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First published online 30 January 2007
doi: 10.1242/jcs.03375

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Synaptotagmins I and IX function redundantly in regulated exocytosis but not endocytosis in PC12 cells

Department of Biochemistry, 433 Babcock Drive, University of Wisconsin, Madison, WI 53706, USA

View larger version (44K): [in this window] [in a new window]   Fig. 1. Syt I and Syt IX colocalize on DCVs in PC12 cells. (A) PC12 cells were fixed and stained with Syt I-specific (red channel) and Syt IX-specific (green channel) antibodies and coimmunolocalization was determined (yellow). 3D reconstruction of deconvolved confocal sections is shown in supplementary material Movie 1. Bar, 5 µm; square insets are 1 µmx1 µm. (B) Syt I and Syt IX colocalization was quantified in deconvolved confocal Z-sections of 12 randomly selected PC12 cells. Bar graphs give the total percent overlap (corrected for random overlap) for Syt I with Syt IX and Syt IX with Syt I as the mean ± s.d. (C) Representative deconvolved Z-section of two PC12 cells showing colocalization (yellow) of Syt I (red channel) and Syt IX (green channel). A 7-µm line was drawn through the edge of one PC12 cell for line-scan analysis. (D) Fluorescence intensity, as arbitrary fluorescence units of Syt I and Syt IX along the 7-µm line shown in C. *, individual DCVs. Fluorescence intensity of a 0.1-µm bead is shown for size comparison.

View larger version (38K): [in this window] [in a new window]   Fig. 2. shRNA knockdown of Syt I and IX abolishes regulated exocytosis. (A) Nucleotide sequence alignment of Syt I and Syt IX in the part of the ORF targeted by shRNA. Predicted structure of the shRNA transcript (Syt I/IX). (B) Clones stably expressing pSHAG-Syt I/IX (Syt I/IX-null) were isolated and an immunoblot of a representative clone is shown using Syt-I-specific and Syt-IX-specific antibodies. The Syt I/IX-null cells were further characterized with Syt-IV-specific and Syt-VII-specific antibodies. SNAP-25 staining was used as a loading control for both immunoblots. (C) Wild-type and Syt I/IX-null cells were transfected with a plasmid encoding hGH, and intact cell secretion assays were conducted. Ca2+-dependent hGH secretion was determined as the amount of hGH secreted in 20 minutes at 37°C, stimulated by depolarization medium (K+-stimulated) minus the amount of hGH secreted in basal medium (basal). Values represent the mean of triplicate determinations, the standard diviation (s.d.) is indicated. Ca2+-dependent DCV exocytosis was similarly abolished in three Syt I/IX-null cell lines and in transient transfections with the Syt I/IX-shRNA plasmid. (D) hGH secretion assays in intact cells were conducted at the indicated time points. Ca2+-dependent hGH secretion (K-stimulated minus basal) was plotted as percent hGH release (total hGH secreted divided by total hGH secreted plus total hGH remaining in the cells x100). The results shown are representative of three independent experiments and similar results were obtained for three Syt I/IX-null cell lines. (E) Time course for basal and K+-stimulated hGH release of wild-type and Syt I/IX-null cells. In Syt I/IX-null cells, release of hGH was the same in the absence or presence of a stimulus, and was significantly reduced compared with hGH release in K+-stimulated wild-type cells. Results are representative of three independent experiments and were similar for three Syt I/IX-null cell lines. (F) Immunocytochemistry of CgB (DCV marker) and transiently transfected hGH in wild-type and Syt I/IX-null cells. hGH is targeted to DCVs in wild-type and Syt I/IX-null cells. The cellular distribution of DCVs was unaffected by the downregulation of Syt I and Syt IX. Bar, 10 µm.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Loss of regulated exocytosis occurs at a late step downstream from Ca2+ entry. (A) Wild-type and clones stably expressing pSHAG-Syt I/IX (Syt I/IX-null) were transfected with a plasmid expressing hGH and permeable cell secretion assays were conducted (see Materials and Methods) in the absence (basal) or presence (Ca2+-stimulated) of 10 µM free Ca2+. Ca2+-dependent hGH secretion was determined as the amount of hGH secreted in 5 minutes at 30°C in Ca2+-stimulated minus basal conditions. Values represent the mean ± s.d. of triplicate determinations. (B) Ca2+ dose-response for exocytosis in wild-type and Syt I/IX-null cells. Secretion assays in permeable cells were conducted at the indicated Ca2+ concentrations in wild-type and Syt I/IX-null cells. hGH released was determined and plotted as the mean ± s.d. of triplicate determinations.

View larger version (36K): [in this window] [in a new window]   Fig. 4. TIRF analysis of ANF-EGFP secretion in wild-type cells and cells lacking Syt I and Syt IX (Syt I/IX-null cells). (A) Representative footprints of wild-type and Syt I/IX-null cells. TIRF images were acquired of live cells resting in basal medium. Square insets are 1 µm2. (B) DCVs in each footprint were counted and divided by the total footprint area. The frequency distribution of DCVs for wild-type and Syt I/IX-null cells was similar. The average number of DCVs per µm2 (± s.d.) was 7.4±1.5 for wild-type and 7.5±1.1 for Syt I/IX-null cells (n=30). (C) Single ANF-EGFP secretion events are shown as sequential images at 4 Hz (bottom) with fluorescence intensity plots (top). The event shown on the left corresponds to full fusion with release of ANF-EGFP whereas the event on the right corresponds to transient fusion event with incomplete ANF-EGFP release; x=2.5 seconds and y=10 arbitrary units. (D) Wild-type and Syt I/IX-null cells expressing ANF-EGFP were stimulated with depolarization medium and images were acquired at 0.25-second intervals. Fusion events were counted manually as a flash (or puff) of fluorescence in wild-type cells or Syt I/IX-null cells and the sum of fusion events in 10-second intervals was plotted (n=21). The number of fusion events was drastically reduced in the Syt I/IX-null cells. (E) Same data as in C plotted as the sum of the average number of fusion events per cell over time (mean ± s.d.).

View larger version (35K): [in this window] [in a new window]   Fig. 5. Downregulation of Syt I or Syt IX alone has no effect on stimulated exocytosis. (A) During isolation of stable cell lines lacking Syt I and Syt IX, clones were isolated in which Syt I was fully downregulated but Syt IX was expressed at wild-type levels (Syt I-null cells). pSHAG-Syt IX downregulated Syt IX without affecting Syt I expression (Syt IX-null cells). Immunoblot analysis was used to analyze Syt I and Syt IX. (B) Wild-type, Syt I-null and Syt IX-null cells were transfected with a plasmid expressing hGH and intact cell secretion assays were conducted. Ca2+-dependent hGH release was unaffected by the downregulation of either Syt I or Syt IX. (C) Wild-type, Syt I-null and Syt IX-null cells, transfected with a plasmid expressing ANF-EGFP, were stimulated in depolarization medium and TIRF images were acquired at 0.25-second intervals. The data were analyzed as in Fig. 4 and plotted as the sum of the events for ten wild-type, ten Syt I-null, ten Syt IX-null and ten Syt I/IX-null cells. DCV exocytosis occurred in the Syt I-null or Syt IX-null cells. (D) The sum of the average number of fusion events per cell over time (mean ± s.d.) for wild-type (n=30), Syt I-null (n=15) and Syt IX-null cells (n=10). values for fusion in wild-type, Syt I-null and Syt IX-null cells were 32.1 seconds, 36.0 seconds and 17.9 seconds, respectively. Differences between Syt IX-null and wild-type or Syt I-null were significant (P<0.001).

View larger version (22K): [in this window] [in a new window]   Fig. 6. Rescue of function by re-expression of Syt I or Syt IX. (A) Immunoblot analysis of wild-type cells, cells that lack Syt I and Syt IX (Syt I/IX-null) and Syt I/IX-null cells that express the pcDNA3-Syt I or pcDNA3-Syt IX rescue vector. (B) Wild-type and Syt I/IX-null cells were transfected with a plasmid expressing ANF-EGFP or Syt I/IX-null cells were co-transfected with a plasmid expressing ANF-EGFP and the pcDNA3-Syt I or pcDNA3-Syt IX rescue vector. Cells were stimulated in depolarization medium and images were acquired at 0.25-second intervals. The data were analyzed as in Fig. 4D and plotted as the sum of the events for ten wild-type, ten Syt I/IX-null, ten Syt I/IX-null + Syt I and ten Syt I/IX-null + Syt IX cells. Re-expression of either Syt I or Syt IX restored Ca2+-dependent DCV exocytosis in the Syt I/IX-null cells. (C) The sum of the average number of fusion events per cell over time (mean ± s.d.) for wild-type (n=40), Syt I/IX-null (n=10), Syt I/IX-null + Syt I (n=20) and Syt IX-null + Syt IX (n=12). values for fusion in wild-type (31.2±9.1 seconds) and Syt I/IX-null + Syt I (15.6±1.9 seconds) cells were significantly different (P<0.001).

View larger version (43K): [in this window] [in a new window]   Fig. 7. Syt I but not Syt IX downregulation inhibits compensatory endocytosis. (A) Wild-type, Syt I-null, Syt IX-null and Syt I/IX-null cells expressing the human Tf receptor were incubated with Texas-Red-conjugated Tf in basal or depolarization medium for 20 minutes. Representative images for the uptake of Tf into the four indicated cells types resting in basal or depolarization medium are shown. Bar, 10 µm. (B) Z-stacks of confocal images were added up to give Tf integrated intensity per cell. The mean Tf integrated intensity ± s.d. is plotted for wild-type, Syt I/IX-null, Syt I-null and Syt IX-null cells (n=15 cells for each cell type) in basal or depolarization medium.