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• W2005972021 abstract "Ca2+-dependent fusion of transport vesicles at their target can be enhanced by intracellular Ca2+ and diacylglycerol. Diacylglycerol induces translocation of the vesicle priming factor Munc13 and association of the secretory vesicle protein DOC2B to the membrane. Here we demonstrate that a rise in intracellular Ca2+ is sufficient for a Munc13-independent recruitment of DOC2B to the target membrane. This novel mechanism occurred readily in the absence of Munc13 and was not influenced by DOC2B mutations that abolish Munc13 binding. Purified DOC2B (expressed as a bacterial fusion protein) bound phospholipids in a Ca2+-dependent way, suggesting that the translocation is the result of a C2 domain activation mechanism. Ca2+-induced translocation was also observed in cultured neurons expressing DOC2B-enhanced green fluorescent protein. In this case, however, various degrees of membrane association occurred under resting conditions, suggesting that physiological Ca2+ concentrations modulate DOC2B localization. Depolarization of the neurons induced a complete translocation of DOC2B-enhanced green fluorescent protein to the target membrane within 5 s. We hypothesize that this novel Ca2+-induced activity of DOC2B functions synergistically with diacylglycerol-induced Munc13 binding to enhance exocytosis during episodes of high secretory activity. Ca2+-dependent fusion of transport vesicles at their target can be enhanced by intracellular Ca2+ and diacylglycerol. Diacylglycerol induces translocation of the vesicle priming factor Munc13 and association of the secretory vesicle protein DOC2B to the membrane. Here we demonstrate that a rise in intracellular Ca2+ is sufficient for a Munc13-independent recruitment of DOC2B to the target membrane. This novel mechanism occurred readily in the absence of Munc13 and was not influenced by DOC2B mutations that abolish Munc13 binding. Purified DOC2B (expressed as a bacterial fusion protein) bound phospholipids in a Ca2+-dependent way, suggesting that the translocation is the result of a C2 domain activation mechanism. Ca2+-induced translocation was also observed in cultured neurons expressing DOC2B-enhanced green fluorescent protein. In this case, however, various degrees of membrane association occurred under resting conditions, suggesting that physiological Ca2+ concentrations modulate DOC2B localization. Depolarization of the neurons induced a complete translocation of DOC2B-enhanced green fluorescent protein to the target membrane within 5 s. We hypothesize that this novel Ca2+-induced activity of DOC2B functions synergistically with diacylglycerol-induced Munc13 binding to enhance exocytosis during episodes of high secretory activity. Ca2+-induced exocytosis is a widely conserved mechanism of importance for a broad range of secretory systems, such as the release of neurotransmitters and neuropeptides in the central and peripheral nervous system. To attain fusion competence, secretory vesicles must first dock at the target membrane and undergo a process called priming. The release of fusion-ready vesicles is coupled to the opening of Ca2+ channels in the plasma membrane, producing a transient high Ca2+ concentration (10-100 μm for ∼1 ms) in close proximity to the channels. The inward Ca2+ current then diffuses into the cytoplasm, resulting in a residual concentration in the range of 1 μm during tens of seconds. It is widely accepted that this residual Ca2+ concentration induces short term changes in exocytotic strength (e.g. see Refs. 1Fisher S.A. Fischer T.M. Carew T.J. Trends Neurosci. 1997; 20: 170-177Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar and 2Zucker R.S. Regehr W.G. Annu. Rev. Physiol. 2002; 64: 355-405Crossref PubMed Scopus (3312) Google Scholar), although the mechanisms that contribute to this phenomenon remain largely unclear. As a result, the secretory strength of neuronal and endocrine release sites is modulated in an activity-dependent way. This phenomenon is important to avoid vesicle depletion during repetitive stimulation and is furthermore considered in neurons to contribute to memory and learning. Although Ca2+ is the most important messenger after high frequency activation of the release site, exocytotic potentiation can also be induced by diacylglycerol (synthesized by members of the phospholipase family) or by its phorbolester analogues. Diacylglycerol has multiple intracellular targets (e.g. protein kinase C isoforms), but the potentiating effect in regulated exocytosis is mediated by the priming factor Munc13-1. Munc13-1-deficient mice die shortly after birth and show a defect in synaptic vesicle exocytosis from glutamatergic synapses, presumably due to an arrest between the docking and fusion steps of the synaptic vesicle cycle (3Augustin I. Rosenmund C. Sudhof T.C. Brose N. Nature. 1999; 400: 457-461Crossref PubMed Scopus (567) Google Scholar). In another study, the Munc13-1 gene was replaced with the allele Munc13-1H567K, encoding a mutant protein that specifically lacks diacylglycerol-binding activity. Hippocampal neurons from homozygous Munc13-1H567K mice show normal evoked postsynaptic currents (4Rhee J.S. Betz A. Pyott S. Reim K. Varoqueaux F. Augustin I. Hesse D. Sudhof T.C. Takahashi M. Rosenmund C. Brose N. Cell. 2002; 108: 121-133Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). However, the readily releasable pool size is diminished, the recovery from high frequency stimulation is slowed down, and the potentiating effect of phorbolesters is blocked (4Rhee J.S. Betz A. Pyott S. Reim K. Varoqueaux F. Augustin I. Hesse D. Sudhof T.C. Takahashi M. Rosenmund C. Brose N. Cell. 2002; 108: 121-133Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). Thus, Munc13-1 may enhance vesicle priming during high frequency stimulation through acting as a diacylglycerol sensor. Several studies have suggested that the potentiating activity of Munc13 involves its interaction with proteins of the DOC2 family, as discussed below. The DOC2 protein family comprises three isoforms, designated DOC2A, -B, and -C. The latter is predominantly expressed in the heart (5Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 2000; 276: 626-632Crossref PubMed Scopus (34) Google Scholar). DOC2A and DOC2B are both expressed in the brain, and in addition DOC2B is found in several peripheral tissues (6Sakaguchi G. Orita S. Maeda M. Igarashi H. Takai Y. Biochem. Biophys. Res. Commun. 1995; 217: 1053-1061Crossref PubMed Scopus (62) Google Scholar, 7Verhage M. de Vries K.J. Roshol H. Burbach J.P. Gispen W.H. Sudhof T.C. Neuron. 1997; 18: 453-461Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). In subcellular fractionations, DOC2A and -B copurify with secretory vesicles, although they lack a transmembrane domain. DOC2 proteins consist of three structural domains: a short N-terminal domain and two C2 domains named C2A and C2B, respectively, similar to those in synaptotagmins and protein kinase C (PKC). 1The abbreviations used are: PKC, protein kinase C; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase; TIRF, total internal reflection fluorescence; PBS, phosphate-buffered saline; MAP2, microtubule-associated protein 2. This presence of these motifs predicts a Ca2+-binding function. For DOC2B, this was experimentally confirmed in vitro; Ca2+ induced binding of the isolated C2A domain to phosphatidylserine-containing liposomes with half-maximal binding at 1 μm Ca2+ (8Kojima T. Fukuda M. Aruga J. Mikoshiba K. J. Biochem. (Tokyo). 1996; 120: 671-676Crossref PubMed Scopus (45) Google Scholar). A positive function in secretion was suggested by the increased release of growth hormone from PC12 cells overexpressing DOC2A (9Orita S. Sasaki T. Komuro R. Sakaguchi G. Maeda M. Igarashi H. Takai Y. J. Biol. Chem. 1996; 271: 7257-7260Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Both DOC2A and -B bind Munc13-1 in a diacylglycerol-dependent manner (10Orita S. Naito A. Sakaguchi G. Maeda M. Igarashi H. Sasaki T. Takai Y. J. Biol. Chem. 1997; 272: 16081-16084Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). This interaction is mediated by the Munc13-interacting domain located within the N-terminal domain of DOC2. Interestingly, diacylglycerol also induces a Munc13-1-dependent translocation of DOC2B to the plasma membrane in HEK293 cells (11Duncan R.R. Betz A. Shipston M.J. Brose N. Chow R.H. J. Biol. Chem. 1999; 274: 27347-27350Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Injection of a synthetic peptide identical to the Munc13-interacting domain region into cholinergic neurons of the superior cervical ganglion was shown to inhibit synaptic transmission in an activity-dependent manner (12Mochida S. Orita S. Sakaguchi G. Sasaki T. Takai Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11418-11422Crossref PubMed Scopus (70) Google Scholar). Similarly, injection of this peptide into the Calyx of Held blocked the potentiating effect of phorbolester administered 5 min after the peptide (13Hori T. Takai Y. Takahashi T. J. Neurosci. 1999; 19: 7262-7267Crossref PubMed Google Scholar). Together, these data suggest that the DOC2-Munc13 interaction contributes to the mechanism of diacylglycerol-induced presynaptic potentiation. DOC2 proteins also bind to Munc18, a syntaxin-binding protein that is required for neurotransmitter secretion (7Verhage M. de Vries K.J. Roshol H. Burbach J.P. Gispen W.H. Sudhof T.C. Neuron. 1997; 18: 453-461Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 14Verhage M. Maia A.S. Plomp J.J. Brussaard A.B. Heeroma J.H. Vermeer H. Toonen R.F. Hammer R.E. van den Berg T.K. Missler M. Geuze H.J. Sudhof T.C. Science. 2000; 287: 864-869Crossref PubMed Scopus (1008) Google Scholar). In chromaffin cells of the adrenal gland, homozygous Munc18 null mice showed a 10-fold reduction in morphologically docked vesicles and a corresponding reduction in Ca2+-dependent exocytosis of large dense core vesicles (15Voets T. Toonen R.F. Brian E.C. de Wit H. Moser T. Rettig J. Sudhof T.C. Neher E. Verhage M. Neuron. 2001; 31: 581-591Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). Many overexpression studies with Munc18 homologs have been reported with seemingly conflicting results, in general supporting the hypothesis that Munc18 exerts its function in the final stages upstream from Ca2+-induced vesicle exocytosis at least in mammalian systems (16Toonen R.F. Verhage M. Trends Cell Biol. 2003; 13: 177-186Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Here we describe a novel mechanism for the translocation of DOC2B to the membrane, triggered by intracellular Ca2+. In contrast to the diacylglycerol-induced translocation, Ca2+-dependent translocation occurs independent of Munc13 binding. Since diacylglycerol and Ca2+ activation generally coincide during episodes of high secretory activity, we hypothesize that both mechanisms may act in concert to increase the exocytotic strength. Antibodies—A polyclonal antibody designated 13.2 was raised against DOC2B by immunizing New Zealand White rabbits with 500 μg of a bacterially expressed polypeptide comprising amino acids 22-116 of DOC2B. The serum recognized a single protein of the expected molecular mass as determined by Western blotting using rat brain homogenate. It also reacted strongly with lysate from HEK293 cells expressing fusion proteins consisting of DOC2B and enhanced green fluorescent protein (EGFP) but not EGFP alone. The mouse monoclonal anti-Myc tag antibody 9E10 and the mouse monoclonal anti-EGFP antibody B34 were purchased from Babco (Richmond, CA). Construction of Expression Vectors—A cDNA fragment encoding full-length wild type DOC2B (GenBank™ accession number U70778) was amplified by polymerase chain reaction with the DNA polymerase PfuI (Stratagene, La Jolla, CA) using the rat cDNA clone pNP3a as template (7Verhage M. de Vries K.J. Roshol H. Burbach J.P. Gispen W.H. Sudhof T.C. Neuron. 1997; 18: 453-461Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The sense primer (5′-ttggtacccgctgacttaccccc-3′) introduces a KpnI restriction site 69 nucleotides upstream of the start codon; the anti-sense primer (5′-gcggatcccgtcgtcgagtacagc-3′) removes the stop codon and introduces a BamHI restriction site. This fragment was introduced into pEGFP-N2 (Clontech) using the corresponding sites. Expression vectors encoding Myc-tagged DOC2B were derived from this vector by subcloning the NheI-BamHI fragment into the corresponding sites of pCDNA3.1-Myc-HisC (Invitrogen). Expression vectors encoding Munc13-1, PKC-α, and PKC-γ were described previously (17Brose N. Hofmann K. Hata Y. Sudhof T.C. J. Biol. Chem. 1995; 270: 25273-25280Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 18Vallentin A. Prevostel C. Fauquier T. Bonnefont X. Joubert D. J. Biol. Chem. 2000; 275: 6014-6021Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 19Sakai N. Sasaki K. Ikegaki N. Shirai Y. Ono Y. Saito N. J. Cell Biol. 1997; 139: 1465-1476Crossref PubMed Scopus (198) Google Scholar). Site-directed Mutagenesis—Complementary DNAs encoding the mutant fusion proteins listed in Table I were generated by site-directed mutagenesis, performed by full-length amplification of the pEGFP-N2-DOC2B expression vector with mutagenic primers. PfuI DNA polymerase (Roche Applied Science) was used for amplification. Template DNA was selectively degraded by incubation with DpnI endonuclease, and the PCR product was introduced into Escherichia coli DH5α. The integrity of all mutant cDNAs was confirmed by sequence analysis of the complete open reading frame on a Beckman CEQ 2000 automated sequencer (Fullerton, CA). All DOC2B variants (wild type and mutant) were also subcloned into pCDNA3.1 and pCDNA3.1-Myc (Invitrogen).Table INames of mutant DOC2B molecules generated by site-directed mutagenesisNameMutationEffectTCTΔ2-9Blocks Tctex-1 bindingM13Substitution of amino acids 15-20 (QEHMAI to YKDWAF)Blocks Munc13 binding Open table in a new tab Cell Culture—HEK293 (ATCC CRL 1573) were cultured at 37 °C in the presence of 5% (v/v) CO2 in Dulbecco's modified Eagle's medium containing sodium pyruvate, pyridoxine, and 1 g/liter glucose (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen), 1 mm glutamine, 1× nonessential amino acids (Gibco), 200 IU/ml penicillin, and 200 μg/ml streptomycin. One day after seeding on glass coverslips in 6-well plates, the cells were transfected with 2.5 μg of vector DNA using the standard calcium phosphate precipitation method. For co-expression of Munc13-1, the expression vector pCDNA3-Munc13-1 was added in a 3-fold molar excess over pEGFPDOC2B. Medium was refreshed at 16 and 40 h, and translocation assays were performed at 48-50 h post-transfection. PC12 cells were cultured on 18-mm glass coverslips in Dulbecco's modified Eagle's medium containing 10% horse serum, 5% fetal calf serum, 4 mm l-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin in the presence of 5% CO2. Cells were transfected with 2 μg of vector DNA using LipofectAMINE 2000 (Invitrogen), and measurements (by total internal reflection fluorescence microscopy) were conducted 6-8 h post-transfection. Neurons were isolated from C57BL/6 mouse embryos at day 18 of gestation and seeded at 25,000 cells/cm2 in 12-well plates on poly-l-lysine-coated coverslips in neurobasal medium (Invitrogen) supplemented with B-27 supplement (Invitrogen). The cells were cultured at 37 °C in a humidified chamber gassed with 5% CO2. Six h after seeding, the neurons were transduced with lentiviral particles using a multiplicity of infection of 1.5-2 infective particles/cell. Western Blotting—Fourty-eight h post-transfection, HEK293 cells were lysed in 10 mm Tris-HCl, pH 7.4, 1 mm EDTA, and 1% Triton X-100. The lysate was cleared by centrifugation for 30 min at 16,000 × g, followed by electrophoresis on a 6-15% Tris-glycine gradient gel. Proteins were transferred to a polyvinylidine difluoride membrane (BioRad). Membranes were incubated for 16 h at 4 °C in 5% blocking agent (Amersham Biosciences) dissolved in TBST (20 mm Tris, 137 mm NaCl, and 0.1% Tween 20), washed, and incubated with anti-GFP (Covance Research Products, Denver, PA; diluted 1:25,000) or anti-Doc2B (13.2; diluted 1:1,000) for 90 min in TBST. The membranes where washed again and incubated for 1 h with alkaline phosphatase-conjugated goat anti-mouse or goat anti-rabbit secondary antibody (Amersham Biosciences) diluted 1:5,000 in TBST. Finally, the membranes were washed, incubated for 5 min in enhanced chemifluorescence substrate (Amersham Biosciences), and analyzed on a FLA5000 fluoroimager (Raytest, Tilburg, The Netherlands). All incubations were carried out at room temperature unless otherwise stated. Immunofluorescence Microscopy—Cells were fixed 48 h post-transfection for 15 min in 2% paraformaldehyde (Merck) in PBS and 15 min in 4% paraformaldehyde. Subsequently, they were incubated in PBS containing 4% fetal calf serum (Invitrogen) for 20 min. Next, the cells were incubated with anti-Doc2B antibody 13.2, diluted 1:4,000 in PBS containing 0.1% Triton X-100. As the secondary antibody we used Alexa546-conjugated goat anti rabbit (Molecular Probes, Inc.) diluted 1:1,000 in PBS containing 0.1% Triton X-100. The cells were mounted in Dabco-Mowiol (Sigma) and analyzed on an LSM 510 confocal microscope (Carl Zeiss BV, Weesp, The Netherlands). Confocal Imaging of Live and Fixed Cells—For imaging of EGFP fluorescence in fixed cells, the cells were incubated for 10 min at 37 °C in basal buffer (25 mm HEPES, pH 7.4, 140 mm NaCl, 4.7 mm KCl, 1.4 mm MgCl2, and 10 mm glucose), in ionophore-containing medium (basal buffer supplemented with 3 mm CaCl2 and 1 μm A23187) or in a depolarizing medium containing 25 mm HEPES, pH 7.4, 90 mm NaCl, 60 mm KCl, 3 mm CaCl2, 1.4 mm MgCl2, and 10 mm glucose. A23187 was purchased from Sigma. In case cells were fixed, this was achieved by adding an equal volume of 4% (w/v) paraformaldehyde to the medium and incubating for 20 min at room temperature. Neurons were then counterstained for microtubule-associated protein 2 (MAP2; a neuron-specific protein located in dendrites) as follows. After fixation, the cells were washed twice with PBS and then incubated for 1 h with a monoclonal antibody recognizing MAP2 (Chemicon; diluted 1:200 in PBS with 0.1% Triton X-100), washed again twice, and incubated in the presence of Alexa546-conjugated goat anti-mouse antibodies (Molecular Probes; diluted 1:000). Confocal imaging was performed with an LSM 510 confocal microscope and a ×63 Plan-Neofluar lens (Carl Zeiss). Fluorescence intensity in regions of interest was quantitated using Multianalyst image analysis software (Bio-Rad). To test statistically if the fluorescence intensity after stimulation was significantly different from that before stimulation, the fluorescence after stimulation was calculated as a percentage of the original value. This was done for each cellular compartment in nine different cells. A two-tailed Student's t test was used to test whether the resulting mean values differed significantly from 100% (p < 0.005). Total Internal Reflection Fluorescence Microscopy—Transfected PC12 cells were incubated in basal solution containing 10 mm HEPES, pH 7.35, 147 mm NaCl, 2.8 mm KCl, 5 mm CaCl2, 1 mm MgCl2, and 10 mm glucose. Depolarizing solution (identical except for NaCl (90 mm) and KCl (60 mm)) was applied using a motor-driven two barrel Piezo perfusion device (Warner Instruments, Hamden, CT), allowing for fast (<10-ms) switching between depolarizing and basal solution. Total internal reflection fluorescence (TIRF) microscopy was performed on a Zeiss inverted microscope with custom built TIRF set up (Till Photonics, Grafelfing, Germany), using a ×100 objective with 1.45 numerical aperture. The depth of the evanescent field was measured to be 128 nm. Images were collected at 30 Hz using an intensified CCD camera (I-Pentamax; Roper Scientific) and analyzed with Metamorph (Universal Imaging Corp., Downingtown, PA). Fluorescence intensity was measured in a region of interest over the cell and corrected for background measured outside of the cell. Expression and Purification of GST-DOC2B—Full-length rat DOC2B cDNA was cloned into the expression vector pGEX4T3 (Amersham Biosciences) to encode a fusion protein of glutathione S-transferase (GST) and DOC2B. To isolate GST-DOC2B, 5 liters of Luria broth medium with 100 μg/ml ampicillin and 0.2% glucose was incubated at 37 °C until the exponential growth phase was reached. The incubation was then prolonged for 4 h in the presence of 100 μm isopropyl-1-thio-β-d-galactopyranoside to induce expression. Bacteria were centrifuged for 15 min at 4,000 × g and resuspended in 100 ml of HBS buffer (140 mm NaCl, 50 mm HEPES, pH 7.4, and protease inhibitor mixture; Roche Applied Science catalog no. 1836170). The cells were lysed by sonication and incubated for 1 h at 4 °C in the presence of 2.5% Triton X-100 for solubilization. After clearing the lysate by centrifugation for 30 min at 15,000 × g and 4 °C, GST-DOC2B was affinity-purified using glutathione-conjugated Sepharose 4B beads (Sigma). The amount of bound protein was determined by eluting GST-DOC2B in HBS plus 10 mm glutathione from a sample of the beads, followed by a Bradford assay using bovine serum albumin as a standard. The final yield of recombinant protein was 1.2 mg/liter of culture volume. Liposome Binding Assay—3H-Labeled liposomes were prepared by drying a mixture of 125 μl of phosphatidylcholine, 50 μl of phosphatidylserine (both 10 mg/ml in chloroform; Sigma), and 20 μl of 1,2-dipalmitoyl, l-3-phosphatidyl [N-methyl-3H]choline (1 mCi/ml; specific activity 83 Ci/mmol; Amersham Biosciences) under a nitrogen gas flow. The phospholipids were suspended in 10 ml of 10 mm HEPES, pH 7.2, and 100 mm NaCl, sonicated for 30 s, and centrifuged for 10 min at 3,000 × g to remove aggregates from the liposome suspension. Buffered Ca2+ solutions were prepared in 50 mm HEPES, pH 7.20, 140 mm NaCl, and 10 mm EGTA (<1 μm free Ca2+) or HEDTA (>1 μm free Ca2+) as a chelator. The concentrations of total Ca2+ were calculated with Win-Maxc version 2.40 (Chris Patton, Stanford, CA; available on the World Wide Web at www.standford.edu/~cpatton). Liposome binding was assayed in each buffer by incubating 50 μl of a 25% Sepharose slurry containing 50 μg of immobilized GST-DOC2B with 100 μl of the 3H-labeled liposome suspension for 1 h at 20 °C in a total volume of 1.5 ml. The beads were collected by centrifugation for 5 min at 1,000 × g and washed with 1.5 ml of the same buffered Ca2+ solution. After five washes, bound 3H-labeled phospholipids were eluted from the beads in 0.5% SDS, and radioactivity was quantitated by liquid scintillation counting in UltimaGold mixture (Packard). To investigate the possibility that DOC2B may function as a Ca2+ sensor in the activity-dependent enhancement of exocytosis, we first used heterologous cell lines to analyze whether Ca2+ influences the cellular distribution of DOC2B. To focus exclusively on Ca2+-dependent mechanisms, we selected HEK293 cells that lack endogenous expression of Munc13, thus excluding the diacylglycerol-induced DOC2B translocation for which Munc13 is required (11Duncan R.R. Betz A. Shipston M.J. Brose N. Chow R.H. J. Biol. Chem. 1999; 274: 27347-27350Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Fusion proteins consisting of full-length DOC2B and EGFP were expressed in HEK293 cells as stable proteins with the expected molecular mass (Fig. 1). The full-length band was also recognized by a polyclonal antibody against DOC2B, whereas this antibody did not recognize EGFP alone (Fig. 1B). To investigate whether the EGFP tag could cause mislocalization of the expressed protein, we compared the cellular distribution of DOC2B-EGFP with that of untagged DOC2B. As shown in Fig. 1, C and F, both proteins produced a similar staining pattern using anti-DOC2B antibody. The immunostaining pattern was also similar to the fluorescence derived directly from the EGFP tag, except that the nuclear staining was relatively weak (Fig. 1, C-E). The antigenic region could be less accessible in nuclear-resident DOC2B-EGFP. As an additional control we replaced EGFP with a Myc tag, resulting in the same distribution under all conditions tested (data not shown). The distribution of DOC2BMyc was again similar to that of the untagged DOC2B variants. From these control experiments, we conclude that the EGFP tag does not cause instability or artifacts in the subcellular localization of DOC2B. Ca2+-dependent Localization of DOC2B-EGFP in Heterologous Cells—Under standard culture conditions, DOC2B-EGFP and DOC2B were homogenously distributed in the cytoplasm and nucleus of transfected HEK293 cells. The fluorescence intensity in the nucleus varied between cells. The same homogenous distribution was observed when the cells were incubated in basal buffer lacking Ca2+. In contrast, when the cells were incubated under conditions that elevate intracellular Ca2+ levels, DOC2B-EGFP showed a clearly distinct distribution characterized by an intense membrane-associated fluorescence and a loss of cytoplasmic fluorescence. This is shown in Fig. 2, where Ca2+ influx was induced by the extracellular application of 3 mm Ca2+ and a 1 μm concentration of the Ca2+ ionophore A23187. Following this treatment, a redistribution of the fusion protein was evident within less than 10 s. After stimulation, the membrane-associated fluorescence was not completely uniform along the cell membrane but showed focal accumulations (Fig. 2, arrowheads). The overall fluorescence intensity in the cytoplasm, membrane, and nucleus was quantitated at different time points in nine different cells. Cytoplasmic fluorescence decreased significantly (p < 0.005) to 44 ± 3% of the original value (mean ± S.E., n = 9), whereas the membrane-associated fluorescence increased significantly (p < 0.005) to 131 ± 6%. The nuclear fluorescence intensity remained constant (mean value 101 ± 1%). We therefore conclude that DOC2B-EGFP migrates from the cytoplasm to the plasma membrane after Ca2+ influx. To exclude a nonspecific activity of the ionophore A23187 or its solvent, the ionophore was administered in the absence of extracellular Ca2+. This treatment did not induce any change in fluorescence distribution (Fig. 3, C and G). When 3 mm extracellular Ca2+ was applied without the ionophore, there was a small but detectable increase in plasma membrane-associated fluorescence (Fig. 3, B and F). This subtle change may reflect a small increase in intracellular Ca2+ as a consequence of the extracellular rise in Ca2+ concentration. Only after application of both components at the same time did a quantitative translocation of the fluorescent protein occur (Fig. 3, D and H). We also investigated whether the translocation was reversible and repeatable (Fig. 3, I-P). Cells expressing DOC2B-EGFP were stimulated as described above. The medium was then replaced to wash out the ionophore, resulting in a reversal of the membrane-associated fluorescence to a homogenous cytoplasmic distribution. An additional replacement with stimulation medium again induced a translocation of DOC2B-EGFP to the plasma membrane. These control experiments indicate that the observed translocation of DOC2BEGFP requires Ca2+ influx and is not caused by irreversible cell damage due to the stimulation treatment. The Ca2+-induced Translocation of DOC2B Is Independent of Munc13 and Tctex-1 Binding—The binding of DOC2B to the plasma membrane could be mediated by accessory proteins or by direct binding of one of the C2 domains to the phospholipid bilayer. To investigate the participation of each functional domain, we introduced targeted mutations in the N-terminal domain to block the interaction with Munc13 and Tctex-1 (a subunit of the cytoplasmic dynein complex), respectively (Fig. 4) (see Table I). All mutants expressed showed the expected molecular mass without visible signs of degradation in Western blotting (Fig. 1). Although HEK293 cells do not express Munc13 to detectable amounts as analyzed by immunoblotting, the above experiments do not exclude the involvement of extremely low amounts of Munc13. Therefore, we introduced a previously characterized mutation in the Munc13-interacting domain of DOC2B that abolishes Munc13 binding (12Mochida S. Orita S. Sakaguchi G. Sasaki T. Takai Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11418-11422Crossref PubMed Scopus (70) Google Scholar) (see Table I). To confirm that this mutation blocks the Munc13-dependent translocation triggered by phorbolesters, we first co-expressed DOC2B-EGFP and Munc13-1 in HEK293 cells. In agreement with previously reported studies (11Duncan R.R. Betz A. Shipston M.J. Brose N. Chow R.H. J. Biol. Chem. 1999; 274: 27347-27350Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), the administration of the phorbolester phorbol 12-myristate 13-acetate resulted in a translocation of DOC2B-EGFP (Fig. 5A). When the same treatment was performed with the mutated DOC2B-EGFP variant (M13), no translocation was observed (Fig. 5B). However, when the same cells were stimulated with 3 mm Ca2+ and 1 μm ionophore, the mutated protein readily translocated to the plasma membrane (Fig. 5C). Having confirmed that the M13 mutation effectively blocks the phorbolester-induced translocation of DOC2B-EGFP, we next expressed mutated DOC2BEGFP without co-expression of Munc13 (Fig. 6). Upon induction of Ca2+ influx, the mutant fusion protein rapidly associated with the plasma membrane in a way that was indistinguishable from wild type DOC2B-" @default.
• W2005972021 created "2016-06-24" @default.
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• W2005972021 date "2004-05-01" @default.
• W2005972021 modified "2023-10-01" @default.
• W2005972021 title "Ca2+-induced Recruitment of the Secretory Vesicle Protein DOC2B to the Target Membrane" @default.
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• W2005972021 doi "https://doi.org/10.1074/jbc.m400731200" @default.
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