The relationship between the cellular Ca2+ signal and secretory vesicle fusion (exocytosis) is a key determinant of the regulation of the kinetics and magnitude of the secretory response. Here, we have investigated secretion in cells where the exocytic response is controlled by Ca2+ release from intracellular Ca2+ stores. Using live-cell two-photon microscopy that simultaneously records Ca2+ signals and exocytic responses, we provide evidence that secretion is controlled by changes in Ca2+ concentration [Ca2+] in relatively large-volume microdomains. Our evidence includes: (1) long latencies (>2 seconds) between the rise in [Ca2+] and exocytosis, (2) observation of exocytosis all along the lumen and not clustered around Ca2+ release hot-spots, (3) high affinity (Kd =1.75 μM) Ca2+ dependence of exocytosis, (4) significant reduction in exocytosis in the prescence of cytosolic EGTA, (5) spatial exclusion of secretory granules from the cell membrane by the endoplasmic reticulum, and (6) inability of local Ca2+ responses to trigger exocytosis. These results strongly indicate that the control of exocytosis, triggered by Ca2+ release from stores, is through the regulation of cytosolic [Ca2+] within a microdomain.

Ca2+-dependent exocytosis is an essential and widespread process (Sudhof, 2004). An increase in cytosolic Ca2+ concentration ([Ca2+]) triggers secretory vesicle fusion with the plasma membrane leading to the release of vesicle cargoes, such as neurotransmitters and proteins, for example, hormones and enzymes. In excitable cells, Ca2+ entry through voltage-gated Ca2+ channels (Rizzuto and Pozzan, 2006) is the major route to elevate cytosolic [Ca2+] and trigger exocytosis. In some excitable cells, Ca2+ channels and exocytic sites are closely apposed; they are positioned within volumes of nanometre dimensions that are called nanodomains (Adler et al., 1991; Bucurenciu et al., 2008; Stanley, 1993) and enable fast, efficient regulation of the secretory response (Stanley, 1993). In other excitable cells, clusters of Ca2+ channels provide a localized ‘cloud’ of Ca2+, triggering exocytosis across microdomains (Beaumont et al., 2005; Borst and Sakmann, 1996; Chow et al., 1994). In the latter case, the secretory response is slower, but precise control of [Ca2+] within the microdomain is used to fine-tune the secretory output (Chow et al., 1994).

The role of Ca2+ release from intracellular stores in triggering exocytosis is less well understood. In cells with voltage-gated Ca2+ channels, Ca2+ release can modulate exocytic responses (Dyachok and Gylfe, 2004; ZhuGe et al., 2006) but in many cell types Ca2+ release is the exclusive source of increase of cytosolic [Ca2+] (Matthews et al., 1973; Tse et al., 1997). How the sites of Ca2+ release from stores are related to the sites of exocytosis and control secretion in these cells is not known.

A good example of secretion regulated by Ca2+ release from Ca2+ stores is in exocrine acinar cells. Here, exocytosis of enzyme containing granules (Chen et al., 2005; Nemoto et al., 2001) is dependent on Ca2+ release through inositol trisphosphate receptors (InsP3Rs) on the endoplasmic reticulum (ER) Ca2+ store (Futatsugi et al., 2005; Ito et al., 1997). This Ca2+ response has complex characteristics in space, time and amplitude (Fogarty et al., 2000a; Kasai and Augustine, 1990; Kasai et al., 1993; Thorn et al., 1993). Through the use of Ca2+ buffers (Kidd et al., 1999) and high-speed imaging (Fogarty et al., 2000b; Kidd et al., 1999) it has been shown that there is one hot spot of Ca2+ release that can act alone, giving a local response or act to initiate larger, global Ca2+ signals (Fogarty et al., 2000b; Shin et al., 2001). This hot spot is likely to represent a site of enrichment of the Ca2+ release apparatus, possibly with more-sensitive isoforms of the InsP3R (Futatsugi et al., 2005; Lee et al., 1997; Nathanson et al., 1994; Park et al., 2008) or with a greater density of IP3Rs (Callamaras et al., 1998). How these complex Ca2+ signals are employed to regulate the exocytic response is not known.

Here, we test the hypothesis that control of exocytosis in acinar cells is through local Ca2+ release that targets high [Ca2+] to close-by sites of exocytosis within nanodomains. We employ high-speed two-photon microscopy to simultaneously measure cytosolic Ca2+ (with Fura-2 and Fura-FF) and exocytosis (with extracellular aqueous dyes) in response to the endogenous agonist cholecystokinin (CCK) and the photoliberation of Ca2+ from o-nitrophenyl (NP) tagged to EGTA (NP-EGTA). Our results show that events of exocytosis are not clustered around hot spots of Ca2+ release and we conclude that Ca2+ release from Ca2+ stores regulates exocytosis through the control of a microdomain. In cells that do not require a rapid secretory response we speculate that fine-tuning of Ca2+ levels within the microdomain gives precise control of secretory output.

Simultaneous recording of cytosolic Ca2+ signals and exocytic responses

Apical-to-basal Ca2+ waves, owing to release of Ca2+ from intracellular stores, are a characteristic feature of responses of pancreatic acinar cell to agonists (Kasai and Augustine, 1990). Fig. 1 shows an example of such a response recorded in freshly isolated tissue fragments loaded with Fura-2 and stimulated with a physiological concentration of 20 pM CCK.

The image sequence in Fig. 1A shows the Ca2+ response in four cells on the edge of the tissue fragment (composed of 10–50 cells); the relative time point of each image is indicated with roman numerals (see Fig. 1B, upper graph). The Fura-2 fluorescence signal was converted into ratios and plotted in pseudocolor in Fig. 1A (upper images). To observe exocytic responses the tissue was bathed in sulforhodamine B (SRB), a fluorescent probe that surrounds the cells and diffuses into the lumens between the cells (Fig. 1A, lower images, colored red) (Nemoto et al., 2001; Thul and Falcke, 2004). Upon granule fusion SRB enters the granules, which seen as the sudden appearance of small spherical objects (~0.8 μm diameter) at the apical pole of the cells (Fig. 1A, lower sequence). The average Fura-2 response in each cell is plotted in Fig. 1B (upper graph) and the time-course of the exocytic responses measured as normalized SRB changes – within regions of interest (ROIs) centered on each exocytic granule are shown in Fig. 1B (lower graph).

These CCK-induced global Ca2+ responses occur asynchronously (Yule et al., 1996); they originate in the apical region spreading across the cell to the basal pole. Fig. 1C shows the image sequence and a graph of average Fura-2 ratio against time of the Ca2+ response for the lower left cell of Fig. 1A. The graph plots average changes in three ROIs spread across the cell and shows the apical to basal spread of the Ca2+ wave. We calculated the velocity of the Ca2+ wave to be 10.5±0.77 μm/second (mean ± s.e.m., n=17), which is comparable to previously published data (Larina and Thorn, 2005).

Response to an exogenous Ca2+ signal – spatial organization of exocytosis

To initially characterize the stimulus-secretion relationship in the absence of the spatial complexities of the agonist-evoked response we induced a cytosolic increase of [Ca2+] by uncaging of Ca2+ from the photolabile Ca2+ buffer NP-EGTA. This method elevates Ca2+ uniformly across the cell. The image sequences in Fig. 2 (and in supplementary material Movie S1) show three cells at the edge of a pancreatic fragment loaded with NP-EGTA (AM ester) and Fura-2. The upper panel in Fig. 2A shows the ratiometric pseudocolor Fura-2 response to a 100-ms UV flash. The lower panel in Fig. 2A shows the induced exocytic response recorded by the entry of SRB into individual granules. The graph of the Fura-2 ratio over time shows a large, rapid rise in [Ca2+] after the UV flash that triggers exocytic activity (Fig. 2B, upper panel), observed as increases in SRB fluorescence in ROIs at each site of exocytosis (Fig. 2B, lower panel).

Fig. 1.

Ca2+ responses within an acinus to CCK stimulation. Cells were loaded with Fura-2 and stimulated by the addition of 20 pM CCK to the bathing solution. (A) Within an acinus, individual cells asynchronously show a rise in [Ca2+]. (Top and middle rows) Top row: Fura-2 response (F0-F/F0 in pseudocolor) at individual time points (i–vii, indicated on the graph in B) throughout the response (blue, low [Ca2+], to red, high [Ca2+]). The cell in the upper left responds first, followed sequentially by the other three cells in the group. Middle row: SRB fluorescence (red) that surrounds the cells and diffuses into the lumen between cells. Individual exocytic fusion events, observed as SRB dye enters the granules are seen in the last three images (v–vii). The boxed area in the first image of the middle row indicates the area that is shown enlarged in the four black and white images (a–d). These images show individual exocytic events at time points a–d (arrowheads), indicated on the bottom graph in B. (B) Exocytic events follow the Ca2+ signals. (Top graph) Average Fura-2 ratiometric response in ROIs within each of the cells (color-coded for each cell, shown in image I in A). (Bottom graph) Time-course of the SRB dye signals in ROIs placed over each individual granule that undergoes fusion (dots above the graph indicate the times of the peaks of each exocytic event). (C) Within a single cell, the Ca2+ response is seen as a wave spreading from apical to basal regions. The enlarged images show a time sequence of images (0.2 second time intervals) from the lower left-hand cell (shown in A). The Ca2+ wave initiates in the apical region (red circle) and then spreads to the basal region (green circle). Scale bar: 10 μm. The graph shows average ratiometric fluorescence changes in each ROI (red, yellow, green) plotted against time. The Ca2+ signal rises first in the apical region.

Fig. 1.

Ca2+ responses within an acinus to CCK stimulation. Cells were loaded with Fura-2 and stimulated by the addition of 20 pM CCK to the bathing solution. (A) Within an acinus, individual cells asynchronously show a rise in [Ca2+]. (Top and middle rows) Top row: Fura-2 response (F0-F/F0 in pseudocolor) at individual time points (i–vii, indicated on the graph in B) throughout the response (blue, low [Ca2+], to red, high [Ca2+]). The cell in the upper left responds first, followed sequentially by the other three cells in the group. Middle row: SRB fluorescence (red) that surrounds the cells and diffuses into the lumen between cells. Individual exocytic fusion events, observed as SRB dye enters the granules are seen in the last three images (v–vii). The boxed area in the first image of the middle row indicates the area that is shown enlarged in the four black and white images (a–d). These images show individual exocytic events at time points a–d (arrowheads), indicated on the bottom graph in B. (B) Exocytic events follow the Ca2+ signals. (Top graph) Average Fura-2 ratiometric response in ROIs within each of the cells (color-coded for each cell, shown in image I in A). (Bottom graph) Time-course of the SRB dye signals in ROIs placed over each individual granule that undergoes fusion (dots above the graph indicate the times of the peaks of each exocytic event). (C) Within a single cell, the Ca2+ response is seen as a wave spreading from apical to basal regions. The enlarged images show a time sequence of images (0.2 second time intervals) from the lower left-hand cell (shown in A). The Ca2+ wave initiates in the apical region (red circle) and then spreads to the basal region (green circle). Scale bar: 10 μm. The graph shows average ratiometric fluorescence changes in each ROI (red, yellow, green) plotted against time. The Ca2+ signal rises first in the apical region.

We then used these Ca2+ responses that were induced by the uncaging process to map sites of exocytosis and to determine whether they are clustered along the lumen. Cells were loaded with NP-EGTA and stimulated with a 100-msecond UV flash. We then measured granule-to-granule distances along the lumen from one granule to all other exocytic granules in the same cell. A frequency histogram showed no evidence of clustering around short granule-to-granule distances (Fig. 3A). However, granule-to-granule distances would be affected by the lengths of lumen in each cell. Therefore, for each cell a scatter plot of granule-to-granule distances was plotted against the lumen length (Fig. 3B). The predicted line – if the granule-to-granule separation were random – shows a close approximation to our data; consistent with a lack of preferential sites of exocytosis along the lumen (Fig. 3C). These data therefore indicate that all regions along the lumen are equally capable of exocytosis.

We applied the same clustering analysis to the exocytic response to 20 pM CCK with similar frequency distributions of granule-to-granule distances (supplementary material Fig. S1A). Since it is known that compound exocytosis (granule-to-granule) fusion is prevalent in this cell type, we extended this analysis to identify the location of primary granules (those fusing directly with the plasma membrane) and secondary granules (those fusing with primary granules). supplementary material Fig. S1B shows that the frequency plot of granule-to-granule distances is very similar for primary and secondary granules.

Response to an exogenous Ca2+ signal – exocytosis has a Ca2+ Kd of 1.75 μM

We also used the responses induced by uncaging Ca2+ to determine the Ca2+ dependence of exocytosis. Here, we varied the duration of the UV flash over a range from 5 ms to 200 ms and, for each duration flash, measured the maximal response to Fura-FF (a low-affinity Fura-2 derivative with a measured in vivo Keff of 1.84 μM for Ca2+, see Materials and Methods) (n=309 cells). The duration of each flash was then calibrated as a Ca2+ change, plotted against the number of exocytic events per cell (by using entry of SRB extracellular dye into fused vesicles). The graph shows a sigmoid relationship with an estimated Kd of 1.75 μM for the Ca2+ dependence of exocytosis (Fig. 4). This is similar to the Kd of 2 μM Ca2+ that is found for enzyme release in these cells (Knight and Koh, 1984). Our Kd value is also comparable to endocrine cells, such as chromaffin cells, where the calculated Kd is 1.6 μM (Augustine and Neher, 1992). In all further experiments, we employed a 100-ms UV flash (3.4 μM Ca2+) to induce maximal exocytic responses.

Fig. 2.

Ca2+ responses within an acinus induced by the uncaging of Ca2+ from NP-EGTA. (A) Cells were loaded simultaneously with Fura 2 and NP-EGTA and stimulated with an UV flash for 100 ms. The example (upper sequence of images) Fura-2 responses (F0-F/F0 ratio in pseudocolor) at individual time points (i-v; see upper graph in B) throughout the response. Cells respond rapidly with no apical-to-basal Ca2+ wave. (Upper sequence of images) Fluorescence of the extracellular SRB dye. Individual exocytic fusion events are seen in the last three images. (B) Upper graph: Average Fura-2 ratiometric response in ROIs within each of the cells (color-coded for each cell). Lower graph: Extracellular SRB dye signals in ROIs placed over each individual granule that undergoes fusion. Diamond symbols above the graph show the time points of the peaks of each exocytic event.

Fig. 2.

Ca2+ responses within an acinus induced by the uncaging of Ca2+ from NP-EGTA. (A) Cells were loaded simultaneously with Fura 2 and NP-EGTA and stimulated with an UV flash for 100 ms. The example (upper sequence of images) Fura-2 responses (F0-F/F0 ratio in pseudocolor) at individual time points (i-v; see upper graph in B) throughout the response. Cells respond rapidly with no apical-to-basal Ca2+ wave. (Upper sequence of images) Fluorescence of the extracellular SRB dye. Individual exocytic fusion events are seen in the last three images. (B) Upper graph: Average Fura-2 ratiometric response in ROIs within each of the cells (color-coded for each cell). Lower graph: Extracellular SRB dye signals in ROIs placed over each individual granule that undergoes fusion. Diamond symbols above the graph show the time points of the peaks of each exocytic event.

In summary, experiments where Ca2+ was uncaged from NP-EGTA show that, in principle, exocytosis can occur all along the lumen and that the exocytic process is relatively sensitive to cytosolic Ca2+. We next set out to determine the spatial relationship between the agonist-evoked Ca2+ signal and the triggered exocytic responses.

Agonist-evoked initiation sites of Ca2+ signals are distant from sites of exocytosis – morphometric analysis

We used first derivatives and region mapping of the Ca2+ response to determine the precise point of origin of the Ca2+ responses to CCK (Fig. 5, n=22 cells). These initiation hot spots of the Ca2+ signal probably correspond to the local enrichment of InsP3Rs in regions below the apical plasma membrane as previously described by Kidd and colleagues (Kidd et al., 1999). Here, we measured changes of Fura-2 intensity in 3×3 μm ROIs, which robustly identified the origin of the Ca2+ responses. In most cases, a single discrete origin was identified (Fig. 5C,D) with the Ca2+ rise occurring in advance of surrounding regions (Fig. 5C). In a small number of cells (<10%) the Ca2+ signal appeared simultaneously across a broader region. We interpret these latter responses to be indicative of the Ca2+ origin lying outside the plane of the two-photon cross-section and did not include these experiments in further analysis.

Fig. 3.

Quantification of the spatial relationships of the exocytic response following uncaging of Ca2+ from NP-EGTA. The granule-to-granule distance was measured using one reference exocytic granule in each cell and determining the distances to all other exocytic granules within the same cell. (A) The frequency distribution has a broad spread of granule-to-granule distances. (B) Same data as in A, but each granule-to-granule distance is plotted against the lumen length in each cell. As expected lumen length is limiting and within each cell there is a wide distribution of granule-to-granule distances. (C) Mean granule-to-granule distances (mean ± s.d., n=24 cells) for each cell plotted against lumen length in that cell. Also shown is the predicted line if numbers of exocytic granules are evenly separated along the lumen.

Fig. 3.

Quantification of the spatial relationships of the exocytic response following uncaging of Ca2+ from NP-EGTA. The granule-to-granule distance was measured using one reference exocytic granule in each cell and determining the distances to all other exocytic granules within the same cell. (A) The frequency distribution has a broad spread of granule-to-granule distances. (B) Same data as in A, but each granule-to-granule distance is plotted against the lumen length in each cell. As expected lumen length is limiting and within each cell there is a wide distribution of granule-to-granule distances. (C) Mean granule-to-granule distances (mean ± s.d., n=24 cells) for each cell plotted against lumen length in that cell. Also shown is the predicted line if numbers of exocytic granules are evenly separated along the lumen.

Fig. 4.

Exocytic response following uncaging of Ca2+ from NP-EGTA. The peak Ca2+ response to the UV photolysis of NP-EGTA was measured with Fura-FF within ROIs in single cells. Each fluorescent response was calibrated according to our in-vitro assay of the Fura-FF Keff.. Changes in the duration of the UV flash varied the amount of Ca2+ liberated. The exocytic response was recorded as the number of fused granules per cell and data are given as the mean ± s.e.m. (n=309 cells, minimum of seven cells) of the exocytic responses plotted against the change in [Ca2+] induced by the UV flashes. The calculated Kdfor theCa2+ sensitivity of exocytosis was 1.75 μM.

Fig. 4.

Exocytic response following uncaging of Ca2+ from NP-EGTA. The peak Ca2+ response to the UV photolysis of NP-EGTA was measured with Fura-FF within ROIs in single cells. Each fluorescent response was calibrated according to our in-vitro assay of the Fura-FF Keff.. Changes in the duration of the UV flash varied the amount of Ca2+ liberated. The exocytic response was recorded as the number of fused granules per cell and data are given as the mean ± s.e.m. (n=309 cells, minimum of seven cells) of the exocytic responses plotted against the change in [Ca2+] induced by the UV flashes. The calculated Kdfor theCa2+ sensitivity of exocytosis was 1.75 μM.

The measured spatial relationship between the site of Ca2+-response initiation and the sites of granule fusion (Fig. 6) did not show any events of granule fusion at the origin of the Ca2+ signal. The numbers of exocytic events increased to a maximum at a distance of 3 μm from the Ca2+ signal origin and then decreased at further distances. These data, therefore, do not support the idea that Ca2+ nanodomains control exocytosis. Instead, they indicate the importance of larger-volume microdomains and even suggest a distinct separation of sites of Ca2+ release from sites of exocytosis.

Ca2+ initiation sites are distant from sites of exocytosis – block of exocytosis by EGTA

EGTA, a Ca2+ chelator with a slow on-rate for binding Ca2+, is often used as an indicator of the spatial extent of a Ca2+ response (Beaumont et al., 2005; Borst and Sakmann, 1996; Bucurenciu et al., 2008; Chow et al., 1994; Stanley, 1993). Since EGTA is unable to act as an effective Ca2+ buffer within nanodomains, a Ca2+ target that is close (<200 nm) to a Ca2+ source will be unaffected by the presence of EGTA (Bucurenciu et al., 2008; Thul and Falcke, 2004). By contrast, EGTA is an effective buffer when the Ca2+ source is further away from the Ca2+ target (<1 μm). We loaded the cells with EGTA-AM and recorded the Ca2+ and the exocytic responses (Fig. 7). The Fura-2 responses decreased with increased duration of EGTA-AM loading, which is as expected because EGTA will compete with Fura-2 for cytosolic Ca2+. The exocytic response was significantly reduced within 30 minutes of EGTA-AM loading (Fig. 7). These data support the idea that exocytosis is regulated by Ca2+ microdomains.

Crowding limits access of secretory granules to the apical plasma membrane

To further investigate the ultrastructural relationship of organelles in the apical region of acinar cells we next used thin-section transmission electron microscopy. The ER surrounds secretory granules in the apical region (Bolender, 1974). We now established that these ER projections extend right up to the apical plasma membrane. Fig. 8 shows examples of typical electron micrographs of the region surrounding the lumen of an acinar endpiece. In all of our electron microscopy sections we found evidence of rough ER lying immediately under the apical plasma membrane, sterically blocking granule access to the apical cell membrane, and measured the distances between the apical plasma membrane and the centre point of the first layer of secretory granules under the membrane. If the granules were tightly packed against the cell membrane then the distance to the centre point of each granule should equal the granule radius. We measured the mean granule diameter as 748.6 ± 11.1 nm (mean ± s.e.m., n=230) and, with tight packing, a normal distribution centered on 374 nm (the radius) is therefore expected, with a further peak at 1122 nm (reflecting another layer of granules). Instead, the frequency-distance plot shows a first peak at 500 nm, indicating that granules are further away from the plasma membrane than predicted (Fig. 8B), and no further peak at greater distances. We therefore conclude that the granules are relatively loosely packed and are separated from each other and from the cell membrane by structures such as the ER.

Fig. 5.

Identification of the site of origin of the Ca2+ wave. Cells stimulated with 20 pM CCK show the Ca2+ signal starting at the apical pole and then spreading out across the cell. (A) Fura-2 responses (F0-F/F0 ratio in pseudocolor) from a CCK-evoked Ca2+ response in a single cell within a pancreatic fragment. (B) Full sequence of still images from a movie (five frames per second) taken as the Ca2+ response spreads across the cell. (C) Same cell as in B, enlarged (at the very first movie frame that shows the start of the Ca2+ response) with four ROIs of 3×3 μm (boxed areas) centered around the origin of the Ca2+ response. (D) The main graph is the magnification of the gray-shaded area in inset graph, showing the average fluorescence within each of the ROIs shown in C. The red ROI rises earlier and faster than the surrounding ROIs; for this cell, this region is therefore identified as the point of origin of the Ca2+ response.

Fig. 5.

Identification of the site of origin of the Ca2+ wave. Cells stimulated with 20 pM CCK show the Ca2+ signal starting at the apical pole and then spreading out across the cell. (A) Fura-2 responses (F0-F/F0 ratio in pseudocolor) from a CCK-evoked Ca2+ response in a single cell within a pancreatic fragment. (B) Full sequence of still images from a movie (five frames per second) taken as the Ca2+ response spreads across the cell. (C) Same cell as in B, enlarged (at the very first movie frame that shows the start of the Ca2+ response) with four ROIs of 3×3 μm (boxed areas) centered around the origin of the Ca2+ response. (D) The main graph is the magnification of the gray-shaded area in inset graph, showing the average fluorescence within each of the ROIs shown in C. The red ROI rises earlier and faster than the surrounding ROIs; for this cell, this region is therefore identified as the point of origin of the Ca2+ response.

Fig. 6.

Exocytic events are excluded from the site of origin of the Ca2+ wave. (A) Shown is the same cell as in Fig. 5. Left image: The frame showing the first sign of a Ca2+ response and the 3×3 μm region identified as the site of origin of the Ca2+ response. Middle and right image: First (showing the lumen) and last SRB images (showing the lumen and all exocytic events, respectively). The location of each exocytic event in the cell of interest was then measured by referring to the centre of the 3×3-μm region of the origin Ca2+. (B,C) Graphs of these distances are shown for the all the cells analysed (n=22) (B) and for this particular cell (C).

Fig. 6.

Exocytic events are excluded from the site of origin of the Ca2+ wave. (A) Shown is the same cell as in Fig. 5. Left image: The frame showing the first sign of a Ca2+ response and the 3×3 μm region identified as the site of origin of the Ca2+ response. Middle and right image: First (showing the lumen) and last SRB images (showing the lumen and all exocytic events, respectively). The location of each exocytic event in the cell of interest was then measured by referring to the centre of the 3×3-μm region of the origin Ca2+. (B,C) Graphs of these distances are shown for the all the cells analysed (n=22) (B) and for this particular cell (C).

Local Ca2+ responses fail to trigger exocytosis

A characteristic of the physiological response to CCK is that the global Ca2+ responses (as focused on in this study) are interspersed with fast local Ca2+ responses that remain within the apical region (Kasai et al., 1993; Thorn et al., 1993). These local responses are thought to represent Ca2+ release from Ca2+ hot spots that fail to propagate across the cell (Thorn et al., 1993). Cytosolic Ca2+ levels within the nanodomain of the hot spot region are transiently expected to be high (>50 μM) (Thul and Falcke, 2004), and sufficient – in principle – to elicit exocytosis within this region. In six independent experiments we compared local and global cytosolic Ca2+ responses in 20 cells stimulated with 10–12 pM CCK. A total of 66 Ca2+ events were observed, 25 of which were local. All global Ca2+ signals induced exocytosis. By contrast, no local Ca2+ signals induced exocytosis (Fig. 9). We therefore conclude that local Ca2+ responses are not sufficient to drive exocytosis, which further supports the notion that nanodomains are not important in the regulation of secretion and that sites of Ca2+ release are further away from sites of exocytosis.

Fig. 7.

Cytosolic EGTA blocks exocytosis. Cells loaded with EGTA-AM were stimulated with 50 pM CCK. Both the Ca2+ response recorded by using Fura-2 and the exocytic response recorded by using extracellular SRB decreased as a function of the EGTA-AM loading time. Comparisons between Fura-2 AM loading and Fura-2 loaded through a patch pipette (Thorn et al., 1993) indicate that, at 30 minutes, intracellular EGTA concentrations reach a concentration of >200 μM. Data were obtained from 3–6 independent experiments per set of data (**P<0.01 compared with controls measured using Student's t-test).

Fig. 7.

Cytosolic EGTA blocks exocytosis. Cells loaded with EGTA-AM were stimulated with 50 pM CCK. Both the Ca2+ response recorded by using Fura-2 and the exocytic response recorded by using extracellular SRB decreased as a function of the EGTA-AM loading time. Comparisons between Fura-2 AM loading and Fura-2 loaded through a patch pipette (Thorn et al., 1993) indicate that, at 30 minutes, intracellular EGTA concentrations reach a concentration of >200 μM. Data were obtained from 3–6 independent experiments per set of data (**P<0.01 compared with controls measured using Student's t-test).

Our study describes the spatial relationship between sites of Ca2+ release and sites of exocytic fusion in a cell type where secretion is dependent on Ca2+ release from Ca2+ stores. On the basis of previous work, with cells dependent on Ca2+ entry to trigger exocytosis, we expected a close apposition between hot spots of Ca2+ release and sites of granule fusion. However, our findings indicate no sites of clustered exocytosis. Instead, we show that exocytosis occurs all along the luminal membrane and is in fact excluded within a region of ~3 μm around the hot spot of Ca2+ release, suggesting that the ER, required for Ca2+ release, is locally obstructing granule access to the plasma membrane. Combined with the observations that EGTA profoundly inhibits exocytosis and that local Ca2+ responses do not trigger exocytosis we conclude that secretion, dependent on Ca2+ release from Ca2+ stores, is controlled by cytosolic Ca2+ microdomains. We suggest that this regulation of the microdomain sacrifices speed of secretory control for precision; small adjustments in [Ca2+] fine tune the secretory output.

Fig. 8.

Organelles spatially compete with granules in the sub-apical region. (A) electron micrographs of he apical region of acinar cells with the lumen (containing microvilli) and the sub-apical region packed with zymogen granules. In all sections the sub-apical region also contained other organelles – such as rough ER – which lay between granules and the apical plasma membrane. (B) Frequency histogram of the distances between the apical membrane and granule centres is not consistent with tight packing of granules against the cell membrane. Instead the data suggest that intercalated organelles, such as the ER, spatially separate granules from each other and from the cell membrane.

Fig. 8.

Organelles spatially compete with granules in the sub-apical region. (A) electron micrographs of he apical region of acinar cells with the lumen (containing microvilli) and the sub-apical region packed with zymogen granules. In all sections the sub-apical region also contained other organelles – such as rough ER – which lay between granules and the apical plasma membrane. (B) Frequency histogram of the distances between the apical membrane and granule centres is not consistent with tight packing of granules against the cell membrane. Instead the data suggest that intercalated organelles, such as the ER, spatially separate granules from each other and from the cell membrane.

Ca2+-release-dependent triggering of exocytosis – a model

The evidence presented here supports the idea that Ca2+ release controls an apical cytosolic microdomain of Ca2+ that in turn triggers exocytosis. Within this microdomain the Ca2+ concentration reflects the activity of many ion channels and will be regulated both by the ensemble control of the Ca2+ release channels and the mechanisms of Ca2+ clearance. In turn this tight regulation of the Ca2+ concentration leads to a precise control of secretion.

By contrast, nanodomain control is found in some neurons; the very local delivery of Ca2+ through ion channels tightly couples the Ca2+ stimulus to secretory output with very short latencies. The limitation of this mechanism is that the stochastic opening and closing of the ion channels produces rapid and extreme local changes in Ca2+ in the cytosolic nanodomain beneath the Ca2+ channel. Active and passive mechanisms of Ca2+ clearance in this nanodomain mean that closure of a Ca2+ channel leads to a rapid drop in [Ca2+]. Thus, the delivery of sufficient Ca2+ levels to trigger exocytosis is dependent on the probabilistic opening of the Ca2+ channel and this can lead to failures to drive exocytosis (Stanley, 1993). Dependence on nanodomains is, thus, necessary to drive fast processes (e.g. neuromuscular control of muscle contraction) but leads to unpredictability in controling the number of exocytosis events.

So how is the microdomain of Ca2+ regulated in the acinar cells? We have previously measured how much Ca2+ is released during responses in acinar cells and have shown that local Ca2+ responses are comparable (slightly larger) to the puffs seen in oocytes (Fogarty et al., 2000b; Xun et al., 1998). Modeling of cytosolic free [Ca2+] suggests that, in the immediate vicinity of a puff site, cytosolic Ca2+ can rise to >50 μM but drops off dramatically further away from the puff site (Thul and Falcke, 2004) where, at distances of 1 μm, the cytosolic Ca2+ is predicted to be <1 μM (Thul and Falcke, 2004). With our estimate of a Kd of 1.75 μM Ca2+ for exocytosis of a secretory response driven solely by Ca2+ release from the hot spot would need to have the exocytic sites within 1 μm of the Ca2+ hot spot. Since we show here that no clustering of exocytosis is evident around hot spots of Ca2+ release, local Ca2+ responses cannot induce exocytosis and EGTA can effectively block exocytosis, a more realistic model is that the explosive Ca2+ release from InsP3Rs within the hot spot recruits further InsP3Rs along the ER (Fogarty et al., 2000a). The summed Ca2+ release from these InsP3Rs thus collectively contributes to microdomain Ca2+ levels that, in turn, control exocytosis.

We also suggest that the regulation of [Ca2+] within a microdomain is the key to integrating convergent cell stimuli on the control of cell secretion. This way, the regulation of InsP3Rs via the CCK and acetylcholine cell-surface receptors is the major mechanism of control (Kasai and Augustine, 1990; Nathanson et al., 1992). This will be modulated by triggering Ca2+ release from ryanodine receptors (Nathanson et al., 1992) through other signaling pathways, such as the secretin-dependent cAMP regulation of InsP3Rs (Giovannucci et al., 2000) and through the regulation of mechanisms of Ca2+ clearance (Camello et al., 1996). Divergent cell stimuli are thus integrated by converging on the regulation of [Ca2+] within the microdomain, which then precisely controls secretory output of the cell.

Mechanisms of regional targeting of the Ca2+ release system and the exocytic machinery

Despite our conclusion that the hot spots of Ca2+ release are only loosely coupled with sites of exocytosis it nevertheless should be recognized that both the Ca2+ release system and the exocytic machinery are precisely localized within the cell by mechanisms that are poorly understood.

Fig. 9.

Local Ca2+ spikes do not induce exocytosis. (A–B) Upper row of images in A: Fura-2 Ca2+ responses (F0-F/F0) following stimulation with CCK taken at different time points (i–v; indicated in B). Shown are two consecutive, short-lasting Ca2+ spikes that are locally restricted to the apical region (red circle). Lower row of images in A: SRB fluorescence (red). Throughout the occurrence of these local Ca2+ spikes there is no change in the SRB signal and no indication of any exocytic events. (B) The large Fura-2 ratiometric changes in the cell from a region of interest placed in the apical pole (red) compared with very little change in a region placed in the basal pole (green). (C) The full sequence of images (and the associated graph of average fluorescent changes) taken over the first local Ca2+ response clearly shows the spatially restricted nature of the signal.

Fig. 9.

Local Ca2+ spikes do not induce exocytosis. (A–B) Upper row of images in A: Fura-2 Ca2+ responses (F0-F/F0) following stimulation with CCK taken at different time points (i–v; indicated in B). Shown are two consecutive, short-lasting Ca2+ spikes that are locally restricted to the apical region (red circle). Lower row of images in A: SRB fluorescence (red). Throughout the occurrence of these local Ca2+ spikes there is no change in the SRB signal and no indication of any exocytic events. (B) The large Fura-2 ratiometric changes in the cell from a region of interest placed in the apical pole (red) compared with very little change in a region placed in the basal pole (green). (C) The full sequence of images (and the associated graph of average fluorescent changes) taken over the first local Ca2+ response clearly shows the spatially restricted nature of the signal.

It is well established that InsP3Rs are enriched beneath the apical plasma membrane of polarized epithelia (Lee et al., 1997; Nathanson et al., 1994; Yule et al., 1997); as shown by immunohistochemistry, the receptors colocalize with apical markers such as the F-actin apical web (Waterman-Storer and Salmon, 1998), and tight-junction markers such as ZO-1 (Larina et al., 2007; Turvey et al., 2005). The mechanisms of this localization are not well understood but disruptions of both the microtubular system (Colosetti et al., 2003) and the F-actin network perturb the generation of Ca2+ signals (Turvey et al., 2005). It has been proposed that the microtubular system acts to position the ER (Fogarty et al., 2000c) and that F-actin is part of a complex that specifically anchors InsP3Rs (Foskett et al., 2007). Functional work has proven that the whole of the ER within these cells forms a single continuous network (Park et al., 2000).

Electron microscopy of the sub-plasmalemmal region under the apical pole has shown that it is enriched in secretory granules with interspersed ER elements (Bolender, 1974) (Fig. 8). Although there is no evidence for close association (docking) of granules at the cell membrane it is clear that there must be mechanisms that moves the granules to the apical region and retain them there. Again, these mechanisms probably depend on the cytoskeleton, a suggestion that is supported by earlier reports that movement of granules depends on kinesin (Marlowe et al., 1998) and myosin 1 (PoucellHatton et al., 1997), and also recently that, in situ, granules are tethered in the apical region (Abu-Hamdah et al., 2006). Most recently a proteomic analysis has shown that myosin Vc is present on zymogen granules (Chen et al., 2006). In terms of the molecular components of exocytosis in non-excitable cells our knowledge lags behind excitable cells. So, whereas soluble NSF attachment protein receptors (SNARE) proteins on the cell membrane and granule membrane have been identified (Cosen-Binker et al., 2008; Gaisano et al., 1996; Hansen et al., 1999), it is still unclear which SNARE proteins are actually involved in exocytosis of zymogen granules at the apical plasma membrane.

Comparison of other measures of secretion

The two-photon method we used here has been proven to reliably identify exocytosis of zymogen granules (Larina et al., 2007; Nemoto et al., 2001; Thorn et al., 2004). Using cell capacitance measurements as a read-out for exocytosis, changes are detected that might not be directly associated with fusion of a zymogen granule. The expectation that fusion of a zymogen granule should lead to rapid, large-step increases in capacitance is seen in parotid cells (Chen et al., 2005) but has rarely been observed in pancreatic acinar cells, in which slower increases are usually seen (Ito et al., 1997; Maruyama and Petersen, 1994). Ito et al. kinetically separated these capacitance signals, and suggested a fast component possibly owing to processes other than the secretion of amylase (fusion of zymogen granules) (Ito et al., 1997). The underlying mechanism used by this fast component is unknown but it might explain why capacitance changes have been seen with local Ca2+ responses (Maruyama and Petersen, 1994); and yet we find no evidence that exocytosis of zymogen granules is induced by local Ca2+ spikes (Fig. 9).

Ca2+ release from Ca2+ stores is either the exclusive regulator of, or a component in, the regulation of exocytosis in many different cell types. Our work described here shows that exocytosis is precisely controlled by regulating Ca2+ release in a microdomain. We further show that, in response to physiological stimuli, exocytosis is only driven by global Ca2+ signals.

Cell preparation

Mice were humanely killed according to local animal ethics procedures. Isolated mouse pancreatic tissue was prepared by a collagenase digestion method in normal NaCl-rich extracellular solution (Thorn et al., 1993) that was modified to reduce the time in collagenase and to limit mechanical trituration. The resultant preparation was composed of pancreatic lobules and fragments (50–100 cells). In the indicated experiments pancreatic fragments were loaded with 2 μM Fura-2 (or Fura-2FF) acetoxymethyl ester (AM) for 30 minutes at 30°C. Fragments were then washed and plated onto poly-L-lysine-coated glass coverslips and used within 3 hours of isolation from the animal. In experiments using NP-EGTA the NP-EGTA-AM (1 μM) was loaded together with Fura-2-AM.

Live-cell two-photon imaging

We used a custom-made, video-rate, two-photon microscope employing a Sapphire-Ti laser (Coherent), with a 60× oil immersion objective (NA 1.42, Olympus), providing an lateral resolution (full width, half maximum) of 0.26 μm and a z-resolution of 1.3 μM (Thorn et al., 2004). We imaged exocytic events using Sulforhodamine B (SRB, 20 μg/ml, Sigma), as a membrane-impermeant fluorescent extracellular marker excited by femtosecond laser pulses at 800 nm, with fluorescence emission detected at 550–700 nm. Fura-2 was excited at the same wavelength with fluorescence emission detected at 450–550 nm. The Fura-2 signal was analysed using the following formula: Fluorescence ratio = (resting fluorescence – signal fluorescence) ÷ resting fluorescence, where the resting fluorescence is taken from an average of images before stimulation. Images, with a resultant capture rate of six frames/second (resolution of 10 pixels/μm, average of five video frames), were analysed with the Metamorph program (Molecular Devices Corporation). Kinetics of exocytic events were measured as changes in SRB fluorescence from ROIs (0.78 μm2, 100 pixels) centered over granules. Traces were rejected if extensive movement was observed. All data are shown as the mean ± s.e.m.

Photoliberation of Ca2+ from NP-EGTA

An epifluorescent mercury light source provided high-intensity ultraviolet (UV) light to uncage Ca2+ from o-nitrophenyl (NP)-EGTA in a ~30-μm diameter field at the image plane. The duration of exposure to UV light was limited by a computer-controlled shutter (Prior) and was varied between 5 and 200 ms.

Fura-FF was calibrated in vivo by loading the cells with 2 μM Fura-FF-AM (for 30 minutes) and then permeabilizing with 500 nM ionomycin in the presence of a range of extracellular solutions of different [Ca2+]. For [Ca2+] of less than 1 μM we used the MAXC chelator program (Patton et al., 2004) to calculate the relative concentrations of Ca2+ and EGTA. For higher [Ca2+] we added Ca2+ directly to the medium. Fura-FF fluorescence was measured after equilibration and the fluorescence-Ca2+ curve fitted using GraphPad Prism giving a Keff of 1.84 μM.

In converting the Fura-FF changes induced by flash photolysis of NP-EGTA we assumed the resting fluorescence was the same as Fmin. Then Fmax was expressed as a fraction of Fmin based on the maximum change of fluorescence induced by ionomycin in the calibration experiments; which was Fmax=0.55Fmin. In this way we used the equation Ca2+ = (Fmin – F) ÷ [(F – (Fmin × 0.55)] × Keff to calculate [Ca2+] reached by each UV flash; Fmin is the fluorescence at minimum [Ca2+], Fmax is the fluorescence at maximum [Ca2+], Keff is for Fura-FF.

Image analysis

All morphometric analysis was performed using the Metamorph imaging suite. Individual cells were readily identified by the outline that is apparent in the extracellular SRB stain. However, some examples had cells lying on top of one another and we used the Fura-2 signal to aid in the identification of single cells. Here, the asynchronous Ca2+ response in each individual cell (as described in Fig. 1) supported unambiguous identification of single cells.

When measuring of distances between granules (Figs 3, 4, 6), we identified the approximate center of all fused granules labeled with SRB and measured granule-to-granule distances parallel to the length of the lumen. The two-photon z-thickness of 1.3 μm approximates to the diameter of the lumens between the cells. Experimentally, we focused through each tissue fragment and selected image planes to optimise the length of lumen observed (because this is where exocytosis exclusively occurs). Most images, therefore, have lumens much longer than the diameter of individual granules, making it simple to measure inter-granule distances parallel to the lumen. By contrast, where the lumen is complex, errors are possible in our estimates of inter-granule distance. However, this did not bias our data analyses because errors of above and below the estimate are equally probable. In calculating the expected granule-to-granule distances (Fig. 3) we assumed to have triggered the maximal exocytic response and a granule diameter of 1 μm, which were to give us the simple linear relationship of: y=0.4x; where y is the granule-to-granule distance and x is the length of the lumen.

In our estimates regarding the focal point of origin of a Ca2+ signal, our experimental approach was – again – to select image planes with long lumens. Knowing that InsP3Rs are located along the lumen (Lee et al., 1997; Nathanson et al., 1994; Yule et al., 1997), we were likely to image the origin of the Ca2+ signal in most recordings. By using the maximal increase of the Fura-2 signal in ROIs along the lumen we believe that, in most cases, were able to identify a single point of origin of a Ca2+ signal. In the few instances where the rate of increase of the Fura-2 signal appeared diffuse along the lumen – indicating a Ca2+-signal-origin outside the plane of focus – records were rejected.

We acknowledge the personnel in the Centre for Microscopy and Microanalysis of the University of Queensland for their help in teaching us electron microscopy. This research was supported by grants from the Australian Research Council Grant (DP0771481) and National Health Medical Research Council (456049).

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