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First published online 14 November 2002
doi: 10.1242/jcs.00186

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Synaptotagmin III is a critical factor for the formation of the perinuclear endocytic recycling compartment and determination of secretory granules size

Elena Grimberg1,2, Ze Peng1,*, Ilan Hammel2 and Ronit Sagi-Eisenberg1,{dagger}

1 Departments of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel
2 Department of Pathology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel
* Present address: Laboratory of Molecular Immunology, NHLBI, National Institutes of Health, Bethesda, MD, USA



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  		Fig. 1. Expression of Syt III protein in RBL cells and brain. (A) Crude brain homogenate (lane 1, 10 µg protein) and whole cell lysates (1x106 cell equivalents) derived from control (lane 2) or Syt III-overexpressing (RBL-Syt III+, lane 3) or Syt III-suppressed (RBL-Syt III-, lane 4) RBL cells were resolved by SDS-PAGE and immunoblotted using a rabbit polyclonal serum directed against the cytoplasmic domain of Syt III (1: 1000 dilution). (B) A whole cell lysate (1x106 cell equivalents) derived from RBL cells transiently transfected with T7-tagged Syt III cDNA were resolved by SDS-PAGE and immunoblotted using anti T7 antibodies. (C) Total cell extracts (500 µg) derived from T7-tagged Syt III expressing cells were subjected to immunoprecipitation (IP) using anti T7 antibodies. The immune complexes were resolved by SDS-PAGE and immunoblotted (IB) with either anti T7 antibodies or anti Syt III antibodies, as indicated. Data represent one of three separate experiments.

 


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  		Fig. 2. Subcellular fractionation of RBL cell lysate. Fractions from a continuous sucrose gradient were collected from the top and assayed for (A) Syt III (anti Syt III serum 1:1000 dilution), Syt II (monoclonal antibodies 1:1000 dilution) and G{alpha}i2 (affinity-purified AS10 antibodies, 1 µg/ml) immunoreactivities. (B) ß-hexosaminidase activity (presented as OD read at 405 nm) ({blacksquare}) and histamine content ({square}). (C) Protein (•) and sucrose density ({circ}). (D) The light density fractions of the gradient were probed for Syt III (polyclonal anti Syt III serum 1: 1000 dilution), Annexin II (monoclonal 1: 5000 dilution), EEA1 (polyclonal anti EEA1 serum 1: 1000 dilution) and syntaxin 7 (polyclonal anti syntaxin 7 antibodies 1: 500 dilution) immunoreactivities. Data represent one of three separate experiments.

 


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  		Fig. 3. Localization of Syt III in RBL cells. RBL cells were processed for immunofluorescent staining and visualized by confocal microscopy, as described under Materials and Methods. Cells were incubated with antibodies directed against Syt III (A,D,G, 1:100 dilution), T7 (B, 1:200 dilution), serotonin (E, 1:25 dilution) followed by rhodamine-conjugated donkey anti-rabbit or FITC-conjugated donkey anti-mouse IgG. To monitor internalized Tfn (H), cells were serum starved for 1 hour before incubation for 10 minutes with FITC-conjugated human Tfn (50 µg/ml). Bars represent 10 µm. Data represent one of three separate expreiments.

 


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  		Fig. 4. Suppression of Syt III expression. Whole cell lysates derived from control (empty-vector-transfected) RBL or RBL-Syt III- cells were resolved by SDS-PAGE, immunoblotted and probed (A) with antibodies directed against Syt III (1:1000 dilution), Syt II (1: 1000 dilution) or anti G{alpha}i2 (affinity-purified AS10 antibodies, 1 µg/ml) as indicated. Sucrose gradient fractions derived from control RBL and RBL-Syt III- cells were resolved by SDS-PAGE, immunoblotted and probed with anti Syt III antibodies (B) or anti Syt II antibodies (C). The first 14 fractions are presented, as indicated. Data represent one of three separate experiments (A) or one of two separate experiments (B,C).

 


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  		Fig. 5. Internalization of FITC-Tfn in control and RBL-Syt III- cells. (A) Control RBL (a,b) or RBL-Syt III- cells (a',b') were grown on glass coverslips, serum starved for 1 hour and incubated with FITC-Tfn (50 µg/ml) for 1 hour at 4°C. Unbound FITC-Tfn was removed by washing with ice-cold PBS and the cells were warmed up to 37°C to allow endocytosis. At time 0 (a and a') and 1.5 minutes (b and b') the cells were placed on ice and subsequently visualized by confocal microscopy as described under Materials and Methods. Bars represent 10 µm. Data represent one of three separate experiments. (B) Biotin-Tfn was allowed to internalize as described under Materials and Methods. At the end of the indicated time periods, cell-surface Tfn was removed and the amount of intracellular biotin-Tfn was determined by subjecting cell lysates to SDS-PAGE and immunoblotting. Blots were probed with HRP-conjugated streptavidin, visualized by ECL and the intensities of the bands corresponding to biotin-Tfn were quantified by densitometry. The amount of cell-associated biotin-Tfn is presented as arbitrary units. Data represent one of three separate experiments.

 


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  		Fig. 6. Recycling of FITC-Tfn in control and RBL-Syt III- cells. Control RBL (a-c) or RBL-Syt III- cells (a'-c') were grown on glass coverslips, serum starved for 1 hour and incubated with FITC-Tfn (50 µg/ml) for 1 hour at 4°C. Unbound FITC-Tfn was removed by washing with ice-cold PBS, and the cells were warmed up to 37°C to allow endocytosis. At the end of 30 minutes, cells were placed on ice and processed for immunofluorescent staining using anti Rab 11 antibodies (polyclonal, 1: 50 dilution). Cells were visualized by confocal microscopy as described under Materials and Methods. Bars, 10 µm. Data represent one of five separate experiments. (B) Tfn recycling was monitored as described in Materials and Methods. The amounts of the extra and intracellular biotin-Tfn were determined by subjecting supernatants and cell lysates to SDS-PAGE and immunoblotting. Blots were probed with HRP-conjugated streptavidin, visualized by ECL, and the intensities of the bands corresponding to biotin-Tfn were quantified by densitometry. The amount of cell-associated biotin-Tfn is presented as a percentage of total biotin-Tfn. The results presented are the average±standard deviations of five independent experiments.

 


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  		Fig. 7. SG in control and RBL-Syt III- cells. (A) Electron micrographs of a control (empty vector transfected) RBL cell and a RBL-Syt III- cell. The arrow points to a `giant' granule depicted in the RBL-Syt III- cell. (B) Distribution of measured granule cross-sectional profile areas of RBL-Syt III- (lower panel) and control cells (upper panel). Both histograms are statistically different based on the Kolmogorov-Smirnov test (P<0.01). A histogram of moving-bin analysis demonstrating multimodal frequency of RBL granule equivalent volumes derived from both cell lines is depicted at the upper inset. The value of this mode is calculated from the inter-modal spacing and is indicated by arrowheads (unit granule volume=0.083 µm3). The second inset (lower panel) represents a scattergram analysis of mean granule profile area and its standard deviation for each cell profile.

 


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  		Fig. 8. A model illustrating Syt III function in endosomal trafficking. According to this model, in control cells (A) Syt III regulates the formation of the endocytic recycling compartment (ERC) and the delivery to this compartment from the early endosome (EE) and the immature granule (ISG) during the process of granule maturation. In the RBL-Syt III- cells (B), suppression of Syt III inhibits the formation of and delivery to ERC from both the EE and the ISG.

 

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