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• W1996681454 abstract "Tomosyn, a soluble R-SNARE protein identified as a binding partner of the Q-SNARE syntaxin 1A, is thought to be critical in setting the level of fusion-competent SNARE complexes for neurosecretion. To date, there has been no direct evaluation of the dynamics in which tomosyn transits through tomosyn-SNARE complexes or of the extent to which tomosyn-SNARE complexes are regulated by secretory demand. Here, we employed biochemical and optical approaches to characterize the dynamic properties of tomosyn-syntaxin 1A complexes in live adrenal chromaffin cells. We demonstrate that secretagogue stimulation results in the rapid translocation of tomosyn from the cytosol to plasma membrane regions and that this translocation is associated with an increase in the tomosyn-syntaxin 1A interaction, including increased cycling of tomosyn into tomosyn-SNARE complexes. The secretagogue-induced interaction was strongly reduced by pharmacological inhibition of the Rho-associated coiled-coil forming kinase, a result consistent with findings demonstrating secretagogue-induced activation of RhoA. Stimulation of chromaffin cells with lysophosphatidic acid, a nonsecretory stimulus that strongly activates RhoA, resulted in effects on tomosyn similar to that of application of the secretagogue. In PC-12 cells overexpressing tomosyn, secretagogue stimulation in the presence of lysophosphatidic acid resulted in reduced evoked secretory responses, an effect that was eliminated upon inhibition of Rho-associated coiled-coil forming kinase. Moreover, this effect required an intact interaction between tomosyn and syntaxin 1A. Thus, modulation of the tomosyn-syntaxin 1A interaction in response to secretagogue activation is an important mechanism allowing for dynamic regulation of the secretory response. Tomosyn, a soluble R-SNARE protein identified as a binding partner of the Q-SNARE syntaxin 1A, is thought to be critical in setting the level of fusion-competent SNARE complexes for neurosecretion. To date, there has been no direct evaluation of the dynamics in which tomosyn transits through tomosyn-SNARE complexes or of the extent to which tomosyn-SNARE complexes are regulated by secretory demand. Here, we employed biochemical and optical approaches to characterize the dynamic properties of tomosyn-syntaxin 1A complexes in live adrenal chromaffin cells. We demonstrate that secretagogue stimulation results in the rapid translocation of tomosyn from the cytosol to plasma membrane regions and that this translocation is associated with an increase in the tomosyn-syntaxin 1A interaction, including increased cycling of tomosyn into tomosyn-SNARE complexes. The secretagogue-induced interaction was strongly reduced by pharmacological inhibition of the Rho-associated coiled-coil forming kinase, a result consistent with findings demonstrating secretagogue-induced activation of RhoA. Stimulation of chromaffin cells with lysophosphatidic acid, a nonsecretory stimulus that strongly activates RhoA, resulted in effects on tomosyn similar to that of application of the secretagogue. In PC-12 cells overexpressing tomosyn, secretagogue stimulation in the presence of lysophosphatidic acid resulted in reduced evoked secretory responses, an effect that was eliminated upon inhibition of Rho-associated coiled-coil forming kinase. Moreover, this effect required an intact interaction between tomosyn and syntaxin 1A. Thus, modulation of the tomosyn-syntaxin 1A interaction in response to secretagogue activation is an important mechanism allowing for dynamic regulation of the secretory response. Regulated neurotransmitter release requires the well orchestrated spatial and temporal actions of many presynaptic proteins (1Jahn R. Lang T. Sudhof T.C. Cell. 2003; 112: 519-533Abstract Full Text Full Text PDF PubMed Scopus (1200) Google Scholar). Although the primary molecular entities in the release path-way have been identified, the exact mechanics of synaptic vesicle fusion and its precise regulation are still not established. Central to the fusion process is the transient formation of SNARE 4The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; cYFP, citrine mutant of yellow fluorescent protein; NSF, N-ethylmaleimide sensitive factor; NEM, N-ethylmaleimide; ROCK, Rho-associated coiled-coil-forming kinase; LPA, lysophosphatidic acid; DMPP, 1,1-dimethyl-4-phenylpiperazinium iodide; PSS, physiological saline solution; EGFP, enhanced green fluorescent protein; ECFP, enhanced cyan fluorescent protein; EcYFP, enhanced citrine mutant of yellow fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; hGH, human growth hormone; GST, glutathione S-transferase. 4The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; cYFP, citrine mutant of yellow fluorescent protein; NSF, N-ethylmaleimide sensitive factor; NEM, N-ethylmaleimide; ROCK, Rho-associated coiled-coil-forming kinase; LPA, lysophosphatidic acid; DMPP, 1,1-dimethyl-4-phenylpiperazinium iodide; PSS, physiological saline solution; EGFP, enhanced green fluorescent protein; ECFP, enhanced cyan fluorescent protein; EcYFP, enhanced citrine mutant of yellow fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; hGH, human growth hormone; GST, glutathione S-transferase. core complexes that include the target membrane SNARE proteins syntaxin 1A and SNAP25 and the vesicle SNARE protein synaptobrevin/VAMP (2Jahn R. Sudhof T.C. Annu. Rev. Biochem. 1999; 68: 863-911Crossref PubMed Scopus (1008) Google Scholar, 3Sudhof T.C. Annu. Rev. Neurosci. 2004; 27: 509-547Crossref PubMed Scopus (1837) Google Scholar, 4Lin R.C. Scheller R.H. Annu. Rev. Cell Dev. Biol. 2000; 16: 19-49Crossref PubMed Scopus (414) Google Scholar). A SNARE core complex is a highly stable, four-α-helix parallel bundle consisting of one SNARE motif from each of syntaxin 1A and synaptobrevin/VAMP, and two SNARE motifs from SNAP25 (5Sollner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1564) Google Scholar, 6Sollner T.H. Rothman J.E. Cell Struct. Funct. 1996; 21: 407-412Crossref PubMed Scopus (27) Google Scholar). Although these proteins alone are sufficient to induce a slow fusion when reconstituted into liposomes (7Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (1989) Google Scholar), additional proteins are necessary to establish the properties that describe fast, Ca2+-dependent neurotransmitter release (8Tucker W.C. Weber T. Chapman E.R. Science. 2004; 304: 435-438Crossref PubMed Scopus (295) Google Scholar). For example, assembly of SNARE core complexes is subject to temporal and spatial regulation by a variety of protein families, including Rab-GTPases (9Geppert M. Sudhof T.C. Annu. Rev. Neurosci. 1998; 21: 75-95Crossref PubMed Scopus (219) Google Scholar, 10Takai Y. Sasaki T. Shirataki H. Nakanishi H. Genes Cells. 1996; 1: 615-632Crossref PubMed Scopus (93) Google Scholar, 11Fischer von Mollard G. Stahl B. Li C. Sudhof T.C. Jahn R. Trends Biochem. Sci. 1994; 19: 164-168Abstract Full Text PDF PubMed Scopus (188) Google Scholar, 12Elferink L.A. Scheller R.H. Prog. Brain Res. 1995; 105: 79-85Crossref PubMed Scopus (30) Google Scholar, 13Sollner T.H. Mol. Membr. Biol. 2003; 20: 209-220Crossref PubMed Scopus (142) Google Scholar), Sec/Munc18s (14Rizo J. Sudhof T.C. Nat. Rev. Neurosci. 2002; 3: 641-653Crossref PubMed Scopus (424) Google Scholar, 15Toonen R.F. Biochem. Soc. Trans. 2003; 31: 848-850Crossref PubMed Google Scholar, 16Weimer R.M. Richmond J.E. Curr. Top. Dev. Biol. 2005; 65: 83-113Crossref PubMed Scopus (25) Google Scholar), exocyst tethering complexes (17Kee Y. Yoo J.S. Hazuka C.D. Peterson K.E. Hsu S.C. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14438-14443Crossref PubMed Scopus (150) Google Scholar, 18Hsu S.C. TerBush D. Abraham M. Guo W. Int. Rev. Cytol. 2004; 233: 243-265Crossref PubMed Scopus (207) Google Scholar, 19EauClaire S. Guo W. Neuron. 2003; 37: 369-370Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 20Lipschutz J.H. Mostov K.E. Curr. Biol. 2002; 12: R212-214Abstract Full Text Full Text PDF PubMed Google Scholar), and Munc13s (21Silinsky E.M. Searl T.J. Br. J. Pharmacol. 2003; 138: 1191-1201Crossref PubMed Scopus (74) Google Scholar, 22Martin T.F. Neuron. 2002; 34: 9-12Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 23Betz A. Thakur P. Junge H.J. Ashery U. Rhee J.S. Scheuss V. Rosenmund C. Rettig J. Brose N. Neuron. 2001; 30: 183-196Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 24Brose N. Rosenmund C. Rettig J. Curr. Opin. Neurobiol. 2000; 10: 303-311Crossref PubMed Scopus (176) Google Scholar). In addition, recent evidence suggests that the temporal and spatial availability of SNAREs for membrane fusion may be subject to precise regulation by the presence of soluble R-SNARE motif-containing proteins, such as amisyn (25Scales S.J. Hesser B.A. Masuda E.S. Scheller R.H. J. Biol. Chem. 2002; 277: 28271-28279Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 26Constable J.R. Graham M.E. Morgan A. Burgoyne R.D. J. Biol. Chem. 2005; 280: 31615-31623Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) and tomosyn (27Fujita Y. Shirataki H. Sakisaka T. Asakura T. Ohya T. Kotani H. Yokoyama S. Nishioka H. Matsuura Y. Mizoguchi A. Scheller R.H. Takai Y. Neuron. 1998; 20: 905-915Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 28Yizhar O. Matti U. Melamed R. Hagalili Y. Bruns D. Rettig J. Ashery U. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2578-2583Crossref PubMed Scopus (85) Google Scholar, 29Hatsuzawa K. Lang T. Fasshauer D. Bruns D. Jahn R. J. Biol. Chem. 2003; 278: 31159-31166Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Tomosyn was originally identified in neurons as a binding partner of the Q-SNARE, syntaxin 1A (27Fujita Y. Shirataki H. Sakisaka T. Asakura T. Ohya T. Kotani H. Yokoyama S. Nishioka H. Matsuura Y. Mizoguchi A. Scheller R.H. Takai Y. Neuron. 1998; 20: 905-915Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar), and belongs to a larger family of proteins that includes the yeast proteins Sro7p and Sro77p, the Drosophila tumor suppressor lethal giant larvae family, and the mammalian MglI family (30Lehman K. Rossi G. Adamo J.E. Brennwald P. J. Cell Biol. 1999; 146: 125-140Crossref PubMed Scopus (173) Google Scholar, 31Kagami M. Toh-e A. Matsui Y. Genetics. 1998; 149: 1717-1727Crossref PubMed Google Scholar, 32Mechler B.M. McGinnis W. Gehring W.J. EMBO J. 1985; 4: 1551-1557Crossref PubMed Scopus (185) Google Scholar, 33Musch A. Cohen D. Yeaman C. Nelson W.J. Rodriguez-Boulan E. Brennwald P.J. Mol. Biol. Cell. 2002; 13: 158-168Crossref PubMed Scopus (160) Google Scholar). Tomosyn homologues also appear in the Fungi and Plantae kingdoms (34Pobbati A.V. Razeto A. Boddener M. Becker S. Fasshauer D. J. Biol. Chem. 2004; 279: 47192-47200Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Structurally, tomosyns are soluble proteins that contain two distinguishable domains. An R-SNARE homology motif near the C terminus defines the primary interaction of tomosyn with the Q-SNARE syntaxin 1A, whereas the remaining N-terminal region contains 7-9 repeating β-transducin-like WD-40 motifs that form additional protein-protein interaction sites (27Fujita Y. Shirataki H. Sakisaka T. Asakura T. Ohya T. Kotani H. Yokoyama S. Nishioka H. Matsuura Y. Mizoguchi A. Scheller R.H. Takai Y. Neuron. 1998; 20: 905-915Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 35Masuda E.S. Huang B.C. Fisher J.M. Luo Y. Scheller R.H. Neuron. 1998; 21: 479-480Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 36Kim Y.S. Chung H.M. Baek K.H. Int. J. Oncol. 2003; 23: 229-233PubMed Google Scholar). In the mouse genome, two paralogous genes for tomosyn exist (tomosyn-1 and -2) and lead to the expression of seven tomosyn isoforms (37Groffen A.J. Jacobsen L. Schut D. Verhage M. J. Neurochem. 2005; 92: 554-568Crossref PubMed Scopus (36) Google Scholar). Variability between these tomosyn isoforms is clustered within a hypervariable domain that separates the N-terminal WD-40 repeats from the C-terminal SNARE domain. Functional actions of tomosyn family members have been ascribed to their interaction with cognate Q-SNAREs; however, as the lethal giant larvae family and Sro7p and Sro77p proteins do not possess a well defined C-terminal R-SNARE homology domain, interactions between these families and their cognate Q-SNAREs have been proposed to involve alternative interaction motifs (30Lehman K. Rossi G. Adamo J.E. Brennwald P. J. Cell Biol. 1999; 146: 125-140Crossref PubMed Scopus (173) Google Scholar, 31Kagami M. Toh-e A. Matsui Y. Genetics. 1998; 149: 1717-1727Crossref PubMed Google Scholar, 38Zhang X. Wang P. Gangar A. Zhang J. Brennwald P. TerBush D. Guo W. J. Cell Biol. 2005; 170: 273-283Crossref PubMed Scopus (82) Google Scholar). Increasing evidence demonstrates that tomosyn and its homologues are critical regulators in vesicular trafficking and membrane fusion processes. Overexpression of tomosyn in PC-12 and adrenal chromaffin cells negatively regulates neurotransmitter secretion (27Fujita Y. Shirataki H. Sakisaka T. Asakura T. Ohya T. Kotani H. Yokoyama S. Nishioka H. Matsuura Y. Mizoguchi A. Scheller R.H. Takai Y. Neuron. 1998; 20: 905-915Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 29Hatsuzawa K. Lang T. Fasshauer D. Bruns D. Jahn R. J. Biol. Chem. 2003; 278: 31159-31166Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), which, in chromaffin cells, results from inhibition in priming of large dense core vesicles and decreased readily releasable pool size (28Yizhar O. Matti U. Melamed R. Hagalili Y. Bruns D. Rettig J. Ashery U. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2578-2583Crossref PubMed Scopus (85) Google Scholar). Tomosyn has also been shown to exert an important role in polarized exocytosis in yeast and epithelial cells (30Lehman K. Rossi G. Adamo J.E. Brennwald P. J. Cell Biol. 1999; 146: 125-140Crossref PubMed Scopus (173) Google Scholar, 39Katoh M. Int. J. Oncol. 2004; 24: 737-742PubMed Google Scholar) to negatively modulate insulin release from pancreatic β-cells (40Zhang W. Lilja L. Mandic S.A. Gromada J. Smidt K. Janson J. Takai Y. Bark C. Berggren P.O. Meister B. Diabetes. 2006; 55: 574-581Crossref PubMed Scopus (47) Google Scholar), and to interact with syntaxin 4 and SNAP23 and inhibit insulin-induced fusion of GLUT4-containing vesicles in 3T3-L1 adipocytes (41Widberg C.H. Bryant N.J. Girotti M. Rea S. James D.E. J. Biol. Chem. 2003; 278: 35093-35101Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Recent genetic studies in Caenorhabditis elegans have also clearly established that Tom-1, the ortholog of mammalian tomosyn, exerts an inhibitory role on neurotransmitter secretion by negatively regulating synaptic vesicle priming (42Dybbs M. Ngai J. Kaplan J.M. PLoS Genet. 2005; 1: 6-16Crossref PubMed Scopus (39) Google Scholar, 43Gracheva E.O. Burdina A.O. Holgado A.M. Berthelot-Grosjean M. Ackley B.D. Hadwiger G. Nonet M.L. Weimer R.M. Richmond J.E. PLoS Biol. 2006; 4: e261Crossref PubMed Scopus (122) Google Scholar). The inhibitory effects of tomosyn have been proposed to result from the formation of specific tomosyn-protein complexes that reduce the availability of interacting proteins to perform functional roles in exocytosis. For example, tomosyn has been shown to compete with Munc18 for binding to syntaxin, and, probably of greater significance, tomosyn competes with synaptobrevin for binding to syntaxin/SNAP25 dimers to form tomosyn-SNARE complexes (27Fujita Y. Shirataki H. Sakisaka T. Asakura T. Ohya T. Kotani H. Yokoyama S. Nishioka H. Matsuura Y. Mizoguchi A. Scheller R.H. Takai Y. Neuron. 1998; 20: 905-915Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). The latter complexes, as shown by their resolved crystal structure, are almost identical to the synaptobrevin/VAMP-containing SNARE complexes (34Pobbati A.V. Razeto A. Boddener M. Becker S. Fasshauer D. J. Biol. Chem. 2004; 279: 47192-47200Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), including a required action by the ATPase NSF for complex disassembly and reuse of the interacting proteins (29Hatsuzawa K. Lang T. Fasshauer D. Bruns D. Jahn R. J. Biol. Chem. 2003; 278: 31159-31166Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). However, whereas tomosyns can participate in the formation of stable tomosyn-SNARE complexes, the absence of a membrane anchor in all tomosyn family members precludes them from acting as fusogenic synaptobrevin/VAMP analogues. Rather, formation of these nonfusogenic tomosyn-SNARE complexes diminishes the availability and formation of fusion-competent SNARE complexes between membrane-anchored SNAREs, and it is this feature that has been proposed to underlie the negative regulation by tomosyn of exocytotic activity. The interaction of tomosyn with syntaxin 1A has recently been reported to be differentially regulated by the Rho/Rho kinase (ROCK) (44Sakisaka T. Baba T. Tanaka S. Izumi G. Yasumi M. Takai Y. J. Cell Biol. 2004; 166: 17-25Crossref PubMed Scopus (61) Google Scholar) and protein kinase A signaling pathways (45Baba T. Sakisaka T. Mochida S. Takai Y. J. Cell Biol. 2005; 170: 1113-1125Crossref PubMed Scopus (81) Google Scholar). Activation of RhoA and its signaling effector ROCK facilitated syntaxin 1A phosphorylation and formation of tomosyn-SNARE complexes at the palms of growth cones in extending neurites in NG108 neuroblastoma cells and cultured neurons (44Sakisaka T. Baba T. Tanaka S. Izumi G. Yasumi M. Takai Y. J. Cell Biol. 2004; 166: 17-25Crossref PubMed Scopus (61) Google Scholar). This resulted in the localized inhibition of functional SNARE complex formation in these areas and spatially directed fusion of plasmalemmal precursor vesicles to the leading edge of growth cones. On the other hand, protein kinase A-catalyzed phosphorylation of tomosyn decreased the interaction of tomosyn with syntaxin 1A and thereby up-regulated SNARE complex formation and enhanced neurotransmitter release in cultured superior cervical ganglion neurons (45Baba T. Sakisaka T. Mochida S. Takai Y. J. Cell Biol. 2005; 170: 1113-1125Crossref PubMed Scopus (81) Google Scholar). Thus, although tomosyn is not essential for neurotransmitter release, its complex regulation suggests that it may play a critical role in integrating multiple receptor-mediated signaling pathways to ultimately achieve a fine modulatory control over the site and extent of secretory responses. To date, there has been no direct evaluation in living cells of the time course or extent to which the assembly/disassembly of tomosyn-SNARE complexes is regulated by secretory demand for neurotransmitter release. Furthermore, although a Rho signaling pathway has been demonstrated to alter tomosyn-SNARE interactions during neurite development, it remains unknown whether this signaling pathway operates to direct tomosyn-SNARE complex assembly during regulated neurotransmitter release. In this paper, we evaluated the spatiotemporal dynamics and regulation of the tomosyn-syntaxin 1A interaction during stimulated secretion in neuroendocrine chromaffin cells. We show that activation of nicotinic acetylcholine receptors, as occurs normally during neurally evoked secretory responses, as well as treatment with lysophosphatidic acid (LPA), activates Rho-GTPase and increases tomosyn-syntaxin 1A complex formation at the plasma membrane in chromaffin cells. These effects of secretagogue stimulation and LPA treatment were inhibited by Y27632, a specific inhibitor of the Rho-GTP effector ROCK. We also show using dynamic FRET measurements between CFP-tomosyn and cYFP-syntaxin 1A that the formation of these complexes is strongly augmented under conditions where NSF action is inhibited, suggesting that a rapid and dynamic cycling of tomosyn-syntaxin 1A interactions occurs in vivo. Finally, we present functional data to demonstrate that LPA activation of the RhoA/ROCK pathway during evoked secretion enhances tomosyn-mediated inhibition of secretion. Chemicals and Expression Constructs—pEGFP-C1 and monomeric mutants (A206K) of pECFP-C1 and pEcYFP-C1 (citrine) vectors containing the LoxP sequence were used as recipient vectors for subcloning using the Cre-recombinase-mediated Creator system (Clontech). Rat syntaxin 1A, m-tomosyn, SNAP25, and Munc18-1 were merged to the C terminus of the EGFP, ECFP, and EcYFP. Effector binding mutants of syntaxin 1A (I209A and I233A), a soluble mutant of SNAP25 (SNAP25c/a, C-80, 88, 90, and 92-A), as well as a carboxyl-terminal deletion of tomosyn-(1-1067) (tomosyn ΔCT, glutamate at residue 1068 changed to stop codon) were constructed using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene). The sequence fidelity of all constructs was confirmed by DNA sequencing (University of Michigan DNA Sequencing Core). RhoA activation was measured using an enzyme-linked immunosorbent assay-based kit (Cytoskeleton). HEK293 cells stably expressing the rat α1B and human β2B and α2δ voltage-gated calcium channel subunits (HEK293-S3 cells) were a gift from D. Rock (Warner-Lambert Parke Davis, Ann Arbor, MI). All other chemicals were obtained from Sigma unless specifically indicated otherwise. Cell Culture and Transfection—HEK293-S3 cells were plated and cultured in RPMI 1640 with l-glutamine supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Invitrogen), 0.4 mg/ml hygromycin, and 0.6 mg/ml Geneticin at 37 °C in 95% O2, 5% CO2 for 2 days on coverslips (thickness 1) attached to the bottom of 35-mm culture dishes before transfection. Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. One hour before transfection, cells were placed into DMEM lacking antibiotics and supplemented with 1% l-glutamine, 1% nonessential amino acids, and 10% fetal bovine serum. 4-6 h after the transfection, cells were returned to the RPMI 1640 medium. Cells were used for imaging 24-48 h after transfection. Chromaffin cells were isolated from bovine adrenal glands using divalent metal ion-free rinse, collagenase digestion, and gradient centrifugation as described previously (46Li Q. Ho C.S. Marinescu V. Bhatti H. Bokoch G.M. Ernst S.A. Holz R.W. Stuenkel E.L. J. Physiol. 2003; 550: 431-445Crossref PubMed Scopus (49) Google Scholar). Cells were cultured in 6-well plates in DMEM/F-12 supplemented with 10% fetal bovine serum, 100 units/ml penicillin/streptomycin, 10 μg/ml gentamicin, and 10 μm cytosine arabinofurosemide. Three days following isolation, cells were transfected using biolistic particle bombardment according to the manufacturer’s instructions with plasmid DNA-laden (2 μg/mg of beads) 1-μm diameter gold beads (Bio-Rad). Cells were replated 4-24 h before imaging onto collagen coated glass coverslips. PC-12 cells were cultured in 10% CO2 in DMEM supplemented with 10% horse serum, 5% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 μg/ml), and 1% gentamicin (10 μg/ml). PC-12 cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. 30 min before transfection, cells were placed in Optimem medium; cells were returned to the DMEM 4-6 h following transfection. Tomosyn Translocation Assay—Cells were serum-starved in DMEM/F-12 medium for 4 h prior to treatment with selected receptor agonists or signaling antagonists. Following the serum-starved period, medium was changed to physiological saline (PSS) containing 140 mm NaCl, 5 mm KCl, 2.2 mm CaCl2, 1mm MgCl2, 10 mm glucose, and 10 mm HEPES (pH 7.4 adjusted with NaOH). Treatments included incubation with LPA (10 μm, Biomol) or the nicotinic acetylcholine receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP; 20 μm), alone or in combination with the Rho kinase inhibitor (Y27632; 20 μm; Calbiochem). In Rho kinase inhibitor experiments, the Y27632 was applied during the serum-deficient preincubation period and to the PSS during agonist treatment. Each treatment was followed by an additional incubation (10 min) with the NSF-alkylating agent N-ethylmaleimide (NEM; 100 μm) prior to cell lysis. Cells were then scraped into the ice-cold lysis buffer containing 2% sucrose, 1 mm EDTA, 20 mm Tris-HCl (pH 7.4), and 100 μm NEM, with a mixture of protease inhibitors (1 μg/μl each of phenylmethylsulfonyl fluoride, leupeptin, pepstatin, aprotinin, and benzamidine). The collected lysis buffer was then subjected to Dounce homogenization (20 strokes) followed by centrifugation (800 × g, 2 min, 4 °C) to remove nuclei. A membrane fraction was then extracted by ultracentrifugation of the samples at 100,000 × g for 30 min at 4 °C, with the pellets resuspended in immunoprecipitation buffer containing 150 mm NaCl, 50 mm Tris (pH 7.4), and 2 mm EDTA, supplemented with 1% Triton and the above mentioned protease inhibitor mixture. Immunoprecipitation using a monoclonal α-tomosyn antibody (Transduction Laboratories) was then performed on all samples that had been initially adjusted to contain equal starting amounts of protein and volume. Immunoprecipitation was carried out by overnight incubation with the antibody at 4 °C with rotation, after which Immunopure protein G beads (Pierce) were added, and the incubation continued another 90 min. The beads were then pelleted by centrifugation (1,000 × g for 2 min at 4 °C) and washed in TNM buffer containing 50 mm NaCl, 50 mm MgCl2, and 50 mm Tris-HCl (pH 7.5). Tomosyn immunoreactivity in the resulting samples was determined by SDS-PAGE fractionation and immunoblotting. Blots were probed with monoclonal α-tomosyn antibody and subsequently with horseradish peroxidase-conjugated goat α-mouse antibody (Developmental Studies Hybridoma Bank, Iowa City, IA). Immunoreactive signals were developed by ECL detection (Amersham Biosciences), visualized using a FluoroMax Imager (Bio-Rad), and quantified from digital images using Quantity One software (Bio-Rad). Rho Activation Assay—Cells in 6-well plates (1.5 × 106 cells/well) were serum-starved in DMEM/F-12 medium for 12 h prior to use. Following the serum-starved period, medium was changed to PSS that contained LPA (10 μm; Biomol), nicotinic acetylcholine receptor agonist DMPP (20 μm), or, for control, PSS alone. Following either a 2- or 10-min period of receptor activation, the medium was rapidly removed, and the cells were rinsed once in ice-cold phosphate-buffered saline. Lysis buffer was then rapidly added, cells were scraped, and lysates were collected and immediately frozen in liquid N2. The relative level of RhoA activation with respect to control conditions was then measured using an enzyme-linked immunosorbent assay-based kit according to the manufacturer’s instructions (Cytoskeleton). Each sample was assayed in duplicate, and each condition was repeated on at least three individual cell preparations. The level of Rho activation was calculated with respect to that of the control condition following subtraction of the assay blank. Human Growth Hormone Secretion Assay—PC-12 cells were plated onto 24-well plates and co-transfected with plasmids coding for human growth hormone (hGH), in addition to full-length tomosyn, tomosyn ΔC, or a neomycin control. The total concentration of DNA was held equal across all treatments. hGH was used as a reporter for regulated secretion specifically from transfected cells (47Wick P.F. Senter R.A. Parsels L.A. Uhler M.D. Holz R.W. J. Biol. Chem. 1993; 268: 10983-10989Abstract Full Text PDF PubMed Google Scholar). Secretion assays were performed 48-72 h following transfection. 16-20 h before the start of the assay, cells were placed in serum-free medium; where applicable, cells were pretreated with Y27632 (20 μm) 4 h prior to the start of the assay. To test secretion, cells were rinsed for 10 min in a physiological saline solution (145 mm NaCl, 5.6 mm KCl, 15 mm NaHEPES, 0.5 mm MgCl2, 2.2 mm CaCl2, 5.6 mm glucose, 0.5 mm sodium ascorbate, 2 mg/ml fatty acid-free bovine serum albumin, pH 7.3) in the presence or absence of Y27632. Cells were then stimulated to secrete by a 6-min treatment with 70 mm K+ (same saline solution but with equimolar substitution of K+ for Na+). Where applicable, 10 μm LPA and/or (20 μm) Y27632 were added to the stimulus solution. The saline solution containing the secreted hGH was collected, and cells were lysed to determine the percentage of total hGH content secreted. hGH content was measured using an hGH enzyme-linked immunosorbent assay kit (Roche Applied Science). Each experiment was performed with quadruplicate replicates for each treatment. GST-Syntaxin 1A-Tomosyn Binding in Vitro and Immunoblotting—Soluble syntaxin 1A (residues 1-264) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. GST was expressed in a similar manner. Both proteins were purified by glutathione-Sepharose (Sigma) binding and extensive washing. The bound GST or GST-syntaxin 1A were then incubated for 16 h at 4 °C with lysates prepared from PC-12 cells expressing EGFP-tomosyn or EGFP-tomosyn ΔCT. The lysis buffer contained 100 mm KCl, 20 mm HEPES-KOH, 2 mm EDTA, 1% Triton X-100, 1 mm dithiothreitol with protease inhibitor mixture (Roche Applied Science; PI Complete). The beads were then collected by centrifugation and washed four times with phosphate" @default.
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• W1996681454 cites W1568777255 @default.
• W1996681454 cites W1574617908 @default.
• W1996681454 cites W1679562638 @default.
• W1996681454 cites W1700565657 @default.
• W1996681454 cites W1861896961 @default.
• W1996681454 cites W1946364275 @default.
• W1996681454 cites W1967395384 @default.
• W1996681454 cites W1971016259 @default.
• W1996681454 cites W1972816955 @default.
• W1996681454 cites W1975364281 @default.
• W1996681454 cites W1977566709 @default.
• W1996681454 cites W1977831677 @default.
• W1996681454 cites W1985302300 @default.
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• W1996681454 cites W2011298151 @default.
• W1996681454 cites W2017237303 @default.
• W1996681454 cites W2021759006 @default.
• W1996681454 cites W2025398161 @default.
• W1996681454 cites W2027775552 @default.
• W1996681454 cites W2029906502 @default.
• W1996681454 cites W2030187168 @default.
• W1996681454 cites W2032069841 @default.
• W1996681454 cites W2040866860 @default.
• W1996681454 cites W2043466785 @default.
• W1996681454 cites W2055384905 @default.
• W1996681454 cites W2058722716 @default.
• W1996681454 cites W2063057624 @default.
• W1996681454 cites W2067206130 @default.
• W1996681454 cites W2068486863 @default.
• W1996681454 cites W2071146563 @default.
• W1996681454 cites W2086442201 @default.
• W1996681454 cites W2090538334 @default.
• W1996681454 cites W2091423236 @default.
• W1996681454 cites W2094131619 @default.
• W1996681454 cites W2096079557 @default.
• W1996681454 cites W2096597301 @default.
• W1996681454 cites W2096791952 @default.
• W1996681454 cites W2105030641 @default.
• W1996681454 cites W2105976709 @default.
• W1996681454 cites W2107335219 @default.
• W1996681454 cites W2108632260 @default.
• W1996681454 cites W2111195613 @default.
• W1996681454 cites W2113679700 @default.
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• W1996681454 cites W2129430733 @default.
• W1996681454 cites W2131092137 @default.
• W1996681454 cites W2131653836 @default.
• W1996681454 cites W2133672958 @default.
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• W1996681454 cites W2165068760 @default.
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