FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2100206769> ?p ?o ?g. }

• W2100206769 endingPage "18111" @default.
• W2100206769 startingPage "18098" @default.
• W2100206769 abstract "Several studies have shown that the neuronal calcium sensor (NCS-1) and phosphoinositol 4-kinase-β (PI4K-β) regulate the exocytotic process of nerve and neuroendocrine cells. The aim of our study was to investigate their possible interaction at rest and during stimulation in living cells and to decipher the role of this interaction in the secretory process. In PC12 cells, we observed a stimulation-induced recruitment of NCS-1 and PI4K-β from the intracellular compartment toward the plasma membrane. This recruitment was highly correlated to the intracellular Ca2+ rise induced by secretagogues. Using fluorescence resonance energy transfer between PI4K-β-ECFP and NCS-1-EYFP, we show that both proteins are interacting in resting cells and that this interaction increases with stimulation. It appears that the membrane insertion of NCS-1 is necessary for the interaction with PI4K-β, since a mutation that prevented the membrane insertion of NCS-1 abolished NCS-1-PI4K-β interaction, as revealed by fluorescence resonance energy transfer analysis. Additionally, the overexpression of mutated NCS-1 prevents the stimulatory effect on secretion induced by PI4K-β, suggesting that the interaction of the two proteins on a membrane compartment is necessary for the secretory function. Moreover, extinction of endogenous PI4K-β by small interfering RNA inhibits secretion and completely prevents the stimulatory effect of NCS-1 on calcium-evoked exocytosis from permeabilized PC12 cells, showing directly for the first time the functional implication of a NCS-1·PI4K-β complex in regulated exocytosis. Several studies have shown that the neuronal calcium sensor (NCS-1) and phosphoinositol 4-kinase-β (PI4K-β) regulate the exocytotic process of nerve and neuroendocrine cells. The aim of our study was to investigate their possible interaction at rest and during stimulation in living cells and to decipher the role of this interaction in the secretory process. In PC12 cells, we observed a stimulation-induced recruitment of NCS-1 and PI4K-β from the intracellular compartment toward the plasma membrane. This recruitment was highly correlated to the intracellular Ca2+ rise induced by secretagogues. Using fluorescence resonance energy transfer between PI4K-β-ECFP and NCS-1-EYFP, we show that both proteins are interacting in resting cells and that this interaction increases with stimulation. It appears that the membrane insertion of NCS-1 is necessary for the interaction with PI4K-β, since a mutation that prevented the membrane insertion of NCS-1 abolished NCS-1-PI4K-β interaction, as revealed by fluorescence resonance energy transfer analysis. Additionally, the overexpression of mutated NCS-1 prevents the stimulatory effect on secretion induced by PI4K-β, suggesting that the interaction of the two proteins on a membrane compartment is necessary for the secretory function. Moreover, extinction of endogenous PI4K-β by small interfering RNA inhibits secretion and completely prevents the stimulatory effect of NCS-1 on calcium-evoked exocytosis from permeabilized PC12 cells, showing directly for the first time the functional implication of a NCS-1·PI4K-β complex in regulated exocytosis. Exocytosis in nerve cells and in neuroendocrine cells is mediated by the exocytotic fusion of synaptic vesicles and secretory granules (dense core vesicles) with the plasma membrane in a process that is largely governed by Ca2+. Formation of the SNARE 3The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; PIP2, phosphoinositol 4,5-diphosphate; PI4K-β, phosphoinositol 4-kinase-β; FRET, fluorescence resonance energy transfer; PBS, phosphate-buffered saline; ROI, region(s) of interest; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; ECFP, enhanced cyan fluorescent protein; EYFP, enhanced yellow fluorescent protein; SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein; siRNA, small interfering RNA; GH, growth hormone. complex between the vesicular synaptobrevin and the plasma membrane syntaxin and SNAP-25 is generally accepted to play a major role in Ca2+-triggered exocytosis. Although the neuronal calcium sensor (NCS-1) (1McFerran B. W. Graham Burgoyne M. E.R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), orthologue of frequenin in invertebrates, and phosphoinositol 4-kinase-β (PI4K-β) (2Rajebhosale M. Greenwood S. Vidugiriene J. Jeromin A. Hilfiker S. J. Biol. Chem. 2003; 278: 6075-6084Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) have been shown to play an important role in regulating synaptic vesicle and secretory granule exocytosis, the underlying molecular mechanisms are still not fully understood. These proteins are mostly found in the cytosol but are also found on the membrane of secretory vesicles (1McFerran B. W. Graham Burgoyne M. E.R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 3Gasman S. Chasserot-Golaz S. Hubert P. Aunis D. Bader M.-F. J. Biol. Chem. 1998; 273: 16913-16920Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). NCS-1 has been shown to modulate Ca2+ membrane conductance through a direct effect on Ca2+ channels (2Rajebhosale M. Greenwood S. Vidugiriene J. Jeromin A. Hilfiker S. J. Biol. Chem. 2003; 278: 6075-6084Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 4Tsujimoto T. Jeromin A. Saitoh N. Roder J.C. Takahashi T. Science. 2002; 295: 2276-2279Crossref PubMed Scopus (184) Google Scholar, 5Rousset M. Cens T. Gavarini S. Jeromin A. Charnet P. J. Biol. Chem. 2003; 278: 7019-7026Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Indeed, the C-terminal part of NCS-1 interacts directly with the β subunit of P/Q type voltage-gated calcium channels (5Rousset M. Cens T. Gavarini S. Jeromin A. Charnet P. J. Biol. Chem. 2003; 278: 7019-7026Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), and a C-terminal peptide of NCS-1 inhibited the facilitation current through P/Q calcium channels after NCS-1 injection (4Tsujimoto T. Jeromin A. Saitoh N. Roder J.C. Takahashi T. Science. 2002; 295: 2276-2279Crossref PubMed Scopus (184) Google Scholar). On the other hand, PI4K-β together with phosphoinositide 5-kinase produces phosphoinositol 4,5-diphosphate (PIP2), which can form phospholipid microdomains (6Eberhard D.A. Cooper C.L. Low M.G. Holz R.W. Biochem. J. 1990; 268: 15-25Crossref PubMed Scopus (214) Google Scholar, 7Martin T.F. Loyet K.M. Barry V.A. Kowalchyk J.A. Biochem. Soc. Trans. 1997; 25: 1137-1141Crossref PubMed Scopus (35) Google Scholar, 8Loyet K.M. Kowalchyk J.A. Chaudhary A. Chen J. Prestwich G.D. Martin T.F. J. Biol. Chem. 1998; 273: 8337-8343Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Besides its effect on calcium channels, NCS-1 has also been shown to regulate PI4K-β activity (2Rajebhosale M. Greenwood S. Vidugiriene J. Jeromin A. Hilfiker S. J. Biol. Chem. 2003; 278: 6075-6084Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 9Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (220) Google Scholar). Indeed, PI4K-β and NCS-1 have been shown to interact in vitro (10Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 4384-4390Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), in Madin-Darby canine kidney (11Weisz O.A. Gibson G.A. Leung S.M. Roder J. Jeromin A. J. Biol. Chem. 2000; 275: 24341-24347Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), COS-7 (12Zhao X. Varnai P. Tuymetova G. Balla A. Toth Z.E. Oker-Blom C. Roder J. Jeromin A. Balla T. J. Biol. Chem. 2001; 276: 40183-40189Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), and PC12 cells (13Taverna E. Francolini M. Jeromin A. Hilfiker S. Rosa Roder. J.P. J. Cell Sci. 2002; 115: 3909-3922Crossref PubMed Scopus (57) Google Scholar). It was first believed that NCS-1 undergoes a Ca2+-induced conformational change, which allows its membrane insertion in secretory granules through its myristoyl residue and the subsequent interaction with PI4K-β (14Zozulya S. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11569-11573Crossref PubMed Scopus (294) Google Scholar, 15Ames J.B. Ishima R. Tanaka T. Gordon J.I. Stryer L. Ikura M. Nature. 1997; 389: 198-202Crossref PubMed Scopus (430) Google Scholar). However, this model has been recently challenged (16McFerran B.W. Weiss J.L. Burgoyne R.D. J. Biol. Chem. 1999; 274: 30258-30265Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 17O'Callaghan D.W. Ivings L. Weiss J.L. Ashby M.C. Tepikin A.V. Burgoyne R.D. J. Biol. Chem. 2002; 277: 14227-14237Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), since the myristoyl group of NCS-1 was reported to be freely accessible, and the membrane association of NCS-1 was independent of intracellular Ca2+ concentration. The aim of our study was to determine the conditions of NCS-1 and PI4K-β type III interaction in living cells and to correlate this interaction with the secretory function. For this purpose, we used living transfected PC12 cells overexpressing PI4K-β-ECFP and/or NCS-1-EYFP as fusion proteins. A stimulation-induced recruitment of NCS-1 and PI4K-β was observed by biochemical and immunofluorescence approaches as well as by time lapse fluorescence imaging and correlated with the intracellular Ca2+ concentration changes. The interaction between NCS-1 and PI4K-β in PC12 cells was demonstrated through fluorescence resonance energy transfer (FRET) between two chromophores and by co-immunoprecipitation. Finally, extinction experiments demonstrated the functional implication of the NCS-1-PI4K-β interaction in dense core secretory granule exocytosis. Materials—Poly-l-ornithine (Mr 30,000–70,000) and the anti-βCOP were from Sigma, and monoclonal anti-NCS-1 antibody was purchased from BD Biosciences. Rabbit anti-human PI4K-β type III antibody was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-SNAP-25 antibody was from Chemicon International (Temecula, CA). Culture medium (RPMI 1640), laminin, nerve growth factor (2.5 S), and horse serum were purchased from Invitrogen. Fetal calf serum was purchased from Dominique Dutscher S.A. (Brumath, France), and type I collagen from rat tail was a product from BD Biosciences. Fura-2-AM was from Molecular Probes, Inc. (Eugene, OR). The transfection reagent (GenePorter) was from Ozyme (St. Quentin/Yvelines, France). Cell Culture and Transfection Procedure—PC12 cells were plated on 35-mm glass bottom plastic Petri dishes (Falcon) coated with collagen and polyornithine (0.1 mg/ml each). They were grown in RPMI 1640 medium containing l-glutamine supplemented with 10% horse serum, 5% fetal calf serum (both decomplemented), 50 units/ml penicillin, and 50 μg/ml streptomycin. They were cultivated for 7 days in a humidified 5% CO2 atmosphere at 37 °C. To induce cell differentiation, 2.5 S murine nerve growth factor (50 ng/ml) was added to the culture medium 24 h after plating. For transfection experiments, we used the following constructs. NCS-1-EYFP construct was generated by subcloning the rat NCS-1 cDNA into pEYFP-N1 (Clontech), as previously described (18Scalettar B.A. Rosa P. Taverna E. Francolini M. Tsuboi T. Terakawa S. Koizumi S. Roder J. Jeromin A. J. Cell Sci. 2002; 115: 2399-2412Crossref PubMed Google Scholar), and the PI4K-β-ECFP construct was generated by subcloning the human PI4K-β cDNA into pECFP-C2 (Clontech), retaining its kinase activity. 4Jean de Barry, A. Janoshazi, J. L. Dupont, O. Procksch, S. Chasserot-Golaz, A. Jeromin, and N. Vitale, unpublished results. After 4 days in culture, 0.5 μg of plasmid of interest was transfected using 5 μl of GenePorter in 1 ml of RPMI 1640 without serum and antibiotics. For co-transfection experiments, 0.5 μg of each plasmid was added to the cells with 10 μl of GenePorter in 1 ml of RPMI 1640. After 4–5 h of incubation, the transfection medium was replaced by full culture medium supplemented with murine nerve growth factor. The cells were used 24–36 h after transfection. Co-transfection efficacy was assessed by spectral fluorescence recording (see below) of single cells. In every co-transfected cell displaying fluorescence, two peaks at 495 and 525 nm were observed. The transfection efficacy for NCS-1-EYFP expression was also compared in monotransfected cells and in co-transfected cells. The fluorescence intensity from single cells (excitation, 460 nm; emission, 525 nm) was 4 times less in co-transfected cells (1090 ± 200; n = 20) than in monotransfected cells (4280 ± 770, n = 20), indicating that co-transfection was effective but affected the level of NCS-1-EYFP expression. For small interfering RNA (siRNA) experiments, a bicistronic mammalian expression vector directing the synthesis of GH and siRNAs targeted against PI4K-β was generated essentially as described previously (19Vitale N. Mawet J. Camonis J. Regazzi R. Bader M.-F. Chasserot-Golaz S. J. Biol. Chem. 2005; 280: 29921-29928Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Two cDNA fragments encoding a 19-nucleotide sequence derived from the target transcript and separated from its reverse 19-nucleotide complement by a short spacer were cloned in front of the H1-RNA promoter. The silencers were generated using the following sequences: rat PI4K-β type III (accession number NM_031083) nucleotides 875–893 (active), nucleotides 1869–1887 (inactive), and the active sequence with the first CA bases changed to GG (mutated)). The specificity of the sequence was verified by BLAST search against the gene data bank. To estimate the silencing effect, the plasmids were electroporated (260 V, 1050 microfarads, for 17 ms) in 107 PC12 cells, and 72 h post-transfection, cells were used for Western blot and immunofluorescence experiments. The transfection efficiency under these conditions was measured by counting GH-positive cells after immunofluorescence using an anti-GH antibody (19Vitale N. Mawet J. Camonis J. Regazzi R. Bader M.-F. Chasserot-Golaz S. J. Biol. Chem. 2005; 280: 29921-29928Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) and was found to be 60 ± 5%. Immunocytochemistry—We used an isoform-specific anti-PI4K-β antibody (20Godi A. Pertile P. Meyers R. Marra P. Di Tullio G. Iurisci C. Luini A. Corda D. De Matteis M.A. Nat. Cell Biol. 1999; 1: 280-287Crossref PubMed Scopus (454) Google Scholar) and an anti-NCS-1 antibody (4Tsujimoto T. Jeromin A. Saitoh N. Roder J.C. Takahashi T. Science. 2002; 295: 2276-2279Crossref PubMed Scopus (184) Google Scholar, 13Taverna E. Francolini M. Jeromin A. Hilfiker S. Rosa Roder. J.P. J. Cell Sci. 2002; 115: 3909-3922Crossref PubMed Scopus (57) Google Scholar, 21Werle M.J. Roder J. Jeromin A. Neurosci. Lett. 2000; 284: 33-36Crossref PubMed Scopus (28) Google Scholar). Cultured cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4), for 20 min. After permeabilization for 10 min in PBS containing 0.1% Triton X-100™, cells were incubated for 1 h in 10% normal goat serum in PBS to block nonspecific binding. This step was followed by 2 h of incubation with primary antibodies in PBS containing 5% normal goat serum at room temperature (anti-PI4K-β, 1:100; anti-NCS-1, 1:100). Goat anti-rabbit or anti-mouse secondary antibody (1:1000 in 5% goat serum) Alexafluor 488™-conjugated (Molecular Probes) or indocarbocyanine-conjugated (Cy3; Jackson Immunoresearch, West Grove, PA) was applied for 1 h in PBS. Cultures were finally mounted in Vectashield™ medium (Vector Laboratories, Burlingame, CA) to reduce photobleaching. Controls were obtained by omitting the primary antibody in the incubation bath, and no staining of the cells was observed in these conditions. Immunostaining was performed as described previously (23Vitale N. Chasserot-Golaz S. Bailly Y. Morinaga N. Frohman M.A. Bader M.-F. J. Cell Biol. 2002; 159: 79-89Crossref PubMed Scopus (103) Google Scholar), and stained cells were visualized using a Zeiss LSM 510 confocal microscope. Quantification was performed using the Zeiss CSLM instrument 3.2 software. Regions of interest (ROI) were selected by taking a similar ellipse (6.5 μm2) with the great diagonal (5 μm) at the plasma membrane. Imaging Experiments in Living Cells—PC12 cells were incubated for 30 min at 37 °C in Krebs medium (106 mm NaCl, 4.5 mm KCl, 1.2 mm MgSO4, 2.5 mm CaCl2, 11 mm d-glucose, 1.2 mm KH2PO4, 25 mm NaHCO3, pH 7.4, equilibrated with 5% CO2) containing 2 μm Fura-2-AM. Cells were then washed and subsequently incubated for 20 min with Krebs medium. After three washes, the cells were placed on an inverted microscope (Axiovert 35M; Zeiss), superfused with Krebs solution (1 ml/min), and alternatively illuminated at 350 ± 10 nm (for Fura-2) or at 490 ± 10 nm (for EGFP). The fluorescent emission was observed using a dichroic mirror at 500 nm and a long pass filter at 510 nm. For each excitation wavelength (350 nm for Fura-2 and 490 nm for EGFP) and every 2.5 s, an image was recorded using an intensified CCD camera (Extended Isis; Photonic Science) and the Fluostar software (Imstar, Paris, France). Drugs were applied by superfusion in Krebs solution for 1 min. Image Analysis and Semiquantification—Image analysis was performed according to Dupont et al. (22Dupont J.L. Janoshazi A. Bellahcene M. Mykita S. de Barry J. Eur. J. Neurosci. 2000; 12: 215-226Crossref PubMed Scopus (16) Google Scholar). Two image series, each corresponding to one excitation wavelength (350 and 490 nm), were recorded for each experiment. After background subtraction from each image series, another image series was calculated by exponential interpolation to assess for the fluorescence base line during the whole recording period, taking into account probe bleaching and possible probe leaks. This interpolation was performed on a pixel-to-pixel basis between the initial recording period and the final recording period. Finally, a third image series was then calculated, dividing experimental images by interpolated base-line images on a pixel-to-pixel basis. This third set of images was called the normalized image series. The normalized values were independent of fluorescence bleaching and probe concentration heterogeneity in the preparation. Image analysis was performed on a DEC-Alpha work station (Digital Co., Boston, MA) using the Khoros function library (Khoral Research Inc.). On the normalized image series of the Fura-2 fluorescence, the data were then semiquantified by defining regions of interest usually corresponding to entire cells in the microscopic field. The average of the normalized values inside these regions was calculated and plotted versus the image record time. The curves displayed peaks corresponding to stimulation-induced increases in intracellular [Ca2+]. On the normalized image series of the EGFP fluorescence, we defined regions of interest corresponding to the periphery and the center of each fluorescent cell body in the microscopic field. For each cell, the average value of the normalized fluorescence intensity of the outer region was divided by the average value of the corresponding center. This ratio was then plotted versus recording time and revealed the protein translocation during the experiment. Fluorescence Resonance Energy Transfer Measurement—For FRET experiments, we used PI4K-β fused with ECFP as a donor and NCS-1 fused with EYFP as an acceptor. PC12 cells monotransfected with plasmids encoding PI4K-β-ECFP or NCS-1-EYFP or co-transfected with both plasmids were placed on an inverted fluorescence microscope (Axiovert 35M; Zeiss; dichroic mirror 470 nm), which was connected by optical fibers with the excitation and the emission monochromators of a spectrofluorimeter (Alphascan, PTI). A single cell was selected in the microscope field. As for imaging experiments, the cells were superfused by Krebs solution (1 ml/min), and drugs were applied by superfusion for 1 min. The cells were illuminated at 430 nm (the excitation wavelength of ECFP), and emission spectra between 480 and 600 nm were recorded every 15 s during the experiment. For FRET measurements, the emission spectra were deconvoluted after base-line subtraction to distinguish donor and acceptor fluorescence intensities. To minimize the bias due to differential expression of the tagged proteins in single cells, donor and acceptor intensities were then normalized by dividing the calculated intensities by the initial measured intensity. These normalized intensities were then plotted versus time. For bleaching resonance energy transfer, the procedure was similar. An initial emission spectrum (excitation 430 nm, the excitation wavelength of ECFP) was recorded, and the preparation was then continuously illuminated at 480 nm (excitation wavelength of EYFP) for 10 min while superfused with Krebs solution. A second emission spectrum (excitation 430 nm) was recorded after illumination. The changes in fluorescence intensities of the donor and the acceptor were calculated after spectral deconvolution. In separate experiments, PC12 cells coexpressing PI4K-β-ECFP and NCS-1-EYFP were fixed with 4% paraformaldehyde in phosphate-buffered saline. They were observed on a confocal microscope (Leica TCS SP2 AOBS), which allows spectral recording for each pixel of the image. The preparation was excited at 405 nm using a diode laser, and the images were spectrally recorded between 440 and 580 nm. The FRET signal was assessed by calculating the intensity ratio I530/I490. FRET images were generated using the Metamorph software (Universal Imaging). Subcellular Fractionation—PC12 cells were washed twice with Locke's solution and then incubated for 10 min with Locke's solution (Resting) or stimulated for 10 min with an elevated K+ solution. Medium was removed, and cells were immediately scraped in 1 ml of 0.32 m sucrose (20 mm Tris, pH 8.0). Cells were broken in a Dounce homogenizer and centrifuged at 800 × g for 15 min. The supernatant was further centrifuged at 20,000 × g for 20 min. The resulting supernatant was further centrifuged for 60 min at 100,000 × g to obtain the cytosol (supernatant) and microsomes. The 20,000 × g pellet containing the crude membrane fraction was resuspended in 0.32 m sucrose (20 mm Tris, pH 8.0), layered on a cushion sucrose density gradient (1–1.6 m sucrose, 20 mm Tris, pH 8.0), and centrifuged for 90 min at 100,000 × g to separate the plasma membrane from secretory granules (23Vitale N. Chasserot-Golaz S. Bailly Y. Morinaga N. Frohman M.A. Bader M.-F. J. Cell Biol. 2002; 159: 79-89Crossref PubMed Scopus (103) Google Scholar). The upper fractions containing SNAP-25 (plasma membrane marker) and the pellet containing chromogranin A (secretory granule markers) were collected and resuspended in buffer (20 mm Tris, pH 8.0, 100 mm NaCl, 1 mm MgCl2) before protein quantification by Bradford measurements. Immunoprecipitation—Cell extracts were prepared by lysing the cells as described previously (19Vitale N. Mawet J. Camonis J. Regazzi R. Bader M.-F. Chasserot-Golaz S. J. Biol. Chem. 2005; 280: 29921-29928Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Lysates were clarified by centrifugation, and 500-μg aliquots were incubated for 24 h at 4 °C with anti-PI4K-β (1:50 dilution). PI4K-β was immunoprecipitated from the supernatant of each sample using protein A-agarose (19Vitale N. Mawet J. Camonis J. Regazzi R. Bader M.-F. Chasserot-Golaz S. J. Biol. Chem. 2005; 280: 29921-29928Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Precipitated proteins were resolved on 12% polyacrylamide-SDS gels and immunoblotted with anti-PI4K-β (1:1000) and anti-NCS-1 (1:1000) antibodies. Blots were processed using the Western-Light Plus chemiluminescent detection system (Tropix, Bedford, MA). Secretion Measurements—PC12 cells were co-transfected with the plasmids of interest together with pXGH5 encoding human growth hormone using GenePorter. GH release experiments were performed 48 h after transfection (23Vitale N. Chasserot-Golaz S. Bailly Y. Morinaga N. Frohman M.A. Bader M.-F. J. Cell Biol. 2002; 159: 79-89Crossref PubMed Scopus (103) Google Scholar). PC12 cells were washed twice with Locke's solution and incubated for 10 min in Ca2+-free Locke's solution (basal release) or with Locke's solution containing either 59 mm KCl and 85 mm NaCl or 30 μm ATP. The supernatant was collected, and the cells were harvested by scraping in 10 mm phosphate-buffered saline. The amount of GH secreted into the medium or present in the cells was measured using a radioimmunoassay kit (Nichols Institute). The amount of GH secretion is expressed as a percentage of total GH present in the cells before stimulation. Localization of PI4K-β and NCS-1—In resting PC12 cells, endogenous PI4K-β immunoreactivity appeared as small patches inside the cytoplasm, and the density of the fluorescence was higher around the nucleus (Fig. 1), probably in the Golgi apparatus, consistent with earlier observations (1McFerran B. W. Graham Burgoyne M. E.R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 3Gasman S. Chasserot-Golaz S. Hubert P. Aunis D. Bader M.-F. J. Biol. Chem. 1998; 273: 16913-16920Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Discrete immunopositive portions of the plasma membrane were also observed, and a careful examination showed that the immunolabel was discontinuous on these portions (Fig. 1A, arrow). In depolarized cells, immunoreactivity appeared further concentrated at the level of the plasma membrane (Fig. 1A). No fluorescence staining of the cells was observed when the primary antibody was omitted, and the distribution pattern of PI4K-β-ECFP was similar to endogenous PI4K-β-immunoreactivity, albeit slightly more fuzzy (see Figs. 2 and 6).FIGURE 2Imaging of NCS-1 and PI4K-β recruitment in KCl-stimulated PC12 cells. A, resting or stimulated PC12 cells expressing either PI4K-β-EGFP or NCS-1-EGFP were fixed and processed for staining with anti-SNAP-25 antibodies. Confocal images in the green (PI4K-β or NCS-1) or red (SNAP-25) were recorded simultaneously in the same optical section by a double exposure procedure. Masks represent the region of colocalization obtained by selecting the double-labeled pixels. Bar, 5 μm. Specific ROI of the plasma membrane were selected (ellipse; see EGFP images). The histogram represents a semiquantitative analysis of the amount of PI4K-β or NCS-1 immunoreactivity colocalized with SNAP-25 in resting (R) and KCl-stimulated (S) cells on the whole cells or in the ROI. (results ± S.E.; n = 38 cells for PI4K-β, and n = 36 cells for NCS-1) Quantification was performed using the Zeiss CSLM instrument 3.2 software (Student's t test with the corresponding resting condition values; *, p < 0.001). B and C, pseudocolor images of living PC12 cells overexpressing either PI4K-β-EGFP (B) or NCS-1-EGFP (C). The micrographs are color-coded according to their normalized fluorescence intensity when illuminated at 490 nm and observed at 520 ± 10 nm. During stimulation with 80 mm KCl for 1 min, both proteins were translocated toward the plasma membrane as revealed by pseudocolor ratio images. This translocation was also revealed by calculating the fluorescence intensity ratio between the outer part of single cells and the corresponding center of the cell body (shown in the graphs). Note that in both cases, the protein translocation during stimulation (solid bars) is transient and that the proteins translocate back to the cytoplasm after stimulus withdrawal (average of five different experiments involving 10–12 cells ± S.E.).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Localization of the FRET signal in resting and stimulated PC12 cells. Confocal micrographs of resting (A) or stimulated (after a 1-min 80 mm KCl application) (B) PC12 cells coexpressing PI4K-β-ECFP and NCS-1-EYFP. The merged images show the colocalization of the two proteins inside the cytoplasm and at the cell periphery (arrows). Pseudocolored FRET efficiency images show a faint FRET signal in resting cells. In stimulated cells, the FRET signal primarily increases in the perinuclear cytoplasm, whereas it seems to decrease at the periphery of the cells (calibration bar, 20 μm). The fluorescence spectrum recorded on the confocal microscope displayed two peaks at 490 and 520 nm, when the preparation was illuminated at 405 nm (continuous line)(C). In cells expressing PI4K-ECFP only a single peak was observed (dotted line)(C). The statistical distribution of the pixel values in FRET signal images shows a single pixel population with low FRET level in resting cells (red columns and dotted line)(D). In stimulated cells (black columns and dashed line)(D), most of the pixels exhibit a high FRET level, but a minor population of low FRET signal pixels is also present. These findings were observed in three distinct cell preparations. Calibration bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) NCS-1 immunoreactivity was partially cytosolic with a higher density of the fluorescence near the nucleus in resting PC12 cells, although we could also observe punctuated immunopositive signal (Fig. 1A) near the plasma membrane and some accumulation of the protein in the distal part of the neurites" @default.
• W2100206769 created "2016-06-24" @default.
• W2100206769 creator A5004837365 @default.
• W2100206769 creator A5021813238 @default.
• W2100206769 creator A5023347751 @default.
• W2100206769 creator A5038071498 @default.
• W2100206769 creator A5066422345 @default.
• W2100206769 creator A5071148207 @default.
• W2100206769 creator A5084738344 @default.
• W2100206769 date "2006-06-01" @default.
• W2100206769 modified "2023-10-14" @default.
• W2100206769 title "Functional Implication of Neuronal Calcium Sensor-1 and Phosphoinositol 4-Kinase-β Interaction in Regulated Exocytosis of PC12 Cells" @default.
• W2100206769 cites W1536403028 @default.
• W2100206769 cites W1568234578 @default.
• W2100206769 cites W1589334735 @default.
• W2100206769 cites W1602043854 @default.
• W2100206769 cites W1670420367 @default.
• W2100206769 cites W1969153940 @default.
• W2100206769 cites W1971742114 @default.
• W2100206769 cites W1975886957 @default.
• W2100206769 cites W1979889913 @default.
• W2100206769 cites W2007667736 @default.
• W2100206769 cites W2010454617 @default.
• W2100206769 cites W2017237303 @default.
• W2100206769 cites W2022425508 @default.
• W2100206769 cites W2026682690 @default.
• W2100206769 cites W2029065061 @default.
• W2100206769 cites W2037140390 @default.
• W2100206769 cites W2047724990 @default.
• W2100206769 cites W2051746145 @default.
• W2100206769 cites W2064731387 @default.
• W2100206769 cites W2069847632 @default.
• W2100206769 cites W2073009787 @default.
• W2100206769 cites W2076801049 @default.
• W2100206769 cites W2076923427 @default.
• W2100206769 cites W2078411393 @default.
• W2100206769 cites W2081211846 @default.
• W2100206769 cites W2081240939 @default.
• W2100206769 cites W2106723144 @default.
• W2100206769 cites W2121079700 @default.
• W2100206769 cites W2135754883 @default.
• W2100206769 cites W2161888613 @default.
• W2100206769 cites W2161986280 @default.
• W2100206769 cites W2200436150 @default.
• W2100206769 cites W2299037128 @default.
• W2100206769 cites W2337043966 @default.
• W2100206769 doi "https://doi.org/10.1074/jbc.m509842200" @default.
• W2100206769 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16638749" @default.
• W2100206769 hasPublicationYear "2006" @default.
• W2100206769 type Work @default.
• W2100206769 sameAs 2100206769 @default.
• W2100206769 citedByCount "53" @default.
• W2100206769 countsByYear W21002067692012 @default.
• W2100206769 countsByYear W21002067692013 @default.
• W2100206769 countsByYear W21002067692015 @default.
• W2100206769 countsByYear W21002067692016 @default.
• W2100206769 countsByYear W21002067692019 @default.
• W2100206769 countsByYear W21002067692020 @default.
• W2100206769 countsByYear W21002067692021 @default.
• W2100206769 countsByYear W21002067692022 @default.
• W2100206769 countsByYear W21002067692023 @default.
• W2100206769 crossrefType "journal-article" @default.
• W2100206769 hasAuthorship W2100206769A5004837365 @default.
• W2100206769 hasAuthorship W2100206769A5021813238 @default.
• W2100206769 hasAuthorship W2100206769A5023347751 @default.
• W2100206769 hasAuthorship W2100206769A5038071498 @default.
• W2100206769 hasAuthorship W2100206769A5066422345 @default.
• W2100206769 hasAuthorship W2100206769A5071148207 @default.
• W2100206769 hasAuthorship W2100206769A5084738344 @default.
• W2100206769 hasBestOaLocation W21002067691 @default.
• W2100206769 hasConcept C12554922 @default.
• W2100206769 hasConcept C169760540 @default.
• W2100206769 hasConcept C175969161 @default.
• W2100206769 hasConcept C178790620 @default.
• W2100206769 hasConcept C184235292 @default.
• W2100206769 hasConcept C185592680 @default.
• W2100206769 hasConcept C41625074 @default.
• W2100206769 hasConcept C519063684 @default.
• W2100206769 hasConcept C55493867 @default.
• W2100206769 hasConcept C86803240 @default.
• W2100206769 hasConcept C95444343 @default.
• W2100206769 hasConceptScore W2100206769C12554922 @default.
• W2100206769 hasConceptScore W2100206769C169760540 @default.
• W2100206769 hasConceptScore W2100206769C175969161 @default.
• W2100206769 hasConceptScore W2100206769C178790620 @default.
• W2100206769 hasConceptScore W2100206769C184235292 @default.
• W2100206769 hasConceptScore W2100206769C185592680 @default.
• W2100206769 hasConceptScore W2100206769C41625074 @default.
• W2100206769 hasConceptScore W2100206769C519063684 @default.
• W2100206769 hasConceptScore W2100206769C55493867 @default.
• W2100206769 hasConceptScore W2100206769C86803240 @default.
• W2100206769 hasConceptScore W2100206769C95444343 @default.
• W2100206769 hasIssue "26" @default.
• W2100206769 hasLocation W21002067691 @default.
• W2100206769 hasOpenAccess W2100206769 @default.
• W2100206769 hasPrimaryLocation W21002067691 @default.
• W2100206769 hasRelatedWork W1588928424 @default.
• W2100206769 hasRelatedWork W1972411901 @default.

• next