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• W2062009818 abstract "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 β-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. 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 β-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. inositol trisphosphate 4,5)P3, inositol 2,4,5-trisphosphate secretory pole basal pole 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.Gray P.C. Scott J.D. Catterall W.A. Curr. Opin. Neurol. 1998; 8: 330-334Crossref Scopus (143) Google Scholar). 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.Fagni L. Chavis P. Ango F. Bockaert J. Trends Neurosci. 2000; 23: 80-88Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). 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.Mays R.W. Beck K.A. Nelson W.J. Curr. Opin. Cell Biol. 1994; 6: 16-24Crossref PubMed Scopus (136) Google Scholar, 4.Caplan M.J. Am. J. Physiol. 1997; 272, 4 Pt 2: F425-F429Google Scholar) 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.Nathanson M.H. Fallon M.B. Padfield P.J. Maranto A.R. J. Biol. Chem. 1994; 269: 4693-4696Abstract Full Text PDF PubMed Google Scholar, 6.Lee M.G. Xu X. Zeng W. Diaz J. Wojcikiewicz R.J. Kuo T.H. Wuytack F. Racymaekers L. Muallem S. J. Biol. Chem. 1997; 272 (15670): 15765Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar), unidirectional Ca2+ waves (7.Kasai H. Augustine G.J. Nature. 1990; 348: 735-738Crossref PubMed Scopus (311) Google Scholar, 8.Nathanson M.H. Padfield P.J. O'Sullivan A.J. Burgstahler A.D. Jamieson J.D. J. Biol. Chem. 1992; 267: 18118-18121Abstract Full Text PDF PubMed Google Scholar), and localized Ca2+ responses (9.Kasai H. Li Y.X. Miyashita Y. Cell. 1993; 74: 669-677Abstract Full Text PDF PubMed Scopus (314) Google Scholar, 10.Thorn P. Lawrie A.M. Smith P.M. Gallacher D.V. Petersen O.H. Cell. 1993; 74: 661-668Abstract Full Text PDF PubMed Scopus (422) Google Scholar). 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.Kasai H. Li Y.X. Miyashita Y. Cell. 1993; 74: 669-677Abstract Full Text PDF PubMed Scopus (314) Google Scholar, 10.Thorn P. Lawrie A.M. Smith P.M. Gallacher D.V. Petersen O.H. Cell. 1993; 74: 661-668Abstract Full Text PDF PubMed Scopus (422) Google Scholar, 11.Thorn P. Moreton R. Berridge M. EMBO J. 1996; 15: 999-1003Crossref PubMed Scopus (53) Google Scholar). 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. 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.Thorn P. Petersen O.H. J. Gen. Physiol. 1992; 100: 11-25Crossref PubMed Scopus (68) Google Scholar). Cells were plated onto poly-l-ornithine (Sigma, Poole, UK)-coated dishes and used within 3 h of isolation. Whole cell patch clamping was performed with an Axopatch 1D (Axon Instruments) patch clamp amplifier. Pipettes had a resistance of 3–5 MΩ (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 MΩ. The pipette solution contained (in millimolar): KCl 140, MgCl21, 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 CaCl2at 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 μminositol 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.Thorn P. Cell Calcium. 1996; 20: 203-214Crossref PubMed Scopus (31) Google Scholar). 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 experiments were performed by inclusion of 40–50 μmCa2+ 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 ΔF/F o images (100 × (F −F o)/F o), whereF is the recorded fluorescence and F owas obtained from the mean of the first 20 acquired frames. 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-α-tubulin or monoclonal mouse anti-α-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.Dictenberg J.B. Zimmerman W. Sparks C.A. Young A. Vidair C. Zheng Y. Carrington W. Fay F.S. Doxsey S.J. J. Cell Biol. 1998; 141: 163-174Crossref PubMed Scopus (407) Google Scholar, 15.Carrington W.A. Lynch R.M. Moore E.D. Isenberg G. Fogarty K.E. Fay F.S. Science. 1995; 268: 1483-1487Crossref PubMed Scopus (298) Google Scholar). 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.Carrington W.A. Lynch R.M. Moore E.D. Isenberg G. Fogarty K.E. Fay F.S. Science. 1995; 268: 1483-1487Crossref PubMed Scopus (298) Google Scholar) 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. 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.Thorn P. Lawrie A.M. Smith P.M. Gallacher D.V. Petersen O.H. Cell. 1993; 74: 661-668Abstract Full Text PDF PubMed Scopus (422) Google Scholar, 16.Kidd J.F. Fogarty K.E. Tuft R. Thorn P. J. Physiol. 1999; 520: 187-201Crossref PubMed Scopus (30) Google Scholar, 17.Fogarty K.E. Kidd J.F. Tuft R.A. Thorn P. Biophys. J. 2000; 78: 2298-2306Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar); 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. 1 A). The secretory pole region of acinar cells, i.e. the region where the Ca2+ spikes are localized, has an extensive network of microfilaments (18.Muallem S. Kwiatkowska K. Xu X. Yin H.L. J. Cell Biol. 1995; 128: 589-598Crossref PubMed Scopus (386) Google Scholar). 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. 1 B). Consistently, we observed that, on addition of 100 μm cytochalasin B to the bathing solution, there was a transient reduction of spike amplitude (Fig. 1 B,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.2 A 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.2 B). Application of the carrier alone (1% Me2SO, n = 3) had no effect on the spikes. 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). 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. 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. 5 A,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.Bergen L.G. Borisy G.G. J. Cell. Biochem. 1986; 30: 11-18Crossref PubMed Scopus (10) Google Scholar). 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 β-lumicolchicine (100 μm; Fig. 5, n = 5), a compound with similar structure to colchicine that has no action on tubulin (20.Salmon E.D. McKeel M. Hays T. J. Cell Biol. 1984; 99: 1066-1075Crossref PubMed Scopus (90) Google Scholar). 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.6 A, n = 6). In some cells taxol led to an immediate transient increase in the Cl(Ca) current before abolition of the response (Fig.6 B, n = 3/5). As with the nocodazole effects, after application of taxol, supramaximal concentrations of carbachol were still able to evoke a response (Fig. 6 B,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. 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.Achler C. Filmer D. Merte C. Drenckhahn D. J. Cell Biol. 1989; 109: 179-189Crossref PubMed Scopus (180) Google Scholar, 22.Marlowe K.J. Farshori P. Torgerson R.R. Anderson K.L. Miller L.J. McNiven M.A. Eur. J. Cell Biol. 1998; 75: 140-152Crossref PubMed Scopus (29) Google Scholar, 23.Kurihara H. Uchida K. Histochemistry. 1987; 87: 223-227Crossref PubMed Scopus (20) Google Scholar) 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-α-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.7 A, 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.7 B, typical results from three preparations). These experiments show that nocodazole does exert significant effects on the microtubular system in our isolated cells. 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.van de Put F.H.M.M. Elliott A.C. J. Biol. Chem. 1996; 271: 4999-5006Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) 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.9 A). Typically, the changes we observed included movement of the unstained region of the nucleus and decreased staining within the secretory pole region (Fig.9 B).Figure 9Two-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 μmnocodazole 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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. 9 C). 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. 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. In many cell types, patterns of IP3-evoked Ca2+ release are dependent on local positioning of Ca2+ release apparatus (25.Berridge M.J. J. Physiol. 1997; 499: 291-306Crossref PubMed Scopus (914) Google Scholar). In acinar cells apical to basal pole waves and local Ca2+ signals are due to polar compartmentalization of IP3-dependent stores (6.Lee M.G. Xu X. Zeng W. Diaz J. Wojcikiewicz R.J. Kuo T.H. Wuytack F. Racymaekers L. Muallem S. J. Biol. Chem. 1997; 272 (15670): 15765Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 7.Kasai H. Augustine G.J. Nature. 1990; 348: 735-738Crossref PubMed Scopus (311) Google Scholar, 13.Thorn P. Cell Calcium. 1996; 20: 203-214Crossref PubMed Scopus (31) Google Scholar). 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.Samson F. Donoso J.A. Heller-Bettinger I. Watson D. Himes R.H. J. Pharm. Exper. Therap. 1979; 208,(3): 411-417Google Scholar) or stabilize (27.Collins C.A. Vallee R.B. J. Cell Biol. 1987; 105, 6 Pt 1: 2847-2854Crossref Scopus (61) Google Scholar, 28.Wilson L. Miller H.P. Farrell K.W. Snyder K.B. Thompson W.C. Purich D.L. Biochemistry. 1985; 24: 5254-5262Crossref PubMed Scopus (78) Google Scholar) microtubule formation. However, colchicine only binds to free tubulin (19.Bergen L.G. Borisy G.G. J. Cell. Biochem. 1986; 30: 11-18Crossref PubMed Scopus (10) Google Scholar, 29.Vandecandelaere A. Martin S.R. Schilstra M.J. Bayley P.M. Biochemistry. 1994; 33: 2792-2801Crossref PubMed Scopus (34) Google Scholar) 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.Kidd J.F. Fogarty K.E. Tuft R. Thorn P. J. Physiol. 1999; 520: 187-201Crossref PubMed Scopus (30) Google Scholar). 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.Graier W.F. Paltauf-Doburzynska J. Hill B.J. Fleischhacker E. Hoebel B.G. Kostner G.M. Sturek M. J. Physiol. 1998; 506: 109-125Crossref PubMed Scopus (87) Google Scholar). 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 t" @default.
• W2062009818 created "2016-06-24" @default.
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• W2062009818 title "Microtubules Regulate Local Ca2+ Spiking in Secretory Epithelial Cells" @default.
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