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First published online 9 September 2003
doi: 10.1242/jcs.00719

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Synaptotagmin IX, a possible linker between the perinuclear endocytic recycling compartment and the microtubules

¹ Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
² Fukuda Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

View larger version (49K): [in a new window]   Fig. 1. PCR amplification of Syt IX cDNA. An agarose gel of the product of the second round of PCR, using for template the PCR product of RBL cell cDNA (2) or no DNA (3). The DNA size markers in bp are shown in (1).

View larger version (39K): [in a new window]   Fig. 2. Expression of Syt IX in RBL cells. (A) Cell extracts (60 µg) derived from Cos-7 cells transfected with empty vector, T7-tagged Syt IX cDNA or Flag-tagged Syt IX cDNA were resolved by SDS-PAGE and subjected to immunoblotting with anti-N-Syt IX (1 µg/ml), anti-T7 or anti-Flag antibodies. (B) Cell extracts (60 µg) derived from Cos-7 cells transfected with empty vector or with Syt I, Syt II, Syt III, Syt V and Syt IX cDNAs were resolved by SDS-PAGE and subjected to immunoblotting with anti-N-Syt IX antibodies. (C) Cell extracts (80 µg) derived from RBL cells stably transfected with either empty vector (1), or with pcDNA3-Syt IX sense cDNA, RBL-Syt IX+ (2) or with pcDNA3-Syt IX antisense cDNA, RBL-Syt IX– (3) were resolved by SDS-PAGE and subjected to immunoblotting with anti-N-Syt IX antibodies in the absence (1-3) or presence (4) of the immunizing peptide (250 ng/ml). (D) Cell extracts (80 µg) derived from RBL-Syt IX+ cells were resolved by SDS-PAGE and subjected to immunoblotting with anti-C2A-Syt IX antibodies (1 µg/ml). The cellular level of actin was determined to judge for equal loading. (E) Cell extracts (80 µg) derived from RBL cells stably transfected with pShooter Syt IX-GFP cDNA were resolved by SDS-PAGE and subjected to immunoblotting with anti-GFP, anti-C2A-Syt IX or anti-N-Syt IX as indicated.

View larger version (36K): [in a new window]   Fig. 3. Subcellular fractionation of RBL and RBL-Syt IX+ cells. Cell homogenates derived from RBL (A,B,D,E) or RBL-Syt IX+ (C) cells were fractionated on continuous sucrose gradients as described under Materials and Methods. Fractions were collected from the top, subjected to SDS-PAGE and immunoblotted with anti-N-Syt IX (A,B), anti-C2A-Syt IX (C) or anti-Gi2 (D) antibodies as indicated. To monitor the distribution of internalized Tfn (E), RBL cells were serum starved for 1 hour followed by 1 hour of incubation with biotin-conjugated Tfn (20 µg/ml) at 37°C before fractionation. Fractions subjected to SDS-PAGE and immunoblotted with HRP-conjugated streptavidin, visualized by ECL and the intensities of the bands corresponding to biotin-Tfn were quantified by densitometry.

View larger version (62K): [in a new window]   Fig. 4. Visualization of Syt IX localization. RBL-Syt IX+ (A), control RBL (B,C) or RBL cells stably transfected with Syt IX-GFP cDNA (D-F) were labeled with anti-C2A-Syt IX (10 µg/ml) (A,D-F) or anti-N-Syt IX (10 µg/ml) alone (B), or together with mouse anti-serotonin (C) followed by rhodamine-conjugated donkey anti-rabbit and FITC-conjugated donkey anti-mouse IgG. Cells were processed for immunofluorescent staining and visualized by confocal microscopy, as described under Materials and Methods. Bars, 5 µm (A,B,D-F); 3 µm (C).

View larger version (93K): [in a new window]   Fig. 5. Association of Syt IX with the ERC. RBL-Syt IX+ cells (A-C,G-I) or control cells (D-F) were allowed to internalize FITC-conjugated Tfn for 30 (A-C) or 15 (D-F) minutes at 37°C, or were transiently transfected with Rab11-GFP cDNA (G-I). Cells were subsequently labeled with anti-C2A-Syt IX (A-C,G-I) or anti-N-Syt IX (D-F), followed by rhodamine-conjugated donkey anti-rabbit IgG. Bars, 5 µm (A-F); 3 µm (G-I).

View larger version (54K): [in a new window]   Fig. 6. Effect of BFA on Syt IX, internalized Tfn and mannosidase localization. RBL-Syt IX+ cells were either untreated (A,C) or treated for 30 minutes at 37°C with BFA (5 µg/ml) (B,D), or allowed to internalize FITC-conjugated Tfn (50 µg/ml) for 1 hour (E,F) without (E) or with BFA (5 µg/ml) added for the last 30 minutes of Tfn internalization (F). Cells were subsequently labeled with either anti-C2A-Syt IX (A,B) or with anti-mannosidase II (C,D), followed by rhodamine-conjugated donkey anti-rabbit antibodies. Bars, 3 µm (A,B); 5 µm (C-F).

View larger version (92K): [in a new window]   Fig. 7. Co-localization of Syt IX and tubulin. RBL-Syt IX+ cells were: left untreated (A-D); treated for 30 minutes with 5 µM taxol (E-H); allowed to internalize FITC-conjugated Tfn (50 µg/ml) for 30 minutes, followed by 30 minutes of treatment with taxol (I); or transiently transfected with Rab 11-GFP cDNA and treated for 30 minutes with taxol (J). Cells were labeled with rabbit anti-C2A-Syt IX (A-H) or mouse anti-ß-tubulin (A-J), followed by rhodamine/FITC-conjugated donkey anti-mouse IgG or rhodamine/FITC-conjugated donkey anti-rabbit IgG as indicated. D and H are the phase-contrast images of A-C and E-G, respectively. Bars, 3 µm (A-H); 2 µm (I,J).

View larger version (37K): [in a new window]   Fig. 8. Binding of tubulin by Syt IX. GST, GST-Syt IX-C2A or GST-Syt IX-C2B (20 µg) immobilized on glutathione sepharose beads were incubated for 4 hours at 4°C with RBL cell extracts (500 µg) as described under Materials and Methods, in the absence or the presence of Ca2+ (3 mM) as indicated. Bound proteins were eluted by sample buffer, resolved on SDS-PAGE and analyzed by either Western blot, using anti-- or anti-ß-tubulin antibodies, or by staining the gel with Coomassie blue as indicated.

View larger version (22K): [in a new window]   Fig. 9. Co-immunoprecipitation of Syt IX and tubulin from intact RBL cells. Immunoprecipitation was performed as described under Materials and Methods, using either the indicated immunoprecipitating antibody (IP Ab) followed by protein A Sepharose or incubated with protein A Sepharose without prior incubation with the primary antibody. Immune complexes were separated by SDS-PAGE and analyzed by Western blot using the indicated antibody (IB Ab).

View larger version (146K): [in a new window]   Fig. 10. Tfn internalization in control, Syt IX-overexpressing and Syt IX-suppressed RBL cells. RBL-Syt IX– (A,D,G), control (B,E,H) and RBL-Syt IX+ (C,F,I) cells were grown on glass coverslips, serum starved for 1 hour and incubated with Texas-Red-conjugated Tfn (TR-Tfn, 50 µg/ml) for 1 hour at 4°C. Cells were subsequently left on ice (A-C) or warmed up to 37°C for 5 (D-F) or 30 (G-I) minutes. Cells were subsequently visualized by confocal microscopy as described under Materials and Methods. Bars, 13 µm.

View larger version (88K): [in a new window]   Fig. 11. Recycling of Tfn in control, Syt IX-overexpressing and Syt IX-suppressed RBL cells. RBL-Syt IX– (A,D,G), control (B,E,F) and RBL-Syt IX+ (C,F,I) cells were grown on glass coverslips, serum starved for 1 hour and incubated with TR-Tfn (50 µg/ml) for 1 hour at 37°C. Cells were subsequently placed on ice (A-C) or washed and subjected to a chase in the presence of unlabeled Tfn (100 µg/ml) and defroxamine mesylate (100 µM) for 30 minutes (D-F) or 1 hour (G-I). Cells were processed and visualized by confocal microscopy, as described under Materials and Methods. Bars, 13 µm.

View larger version (11K): [in a new window]   Fig. 12. Quantitative analysis of fluorescence images of Tfn recycling in control, Syt IX-overexpressing and Syt IX-suppressed RBL cells. The average fluorescence intensity per cell was measured for more than 100 cells per each condition described under Fig. 11.