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doi: 10.1242/10.1242/jcs.00072

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Neuronal calcium sensor 1 and phosphatidylinositol 4-OH kinase ß interact in neuronal cells and are translocated to membranes during nucleotide-evoked exocytosis

¹ CNR, Institute of Neuroscience, Cellular and Molecular Pharmacology, Center of Excellence on Neurodegenerative Diseases, Department of Medical Pharmacology, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy
² Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada M5G 1X5
³ School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK

View larger version (80K): [in a new window]   Fig. 1. Characterization of anti-NCS-1 antibodies. Total homogenates (40 µg) from rat brain (R.B.) and human cerebellum (H.C.) were analyzed by western blots using polyclonal (A, 44162; diluted 1:1000) or monoclonal antibodies (C, 3D5; diluted 1:5000) raised against recombinant NCS-1. Parallel samples were immunostained with preimmune rabbit or mouse IgG (IgG in A and C) or with the anti-mouse IgG, omitting the primary monoclonal antibody (C, no Ab). In B, proteins from rat brain total homogenate (R.B.) were labeled with anti-NCS-1 (44162) with (+) or without (-) pre-adsorption with 1 µg of purified recombinant NCS-1. In C, the arrow indicates the position of NCS-1; the bands of 55 and 28 kDa correspond to rat Ig heavy and light chains, respectively, which were present in the brain extracts and were recognized by the secondary antibodies (no Ab).

View larger version (65K): [in a new window]   Fig. 2. Distribution of NCS-1 and PI4Kß in subcellular fractions of rat brain. (A) Differential centrifugation of rat brain total homogenates. Equal amounts of proteins (12 µg) from each fraction (defined as described in Materials and Methods) were analyzed by SDS-PAGE and western blotting. The blots were probed with antibodies directed against PI4Kß (PI4K, diluted 1:4000), NCS-1, synaptophysin (Syn, diluted 1:2000) or synaptotagmin (Syt, diluted 1:1000). Note the presence of small amounts of NCS-1 and PI4Kß in a fraction of purified synaptic vesicles (SG-V). Both proteins are present in large amounts in P3 containing membrane-bound organelles of perikarya. (B) Velocity sucrose gradient centrifugation of the P3 (see A). Equal volumes (250 µl) of each fraction of the velocity gradients (fraction 1=top) were analyzed by western blotting using antibodies raised against PI4Kß (PI4K), NCS-1, calreticulin (Cal, diluted 1:2000), ribophorin (Ribo, diluted 1:250), TGN38 (diluted 1:1000) and syntaxin 1 (Syx, diluted 1:4000). (C) Fractionation of the S2 on discontinous sucrose density gradients. Equal amount of proteins from fractions 1, 2 and pellet (3) were analyzed by immunoblotting using anti-NCS1, PI4Kß, ribophorin, TGN38 and GOS-28 (GS28, 1:100) antibodies.

View larger version (169K): [in a new window]   Fig. 3. NCS-1 immunoreactivity is associated with the ER. Ultrathin frozen sections from rat brain cortex (A,C,D) and hippocampus (B) were immunostained for NCS-1 (A,B) or double immunolabeled (C,D) for NCS-1 (18 nm gold particles) and PDI (12 nm gold particles). NCS-1 immunoreactivity is visible in the cytoplasm and, to a lesser extent, on tubulovesicular structures (A-D, arrowheads). In these structures, NCS-1 (arrowheads) colocalizes with the ER marker PDI. No labeling is detectable on mitochondria (m), lysosmes (L) or nuclei (n). Bars, 200 nm.

View larger version (207K): [in a new window]   Fig. 4. NCS-1 immunoreactivity in the Golgi area. Ultrathin frozen sections from rat brain cortices were labeled for NCS-1 (A) and TGN38 (C) or double-immunostained (D) for NCS-1 (6 nm gold particles, arrowheads) and TGN38 (12 nm gold particles, arrows). In B, a section immunolabeled with rabbit IgG (control) is shown. NCS-1 immunoreactivity is present in the area of the Golgi complex (gc). The anti-TGN38 antibody specifically immunostains tubular-like elements corresponding to the TGN (tgn, C). Some labeling for NCS-1 (D, arrowheads) is detectable on the tubular-like structures immunolabeled with the anti-TGN38 antibodies (D, arrows). Note the lack of labeling when non-immune rabbit IgGs are used for immunostaining (B). Bars, 200 nm.

View larger version (18K): [in a new window]   Fig. 5. Quantitation of NCS-1 in total synaptic profiles and presynaptic terminals. Thin sections were immunolabeled with rabbit IgGs or anti-NCS-1 antibodies, followed by anti-rabbit IgG conjugated to 12 nm gold particles. The gold particles were counted over 200 or 300 synaptic profiles, respectively. The values are expressed as the number of gold particles per percentage of synapses (A) and presynaptic profiles (B).

View larger version (153K): [in a new window]   Fig. 6. Localization of NCS-1 in axon terminals. Ultrathin frozen sections were labeled with anti-NCS-1 antibody (A,B) or rabbit IgGs (C). NCS-1 immunolabeling is detectable in the presynaptic region. Note the presence of gold particles on synaptic vesicles (arrowheads); m, mitochondria; ps, postsynaptic region. Bars, 100 nm.

View larger version (60K): [in a new window]   Fig. 7. PI4Kß in hippocampal neurons. The distribution of PI4Kb in 15-day-old neurons was analyzed by confocal fluorescence microscopy. (A) Neurons immunolabeled with anti-PI4Kß antibodies. (B-D) Superimposed images (merge) of optical sections collected from neurons doubly immunostained for PI4Kß (red, B-D) and TGN38 (green, B), PDI (green, C) or synaptobrevin 2 (green, D). Note that immunolabeling for PI4Kß partially overlaps with that for the Golgi complex marker TGN38 or for the ER marker PDI (B,C). Bars, 10 µm.

View larger version (30K): [in a new window]   Fig. 8. Association of NCS-1 with PI4Kß. Triton X-100 rat brain (600 µg) or PC12 cell (1 mg) extracts were immunoprecipitated (IP) with antibodies against NCS-1, PI4Kß (PI4K) or non-immune rabbit IgG (IgG). The immunoprecipitated proteins were analyzed by western blotting using rabbit anti-PI4Kß IgG or polyclonal anti-NCS-1 antibodies.

View larger version (37K): [in a new window]   Fig. 9. Association of NCS-1 and PI4Kß occurs in the cytosol and membrane fractions and is not influenced by Ca2+. In A, equal proportions of Triton X-100 extracts prepared from total rat brain postnuclear supernatant (S1), membrane (Mem) or cytosol (Cyt) were immunoprecipitated (IP) with anti-NCS-1 antibodies, or rabbit IgG (IgG). In B, equal amounts (600 µg proteins) of rat brain extracts (Con) or rat brain extracts supplemented with either 1 mM Ca2+ (+Ca) or 5 mM EGTA were immunoprecipitated (IP) with anti-NCS-1 antibodies or rabbit IgG (IgG). The immunoprecipitates were analyzed by western blotting using anti-PI4Kß or anti-NCS-1 antibodies.

View larger version (38K): [in a new window]   Fig. 10. NCS-1 distribution in UTP-stimulated PC12 cells. The cells grown on petri dishes were incubated for 3 minutes at 37°C in KBR in the absence (CON) or presence of 300 µM UTP (UTP) or in KBR minus Ca2+ supplemented with 2 mM EGTA and UTP (UTP-Ca++OUT). The cells were homogenized and the and cytosolic (C) and membrane (M) fractions were obtained. Equal volumes of the fractions were analyzed by western blotting using anti-NCS-1 or anti-tubulin antibodies and the amount of NCS-1 in the cytosol or in the membrane were quantified. (A) Data are expressed as a percentage of total NCS-1 measured in the cytosolic and membrane fractions after no treatment (CON) or treatment with UTP or UTP minus Ca2+. The results represent the mean±s.e.m. (*P<0.001 compared with control). (B) Immunoblots showing the relative membrane/cytosol distribution of NCS-1 and tubulin (TUB) in membranes and cytosol of control and stimulated cells in the presence or absence of Ca2+.

View larger version (28K): [in a new window]   Fig. 11. PI4Kß distribution in UTP-stimulated PC12 cells. The cells grown on petri dishes were incubated for 3 minutes at 37°C in KBR in the absence (CON) or presence of 300 µM UTP (UTP) or in KBR minus Ca2+ supplemented with 2 mM EGTA and UTP (UTP-Ca++OUT). The cells were homogenized and cytosolic (C) and membrane (M) fractions were obtained. Equal volumes of the various fractions were analyzed by western blotting using anti-PI4Kß. (A) Immunoblots showing the distribution of PI4Kß in the membranes and cytosol of control and stimulated cells in the presence or absence of Ca2+. (B) The amounts of PI4Kß in the membrane fractions were analyzed by immunoblotting and the signals for PI4Kß were normalized to those of synaptophysin present in the same blots. The results are expressed as a percentage of the control and represent the mean±s.e.m. (n=14, *P<0.005 compared with control).

View larger version (30K): [in a new window]   Fig. 12. PI4Kß distribution in NCS-1-overexpressing PC12 cells. Cells were electroporated with cDNAs coding for NCS-1 and GFP (GFP+NCS-1) or GFP alone (GFP). 48 hours after transfection, the cells were incubated for 3 minutes at 37°C in KBR in the absence or presence of 300 µM UTP (+UTP) and then homogenized. The cytosolic (C) and membrane (M) fractions were prepared and analyzed by western blotting using anti-PI4Kß or NCS-1 antibodies. In A, the amounts of PI4Kß in the membrane fractions were quantified as described in Fig. 11 and the results are expressed as a percentage of the control (GFP-transfected cells) and represent the mean±s.e.m. (n=6, *P<0.002 compared with GFP-transfected cells). (B) Immunoblots showing the distribution of NCS-1 in the membranes and cytosol of GFP and NCS-1+GFP-transfected cells.