarly imaged using the three dyes. CCK (CCK octapeptide;
Peptide Institute, Osaka, Japan) was dissolved in extracelullar solution and applied to cells through a
glass pipette. To compare two-photon excitation and confocal imaging, acinar preparations were
loaded with Oregon green 488 BAPTA-1-AM (Molecular Probes). For simultaneous imaging of Ca2+
and exocytosis, acini were preloaded with fura-2-AM or fura-2FF-AM (10–20 µM; Tef Lab, Dallas,
Texas) for 30 min. Unless otherwise stated, experiments were carried out at room temperature
(22–25 °C).
Multiphoton excitation imaging.
Multiphoton excitation imaging of acini was carried out using an inverted microscope (IX70,
Olympus) with a water-immersion objective lens (UApo ×60 water/IR). A mode-locked femtosecondpulse Ti:sapphire laser (Tsunami, Spectra Physics, Mountain View, California) with an original pulse
duration of 70–100 fs was attached to the laser port of a laser-scanning microscope (FluoView,
Olympus). The group velocity dispersion of the microscope was compensated for by chirp compensation optics. The laser power at the specimen was 3–20 mW. SRB (or Texas Red) and fura-2 (or fura2FF or Oregon green 488 BAPTA-1) were excited at 830 nm, whereas Lucifer yellow and ultraviolet
autofluorescence of acini were excited at 720 nm. The fluorescence of SRB (or Texas Red), lucifer yellow, and fura-2 (or fura-2FF) was measured with a photomultiplier of the laser-scanning microscope
at wavelengths of 580–620 nm, 420–650 nm and 420–560 nm, respectively. Corresponding fluorescence
images were acquired every 1–3 s. Cellular ultraviolet autofluorescence was measured directly with a
photomultiplier attached to the back port of the inverted microscope, without descanning; these
images were acquired every 30 s.
[Ca2+]i was calculated from the ratio of fura-2 or fura-2FF fluorescence during stimulation (F) to
that obtained before stimulation (F0) according to equation (1).
F/F0 –
[Ca2+]i = K
1 + [Ca2+]0/K
1 + (Fmin/Fmax)[Ca2+]0/K
1 + [Ca2+]0/K
– F/F0
(Fmax/Fmin) + [Ca2+]0/K
(1)
where [Ca2+]0 is assumed to be 0.1 µM, and the affinity of Ca2+ for fura-2 and fura-2FF, K, is assumed
to be 0.2 and 40 µM, respectively. Fmax and Fmin represent fluorescence values for the Ca2+-free and
Ca2+-bound forms of the indicators, respectively, and Fmin/Fmax were estimated in vivo as 0.29 for fura-2
and 0.15 for fura-2FF. Values were obtained by dividing the fluorescence of resting cells by that of cells
treated with 8-bromo-A23187 and 10 mM CaCl2 for 3 min. [Ca2+]i is represented in Ca2+ images by a
pseudocolour coding, in which 0.1, 0.3, 1, 3 and 10 µM are expressed as blue, sky blue, green, yellow
and red, respectively (Fig. 1c).
RECEIVED 23 MAY 2000; REVISED 11 AUGUST 2000; ACCEPTED 10 OCTOBER 2000;
PUBLISHED 7 FEBRUARY 2001.
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ACKNOWLEDGMENTS
This work was supported by Core Research for Evolutional Science and Technology (CREST) of the
Japan Science and Technology Corporation (JST), and by the Research for the Future program of the
Japan Society for the Promotion of Science (JSPS), of which K.I. is a research fellow.
Correspondence and request for materials should be addressed to H.K. Supplementary Information is
available on Nature Cell Biology’s website (http://cellbio.nature.com) or as paper copy from the
London editorial office of Nature Cell Biology.
NATURE CELL BIOLOGY VOL 3 MARCH 2001 http://cellbio.nature.com
© 2001 Macmillan Magazines Ltd
supplementary information
Figure S1 Comparison of depth penetration between two-photon excitation
imaging and confocal imaging. a, b, Images of an acinar preparation stained
with Oregon green 488 BAPTA-1-AM, acquired by x–z scanning with two-photon
laser-scanning microscopy (a) or confocal laser-scanning microscopy (b). Luminal
structures are apparent in the two-photon excitation image, but not in the confocal
image. Confocal imaging (Fig. 1b, d) was carried out using the same scanning
microscope and objective lens, but an argon laser was used for excitation at 488
nm and fluorescence of Oregon green 488 BAPTA-1 was measured at 500–600 nm
through a pinhole (Olympus, CA2). c, Stereo-pair of two-photon images, obtained by
x–y–z scanning of an acinar preparation that had been immersed in 0.5 mM SRB
for 20 min and stimulated with 10 pM CCK for 5 min. Many Ω-shaped profiles of
fused zymogen granules are visible adjacent to lumens, but not to basolateral
membranes.
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© 2001 Macmillan Magazines Ltd
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