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J. Biol. Chem., Vol. 275, Issue 29, 22487-22494, July 21, 2000

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Microtubules Regulate Local Ca²⁺ Spiking in Secretory Epithelial Cells^*

Kevin E. Fogarty^§, Jackie F. Kidd^§¶, Angelina Turner^¶, Jeremy N. Skepper^**, Jeff Carmichael, and Peter Thorn

From ^¶ The Department of Pharmacology, Cambridge University, Cambridge CB2 1QJ, United Kingdom, the  Biomedical Imaging Group, Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01650, and the ^** Multi-Imaging Centre, Department of Anatomy, Cambridge University, Cambridge CB2 3DY, United Kingdom

Received for publication, November 29, 1999, and in revised form, April 19, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The role of the cytoskeleton in regulating Ca²⁺ release has been explored in epithelial cells. Trains of local Ca²⁺ spikes were elicited in pancreatic acinar cells by infusion of inositol trisphosphate through a whole cell patch pipette, and the Ca²⁺-dependent Cl current spikes were recorded. The spikes were only transiently inhibited by cytochalasin B, an agent that acts on microfilaments. In contrast, nocodazole (5-100 µM), an agent that disrupts the microtubular network, dose-dependently reduced spike frequency and decreased spike amplitude leading to total blockade of the response. Consistent with an effect of microtubular disruption, colchicine also inhibited spiking but neither Me₂SO nor -lumicolchicine, an inactive analogue of colchicine, had any effect. The microtubule-stabilizing agent, taxol, also inhibited spiking. The nocodazole effects were not due to complete loss of function of the Ca²⁺ signaling apparatus, because supramaximal carbachol concentrations were still able to mobilize a Ca²⁺ response. Finally, as visualized by 2-photon excitation microscopy of ER-Tracker, nocodazole promoted a loss of the endoplasmic reticulum in the secretory pole region. We conclude that microtubules specifically maintain localized Ca²⁺ spikes at least in part because of the local positioning of the endoplasmic reticulum.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The localization of signaling complexes is important for the specificity of action of signals within a cell. For example, the tethering of protein kinase A to protein kinase A-associated proteins is used to direct global cAMP signals to specifically regulate proteins linked to protein kinase A-associated protein (1). Another example is Homer, a protein that anchors intracellular release channels close to metabotropic glutamate receptors, and so functionally couples local inositol trisphosphate (IP₃)¹ production with local IP₃ receptors (2). The cytoskeleton is thought to play a role in the cellular positioning of these signaling complexes. In our experiments we sought to determine a role for the cytoskeleton in regional positioning of the Ca²⁺ release apparatus in polarized epithelial cells.

The cytoskeleton maintains the polarization observed in many epithelial cells (3, 4) and, therefore, might be expected to play a role in second messenger signaling cascades. Many epithelia exhibit polarization of Ca²⁺ signaling pathways, including the differential distribution of IP₃ receptors (5, 6), unidirectional Ca²⁺ waves (7, 8), and localized Ca²⁺ responses (9, 10). However, to date, there have been no direct experiments to investigate the role of the cytoskeleton in shaping these signaling elements.

In this study we have used acutely isolated mouse pancreatic acinar cells and established trains of Ca²⁺-dependent current spikes by the infusion of IP₃ through a whole cell patch pipette. These spikes have previously been shown to be due to localized Ca²⁺ release in the secretory pole region (as identified by the clustering of secretory granules) (9-11). During the trains of IP₃-induced spikes, we tested the effects of agents that affect microfilaments and microtubules. Microfilament disruption transiently affected the response, whereas agents that act on microtubules specifically inhibited the local Ca²⁺ spikes but left the responses to supramaximal carbachol concentrations intact even after an extended time period (up to 1.5 h). We determined that microtubule disruption led to a redistribution of the endoplasmic reticulum away from the secretory pole region. We conclude that microtubules are essential in maintaining local Ca²⁺ spikes, at least in part by locally positioning the endoplasmic reticulum.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Preparation-- Fresh isolated mouse pancreatic acinar cells were prepared by collagenase (CLSPA, Worthington, Lakewood, NJ) digestion at 36 °C for 7 min as described previously (12). Cells were plated onto poly-L-ornithine (Sigma, Poole, UK)-coated dishes and used within 3 h of isolation.

Patch Clamp-- Whole cell patch clamping was performed with an Axopatch 1D (Axon Instruments) patch clamp amplifier. Pipettes had a resistance of 3-5 M (pipette puller; Brown and Flaming, Sutter Instruments, Novato, CA) and, after breaking through to whole cell had a measured, but uncompensated series resistance of 10-20 M. The pipette solution contained (in millimolar): KCl 140, MgCl₂ 1, EGTA or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid 0.5, KOH-HEPES 10, ATP 2, pH 7.2, inositol 2,4,5-trisphosphate (Ins(2,4,5)P₃) 0.01, with the free [Ca²⁺] fixed at 50 or 100 nM by the addition of CaCl₂ at appropriate concentrations (MAXC; Chris Patten, Pacific Grove, CA). The extracellular solution contained (in millimolar): NaCl 135, KCl 5, MgCl₂ 1, CaCl₂ 1, glucose 10, NaOH-HEPES 10, pH 7.4. Drugs (all obtained from Sigma) were bolus-applied to the bathing solution, and all experiments were conducted at room temperature (~21 °C). The inclusion of 10-12 µM inositol 2,4,5-trisphosphate (gift from Professor R. Irvine) in the pipette solution elicited a train of short lasting Ca²⁺-dependent current spikes, previously shown to be a good correlate of localized Ca²⁺ release in the secretory pole of acinar cells (13). The spikes were recorded on a computer using an analogue/digital interface (National Instruments, Austin, TX) and a data acquisition program (J. Dempster). Current amplitudes and current frequency were determined and analyzed with an Excel spreadsheet (Microsoft, OR). In the experiments of Fig. 3 (inset), the pipette solution contained (in millimolar): NMDGCl 40, calcium gluconate 1.71, MgCl₂ 6.77, N-(2-hydroxyethyl)-ethylenediamine-triacetic acid, 10, calculated to give a final free [Ca²⁺] of 448 nM using the computer algorithm MAXC. The osmolarity was adjusted with mannitol to 300 mOsm. In the experiments of Fig. 3 (inset), cells were whole cell voltage-clamped at a potential of 38 mV and voltage steps made in 10 mV increments between 68 and +82 mV. Currents were sampled at 2 kHz, and the peak current amplitudes for each voltage step were recorded as the mean over a 100-ms period at the end of the 2.5-s pulse.

Ca²⁺ Imaging-- Ca²⁺ imaging experiments were performed by inclusion of 40-50 µM Ca²⁺ Green (Molecular Probes, Eugene, OR) in the pipette solution. Cells were illuminated with a visible laser (Annova 70; Coherent, Santa Clara, CA) at 488 nm and imaged through a Nikon 40× UV, 1.2 numerical aperature, oil immersion objective. Full-frame images (128 × 128 pixels) were captured on a cooled charge-coupled device camera (70% quantum efficiency, 5 electrons of readout noise; Lincoln Laboratories, Massachusetts Institute of Technology, Cambridge, MA) with a pixel size of 200 nm at the specimen and at frame rates of up to 500 Hz. After recording on the computer, the data were analyzed with custom software with bleach correction routines and appropriate smoothing. Data was recorded as F/F_o images (100 × (F  F_o)/F_o), where F is the recorded fluorescence and F_o was obtained from the mean of the first 20 acquired frames.

Immunohistochemistry-- Cells were prepared as for the patch clamp experiments and plated onto glass coverslips. Next the cells were incubated for 15-20 min in control extracellular solution, solution containing 1% Me₂SO, or solution containing 100 µM nocodazole. At the end of the incubation period the cells were fixed in 2% paraformaldehyde for 15 min and then quenched with ethanolamine, permeabilized with 0.1% Triton, and washed with phosphate-buffered saline. Primary antibodies, either polyclonal rabbit anti--tubulin or monoclonal mouse anti--tubulin (Sigma), were incubated for 1 h at room temperature (with 3% bovine serum albumin). The cells were then washed three times before addition of either donkey anti-rabbit or goat anti-mouse secondary antibody conjugated to Oregon Green for 1 h at 4 °C. The cells were then washed three times and mounted. The cell fluorescence was imaged in three dimensions and restored as described previously (14, 15).

Visualizing the Endoplasmic Reticulum-- We used two methods to observe the endoplasmic reticulum, both using the Dapoxyl probe ER-Tracker (Molecular Probes). The first method used three-dimensional image reconstruction techniques as described (15) with a microscope (Olympus IX70; Melville, NY), an Olympus PL APO 60 × 1.4 numerical aperature oil immersion objective and a 0.25 µm Z section resolution. After cell preparation we incubated the cells in 100-200 nM ER-Tracker for 20-30 min. The cells were then centrifuged, resuspended in normal extracellular solution, and plated onto glass coverslips. These were then treated with drugs before fluorescence microscopy analysis.

In the second method the cells were prepared in exactly the same way but two-photon excitation microscopy (model TCS-SP-MP; Leica Microsystems, Heidelberg, Germany) was used to record the fluorescence signal. Small groups of cells were selected in phase contrast using an infinity-corrected, 63× water immersion, 1.2 numerical aperature, plan apochromatic lens with a cover glass correction collar and a 225-µm working distance. The ER-Tracker was excited by laser light from a solid state Millenia V-pumped Tsunami Ti/sapphire laser tuned to 800 nm, with a pulse width of 1.3 ps and a repetition rate of 82 MHz. Emitted light was captured with a spectrophotometer detector using a window of 450-700 nm. A series of optical sections, with 1-µm increments between images, was taken through the cells to build up a three-dimensional picture of the fluorescence distribution. Drugs were bath-applied after the first series of optical sections had been captured, and further series were captured every 5 min for up to 40 min.

Image analysis was performed using the computer program Lucida (Kinetic Imaging, Liverpool, UK). We measured the average fluorescence in secretory pole (SP) and basal pole (BP) regions (within regions of about 5-µm diameter) and expressed them as a ratio (SP/BP). For each cell, all values were expressed as a percentage of the initial ratio obtained at time 0. The SP/BP ratio, obtained from the same regions, was then followed over time to give an indication of regional changes in fluorescence.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We whole cell patch clamped single mouse pancreatic acinar cells and established a train of Ca²⁺ spikes by the infusion of 10-12 µM Ins(2,4,5)P₃ through the pipette solution. Previous work has shown that the activation of Cl_(Ca) current spikes are a faithful record of a local secretory pole Ca²⁺ signal (10, 16, 17); therefore, we recorded the whole cell currents as a convenient measure of the regional Ca²⁺ spikes. The injection of Ins(2,4,5)P₃ circumvents cell surface receptors and allows the direct study of the mechanisms of IP₃-dependent Ca²⁺ release. Typically, once the whole cell has been established and, after a short period of equilibration (~1 min), a train of Cl_(Ca) spikes are established that continue for the lifetime of the whole cell (up to 40 min, Fig. 1A).

View larger version (55K): [in this window] [in a new window]   Fig. 1.   Whole cell Cl(Ca) current spikes induced by Ins(2,4,5)P3 (10 µM) infusion into a single pancreatic acinar cell. A, cells were voltage-clamped to 30 mV, and the downward deflection of current (spikes) is due to the activation of Cl(Ca) channels. The spikes have a duration of 2 s (see inset) and have previously been shown to be specifically associated with a local Ca2+ signal in the secretory pole region. The horizontal line on the left of the current records in this and other figures is the zero current line. Small changes in the baseline of the current signal are a reflection of changes in pipette seal resistance or possibly small, slow fluctuations in intracellular Ca2+. Spike activity continued for the length of the whole cell recording with little change in the characteristics of spike amplitude and frequency. These trains of whole cell current spikes were used to test the effects of agents that affect the cytoskeleton. B, the addition of cytochalasin B (100 µM) to the bathing solution temporarily decreased the amplitude of the spikes, which thereafter resumed a similar pattern of activity to the control period.

The secretory pole region of acinar cells, i.e. the region where the Ca²⁺ spikes are localized, has an extensive network of microfilaments (18). To test for a role of microfilaments in the mechanism of the generation of the Ca²⁺ spikes, we applied cytochalasin B during an IP₃-induced spike train (Fig. 1B). Consistently, we observed that, on addition of 100 µM cytochalasin B to the bathing solution, there was a transient reduction of spike amplitude (Fig. 1B, n = 3). However, once resumed, the spike activity was apparently no different from the control period before addition of the drug. We conclude that, although the transient inhibition suggests some role, microfilaments are not essential for the maintenance of the local Ca²⁺ spikes.

We then tested the effects of agents known to act on microtubules. Fig. 2A shows that the application of nocodazole, an agent known to promote microtubule depolymerization, to the bathing solution led to a cessation of spiking characterized by an initial decrease in spike amplitude followed by a decrease in frequency (n = 11). Lower concentrations of nocodazole did not have such rapid effects but instead led to a slower dose-dependent decrease in spike amplitude (Fig. 2B). Application of the carrier alone (1% Me₂SO, n = 3) had no effect on the spikes.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   A, after establishment of a train of spikes the bath application of 100 µM nocodazole led to a rapid decrease in spike amplitude followed by a decrease in frequency. B, the effects on spike amplitude were measured by cumulative dose-response experiments. In these experiments nocodazole was applied and the amplitude of the spikes recorded. After they had reached a stable level, the spike amplitude was measured. Then higher concentrations of nocodazole were added, and, again after reaching a stable amplitude, the spike size was measured. The results showed a dose-dependent effect of nocodazole on spike amplitude with an approximate IC50 of 10.4 µM and a Hill coefficient of 2.1.

Given the widespread importance of the microtubular network in cell physiology, the effect of nocodazole treatment might be nonspecific and reflect a general compromise of cell function. However, we showed that, after nocodazole had completely abolished the IP₃-induced spikes, the cells were still able to respond to a supramaximal concentration of carbachol (Fig. 3, 1 mM carbachol, n = 3). In fact, we found in other experiments that this supramaximal carbachol response, measured using Ca²⁺ fluorescence techniques, was still maintained after 1.5 h (maximum tested) of nocodazole treatment (n = 4/5 cells; 1 cell showed no response, data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Application of nocodazole (100 µM) abolished the train of IP3-induced spikes, but the subsequent bath application of a supramaximal concentration of the muscarinic agonist carbachol (1 mM) led to a rapid and reversible Cl(Ca) current response. Inset, current-voltage relationships obtained from three cells demonstrate that nocodazole has no direct effect on the Cl(Ca) current. The graph shows the mean and the standard error (downward error bars for nocodazole data, upward for control data).

The above experiments indicate a specific effect of nocodazole, but in our experiments nocodazole might be directly affecting the Cl_(Ca) currents and not the underlying Ca²⁺ signal. We addressed this issue in two ways. First, we directly activated the Cl_(Ca) current by the infusion of an intracellular solution containing 448 nM free Ca²⁺ via the whole cell patch pipette. The current-voltage relationships obtained before and after 100 µM nocodazole (Fig. 3, inset, n = 3) showed no difference in amplitude. Second, we combined patch clamp and Ca²⁺ imaging experiments and directly measured the local secretory pole Ca²⁺ response. Nocodazole (25 µM) reduced the Cl_(Ca) current spike amplitude, and this was associated with a reduction in the cytosolic Ca²⁺ rise time and amplitude (Fig. 4, n = 3). We conclude that nocodazole specifically affects the local Ca²⁺ spike and not the Cl_(Ca) current.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Simultaneous measurement of the secretory pole Ca2+ signal (using Calcium Green) with the Cl(Ca) whole cell current. The control experiment in A shows that the Cl(Ca) current spike is associated with a rise in the Ca2+ signal recorded in the secretory pole. B, after treatment with nocodazole (25 µM) the Cl(Ca) current was reduced in amplitude as was the rate of rise and amplitude of the Ca2+ response. In cells where the Cl(Ca) current was abolished by nocodazole, no Ca2+ spikes were observed.

These experiments indicate that nocodazole acts on the mechanism of generation of the local secretory pole Ca²⁺ spike. From the known actions of nocodazole it is implied that its effects are mediated by disruption of the microtubular system. To test this we looked for consistency of action of other agents known to act on microtubules. Colchicine application consistently led to a decrease in spike amplitude (Fig. 5A, n = 4), and in two cells a decrease in frequency resulted. The effects of colchicine were slower in onset than nocodazole, with observable effects of colchicine on spike amplitude found at around 3 min after drug application. This is probably a reflection of the action of colchicine, which only binds to free tubulin and does not act directly on tubulin polymerized within microtubules (taxol and nocodazole act on polymerized tubulin) (19). A higher concentration of colchicine (200 µM) blocked the spikes (n = 3). A demonstration that these effects were likely to be specific to an action on the microtubular system is shown by the lack of effect of -lumicolchicine (100 µM; Fig. 5, n = 5), a compound with similar structure to colchicine that has no action on tubulin (20).

View larger version (53K): [in this window] [in a new window]   Fig. 5.   A, bath application of colchicine (100 µM) led to a reduction in spike amplitude and frequency. By comparison to the effects of nocodazole, these effects were delayed in onset. B, this effect was not observed in control experiments with -lumicolchicine, an analogue of colchicine inactive at microtubules.

Another agent that affects microtubules is taxol. Taxol binds to, and stabilizes, microtubules, and we might therefore expect some effect on the Ca²⁺ signal. The addition of 10 µM taxol to the bathing solution led to a loss of spiking (Fig. 6A, n = 6). In some cells taxol led to an immediate transient increase in the Cl_(Ca) current before abolition of the response (Fig. 6B, n = 3/5). As with the nocodazole effects, after application of taxol, supramaximal concentrations of carbachol were still able to evoke a response (Fig. 6B, n = 3), indicating the cells were still viable. Furthermore, the current-voltage relationships obtained before and after 10 µM taxol (n = 3, data not shown) application showed no difference in amplitude.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   A, bath application of taxol (100 µM) rapidly blocked the train of Cl(Ca) current spikes. In other cells taxol led to a transient increase in spike activity and an increase in the baseline current (B) before attenuating spiking. B, as with the nocodazole effects we could show that carbachol responses were still maintained after spikes had been abolished.

Our data are therefore consistent with a role for microtubules in the mechanism of local IP₃-dependent Ca²⁺ release from Ca²⁺ stores. Although the microtubular network has been described for pancreatic acinar cells (21-23) from slices of pancreas, it not been shown in the type of isolated cell preparations we used. Therefore, we performed immunolocalization experiments, using an anti--tubulin antibody, on isolated cells that were prepared in the same way as for the previous electrophysiological experiments. The results show a complex network of microtubules throughout the cell (Fig. 7A, typical of five preparations). In cells that had been treated with nocodazole for 15 min, the microtubular network was less abundant and showed evidence for truncated tubules, rather than continuous microtubule strands (Fig. 7B, typical results from three preparations). These experiments show that nocodazole does exert significant effects on the microtubular system in our isolated cells.

View larger version (62K): [in this window] [in a new window]   Fig. 7.   Immunolocalization of the microtubules using an antibody raised against -tubulin. 36 serial z-sections were taken through the cell and then projected onto the single images shown. A, a typical example of a two-cell cluster, a dense network of microtubules with some concentration around the secretory pole. B, different cells obtained from the same preparation showed that after treatment with nocodazole the number of microtubules was reduced and those remaining were broken and truncated. Scale bar, 10 µm.

We next explored the possible relationship between the microtubule system and the Ca²⁺ release apparatus. It is well known that microtubules are associated with the organization of the endoplasmic reticulum, and this action may be the source of the functional effects we observe. We visualized the endoplasmic reticulum distribution with the specific probe, ER-Tracker. As described previously, the endoplasmic reticulum was distributed throughout the cell (24) but was excluded from the nucleus and the secretory granules (Fig. 8). In control experiments we also studied the distribution of the endoplasmic reticulum resident proteins, calreticulin and BiP, using immunolocalization techniques. Both proteins had a similar apparent cellular distribution to ER-Tracker (data not shown). Two-photon fluorescence imaging methods were used to visualize the endoplasmic reticulum during drug application, using the ER-Tracker dye. We observed changes in the distribution of the endoplasmic reticulum after treatment with nocodazole (100 µM, up to 40 min, n = 7/9 cells; 2 cells showed no apparent change) compared with controls (no drug added, n = 5/6 cells; 1 cell showed small changes in the secretory pole; Fig. 9A). Typically, the changes we observed included movement of the unstained region of the nucleus and decreased staining within the secretory pole region (Fig. 9B).

View larger version (91K): [in this window] [in a new window]   Fig. 8.   Using three-dimensional image reconstruction techniques, the fluorescence signal from the endoplasmic reticulum probe ER-Tracker was localized within a two-cell cluster of acinar cells. Serial optical sections 1.25 µm apart (identified on the figure) were taken through the cells. The endoplasmic reticulum was found throughout the cell except within the nuclear region and secretory granules.

View larger version (53K): [in this window] [in a new window]   Fig. 9.   Two-photon images of ER-Tracker fluorescence taken from a mid section through an acinar cell cluster. The endoplasmic reticulum is widely distributed throughout the cell but, as expected, fluorescence is excluded from the nucleus and the secretory granules. Under control conditions (A) the pattern of staining did not change when recording over time. In B, control images were obtained at 0 min, after which 100 µM nocodazole was added to the bath. Subsequently images were captured at 5 and 20 min after nocodazole treatment. The pattern of fluorescence was observed to change in the secretory pole region and in the position and shape of the excluded fluorescence in the nuclear region (n = 7/9, with two cells showing no apparent changes). C, the ratio of the mean fluorescence intensity within a 5-µm diameter area in the secretory pole (SP) against a spot of 5-µm diameter in the basal pole (BP) was measured in the same regions over time. Within a single cell, all values were expressed as a percentage of the ratio obtained at time 0. The ratios do not change in control conditions but show a decrease over time in the presence of nocodazole. This decrease is significant (at p < 0.05, Student's t test) at times 10 and 20 min after the addition of nocodazole, compared with controls.

To quantify these fluorescence changes, we measured the average signal intensity in a region within the secretory pole (~5-µm diameter) and a region in the basal pole (~5-µm diameter chosen to be away from the nucleus). The ratio of the secretory pole to basal pole signal (SP/BP ratio) was then used as a measure of changes in endoplasmic reticulum distribution. In control conditions we observed no change in the SP/BP ratio over time (Fig. 9C). After treatment with nocodazole the ratio decreased significantly (p < 0.05 at 10 and 20 min after drug treatment, compared with controls) indicating a reorganization of the endoplasmic reticulum away from the secretory pole.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We show agents that act on microtubules have a specific and rapid effect in attenuating IP₃-evoked local Ca²⁺ spikes. Responses to supramaximal agonist concentrations were still observed, even after prolonged treatment with nocodazole, indicating that cell function was still retained and that the microtubular cytoskeleton is not critical for these global Ca²⁺ signals. Microtubular disruption induced a specific decrease of the endoplasmic reticulum in the secretory pole, as visualized by a local loss of ER-Tracker fluorescence. This loss was significant at 10 min after nocodazole treatment, a time course consistent with the effects of nocodazole on the Ca²⁺ spikes. We conclude that the microtubular network specifically maintains local Ca²⁺ responses possibly by local positioning of the endoplasmic reticulum.

There are now a number of recent studies that indicate that the cytoskeleton may play a role in Ca²⁺ signaling processes. Although the cell type and stimulus and signal responses are diverse in these reports, the common thread is a cytoskeletal involvement in signal compartmentalization.

IP3-evoked Release Compartment-- In many cell types, patterns of IP₃-evoked Ca²⁺ release are dependent on local positioning of Ca²⁺ release apparatus (25). In acinar cells apical to basal pole waves and local Ca²⁺ signals are due to polar compartmentalization of IP₃-dependent stores (6, 7, 13). Our work now shows that the functionality of the apical compartment, which generates the local Ca²⁺ spike, is maintained by the microtubular system. This conclusion is based on the consistency of action of agents that target microtubules. Both nocodazole and taxol bind to polymerized tubulin and, respectively, prevent (26) or stabilize (27, 28) microtubule formation. However, colchicine only binds to free tubulin (19, 29) and secondarily interferes with microtubule polymerization. The fact that all these agents inhibit local Ca²⁺ spiking strongly argues for a crucial role of the microtubular system in maintaining the function of Ca²⁺ release sites within the secretory pole region.

If the function of the Ca²⁺ release apparatus is dependent on microtubules, why do we see effects on the local Ca²⁺ spike and not on the carbachol-induced global Ca²⁺ signal? We know that the local Ca²⁺ spike, which we elicited at low IP₃ concentrations (just above threshold), is the reflection of multiple sites of Ca²⁺ release within the apical region that are coordinated together by the action of cytosolic Ca²⁺ (16). Therefore, the architectural arrangement of these release sites within the cell might be an important parameter in the production of the local Ca²⁺ spike. Microtubules could act to position the release sites, and microtubule reorganization might move the sites far enough apart such that Ca²⁺ release from one site would not be able to act on an adjacent site to coordinate the signal response via Ca²⁺-induced Ca²⁺ release. In contrast, the Ca²⁺ release elicited by high agonist concentrations reflects a synchronized global response to saturating IP₃ concentrations. The precise position of Ca²⁺ release sites within the cell would therefore be expected to be of less importance.

A few studies suggest that microtubules underlie the location of Ca²⁺ release within a cell (e.g. see Ref. 30). Specific support for a role of microtubules in precisely and locally positioning Ca²⁺ release sites comes from work on endothelial cells. In these cells colchimid treatment for 24 h led to a cellular relocation of caveolin, a protein associated with caveolae, from the cell surface to the interior. Associated with the movement of caveolin was a parallel relocation of sites of Ca²⁺ release (31).

Store-operated Ca²⁺ Entry: Endoplasmic Reticulum Compartment-- Modification of the cytoskeleton has been shown to attenuate store-operated Ca²⁺ entry. These experiments have been used as an argument to support a conformational coupling between the IP₃ receptor and the Ca²⁺ entry channel. However, it is not clear at the moment if microtubules or microfilaments play a specific role in this process or if their action is an indirect effect of changes in cell shape. In type 1 astrocytes, microfilament or microtubule disruption abolished a cAMP-mediated up-regulation of Ca²⁺ entry (32), and cytoskeletal disruption was associated with changes in the endoplasmic reticulum, as visualized with ER-Tracker. Abolition of store-operated Ca²⁺ entry has also been seen in HEK293 cells by a calyculin A-mediated production of a cortical actin network (33). In support of a role for microfilaments, their disruption abolishes Ca²⁺ entry into endothelial cells (34). In complete contrast to the above work, Ribeiro et al. (35) show that neither microfilament or microtubule disruption affect store-dependent Ca²⁺ entry in fibroblasts, even though they observed dramatic changes in cell shape and endoplasmic reticulum distribution.

What Ribeiro et al. (35) have shown is that that 1-h treatment with cytochalasin D or nocodazole abolished agonist-evoked global Ca²⁺ signals in fibroblasts. This effect was not due to a reduction in IP₃ production, a loss of IP₃-evoked Ca²⁺ release, or an effect on Ca²⁺ influx. They explained their data in terms of a role for the cytoskeleton in maintaining a subplasma membrane compartment where phospholipase C, and therefore IP₃ production, is positioned close to the IP₃ receptors. In their view, disruption of the cytoskeleton moves these components far enough apart such that IP₃ degradation becomes significant. Consistent with the idea of a local compartment of IP₃ production, work in polarized epithelial cells suggests that phospholipase C activation in the apical or basal membrane leads to domain-specific responses (36). However, in other cells this is not the case and here it can be shown that IP₃ acts as a global messenger (37). For example, in acinar cells it is clear that agonist action at receptors on the basal pole leads to global elevation in IP₃ and a primary effect on Ca²⁺ release sites at the opposite, secretory (apical) pole (7). In fact, in contrast to Ribeiro et al., evidence from endothelial cells (30, 31, 34), type 1 astrocytes (32), and the work we now present in acinar cells (studying the global Ca²⁺ response) shows that agonists are capable of inducing responses after cytoskeletal disruption.

With reference to our own work, the local Ca²⁺ responses in acinar cells that we have recorded show little dependence on Ca²⁺ influx (38). In our experiments (data not shown) we found that removal of extracellular Ca²⁺ had no acute effects on Ca²⁺ spiking. This indicates that the effects of microtubular disruption we observe are not due to effects on a Ca²⁺ entry mechanism.

Mechanism of Action of Microtubules on Local Ca²⁺ Release in Acinar Cells-- The effects we observe indicate that microtubules are important in maintaining the local Ca²⁺ response. However, the link between microtubules and the Ca²⁺ release apparatus is not clear. We consider here two likely potential components of the Ca²⁺ release apparatus, the endoplasmic reticulum and the IP₃ receptor, that might interact with microtubules.

It is well known that endoplasmic reticulum is associated with the microtubular network (39, 40) and potentially involves multiple transport and localization mechanisms (41). The mechanisms for this association remain unclear (42, 43) but lead to the movement of endoplasmic reticulum vesicles along microtubule tracks and association of endoplasmic reticulum with growing plus ends of microtubules (41). These processes probably do play a role in positioning the endoplasmic reticulum in acinar cells. In support of this, after nocodazole treatment, we show a specific regional reorganization of the endoplasmic reticulum, which we have quantified as a loss of the endoplasmic reticulum in the secretory pole. Furthermore, our study shows that significant effects are seen after 10 min of nocodazole treatment, which is consistent with the rapid time course of the loss of the Ca²⁺ spikes we observe.

Despite the fact that we do observe changes in the distribution of the endoplasmic reticulum, it is difficult to conceive of a microtubule-endoplasmic reticulum interaction, which alone could explain the establishment of local Ca²⁺ responses in acinar cells. The endoplasmic reticulum is widespread throughout the acinar cell with a predominance in the basal pole. Functionally, we know that the endoplasmic reticulum is heterogeneous with local regions of protein exported to the Golgi (44, 45) and other regions specialized for Ca²⁺ release (25). A mechanism for positioning of the Ca²⁺ release apparatus in the apical pole would therefore have to distinguish between endoplasmic reticulum destined for each of the two poles.

In terms of the generation of the local Ca²⁺ signal, interaction between IP₃ receptors and microtubules could provide a direct way for the cell to position the secretory pole Ca²⁺ release apparatus. Disruption of the microtubular network might affect the IP₃ receptor in two ways. First, untethering the IP₃ receptors may lead to a spatial disorganization in the secretory pole region. As speculated above, positioning of Ca²⁺ release sites may be a critical factor in maintaining the local Ca²⁺ spike. Second, uncoupling the IP₃ receptor from the microtubules may more directly affect IP₃ receptor function. In support of an effect on IP₃ receptor function, it has been shown, in Xenopus oocyte microsomes, that taxol (but not nocodazole) reduced the effectiveness of IP₃ to evoke Ca²⁺ release (46). This work implies that taxol modifies the function of the IP₃ receptor. Further support for a direct interaction between microtubules and IP₃ receptors comes from experiments in mast cells that showed that the agonist-evoked Ca²⁺ signal was lost after colchicine treatment. This effect was demonstrated to be due to a direct block at the IP₃ receptor (47). Although we did not see block of the agonist-evoked global signals after nocodazole treatment, it is possible that the behavior of the IP₃ receptor (such as affinity for IP₃) may have changed in our experiments. This might lead to our observations of a block of the local response while sparing the global signal.

Microtubule Dynamics during Agonist-evoked Responses-- Agonists have been shown to induce changes in the cytoskeleton of acinar cells and play a role in modulating the secretory response (18, 48). These effects may be mediated by a variety of mechanisms. For example, it is known that receptor-dependent activation of cytoskeleton-directed kinases, such as p38 mitogen-activated protein kinase, can modulate the actin microfilament network (49). In addition, second messenger signals may regulate the cytoskeleton. For example, Ca²⁺ (50) or Ca²⁺-calmodulin can lead to microtubule disassembly (51), an effect that may be modulated by specific microtubule-associated proteins (52). This potential feedback process of Ca²⁺ on the cytoskeleton might be important for microtubule localization in the secretory pole region. Indeed, it is known that calmodulin does translocate to the secretory pole region (53) and, therefore, may well play a role in local microtubule dynamics.

Conclusion-- Our data provide functional evidence that the microtubular network plays an important and specific role in the maintenance of local Ca²⁺ spikes. We suggest that this network maintains the position of the Ca²⁺ release apparatus via a local organization of the endoplasmic reticulum.

	ACKNOWLEDGEMENT

The in vitro two-photon imaging studies were carried out in the Multi-Imaging Center on equipment generously provided by the Wellcome Trust.

	FOOTNOTES

^* This work was supported by The Wellcome Trust (Biomedical Research Collaboration Grant to P. T. and R. A. T.), The Wellcome Trust Showcase Award (to P. T. and A. T.), the Medical Research Council (project grant to P. T. and Dr. T. R. Cheek), The Royal Society (project grant to P. T.), National Science Foundation Grants DBI-9200027 and DBI-9724611, and National Institutes of Health Grant R01-5RR09799 (to Walter Carrington supporting K. E. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ These authors contributed equally to this work.

Recipient of a Biotechnological and Biological Sciences Research Council Studentship.

To whom correspondence should be addressed: Dept. of Pharmacology, Cambridge University, Tennis Court Rd., Cambridge CB2 1 QJ, United Kingdom. Tel.: 01223-334017; Fax: 01223-334040; E-mail: pt207@cus.cam.ac.uk.

Published, JBC Papers in Press, May 8, 2000, DOI 10.1074/jbc.M909402199

	ABBREVIATIONS

The abbreviations used are: IP₃, inositol trisphosphate; Ins(2, 4,5)P₃, inositol 2,4,5-trisphosphate; SP, secretory pole; BP, basal pole.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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