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Synaptotagmin VII restricts fusion pore expansion during lysosomal exocytosis.

Jaiswal JK, Chakrabarti S, Andrews NW, Simon SM - PLoS Biol. (2004)

Bottom Line: Synaptotagmin is considered a calcium-dependent trigger for regulated exocytosis.These observations indicate that Syt VII does not function as the calcium-dependent trigger for lysosomal exocytosis.Instead, it restricts the kinetics and extent of calcium-dependent lysosomal fusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biophysics, Rockefeller University, New York, New York, USA.

ABSTRACT
Synaptotagmin is considered a calcium-dependent trigger for regulated exocytosis. We examined the role of synaptotagmin VII (Syt VII) in the calcium-dependent exocytosis of individual lysosomes in wild-type (WT) and Syt VII knockout (KO) mouse embryonic fibroblasts (MEFs) using total internal reflection fluorescence microscopy. In WT MEFs, most lysosomes only partially released their contents, their membrane proteins did not diffuse into the plasma membrane, and inner diameters of their fusion pores were smaller than 30 nm. In Syt VII KO MEFs, not only was lysosomal exocytosis triggered by calcium, but all of these restrictions on fusion were also removed. These observations indicate that Syt VII does not function as the calcium-dependent trigger for lysosomal exocytosis. Instead, it restricts the kinetics and extent of calcium-dependent lysosomal fusion.

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Related in: MedlinePlus

Fate of Lumenal Content during Lysosomal FusionMEFs were incubated for 2 h with 70 kDa FITC–dextran followed by more than 3 h in dextran-free media to chase the dextrans into the lysosomes. These cells were then treated with calcium ionophore (A23187) to trigger exocytosis of lysosomes.(A and B) The middle panels are images of lyosomes undergoing complete (A) and partial (B) exocytosis. Intensity plots for the regions in these images marked by dotted circles are shown in the lower panel. The top panel shows a schematic representation of these different stages.(C) Schematic fluorescence intensity plots for lysosomes undergoing partial (red) or complete (green) fusion. Owing to the exponential decay of the evanescent field (blue; top panels in [A] and [B]) away from the coverslip, a lysosome that is more than 150 nm from the cell membrane (black line in top panels in [A] and [B]) is not fluorescent. As this lysosome moves closer (labeled as “entry into evanescent field”), its fluorescence intensity increases. Since the lumen of lysosome is acidic, it quenches FITC fluorescence. As soon as the fusion pore is formed, the lysosomal lumen is rapidly alkalinized resulting in an increase of FITC–dextran fluorescence (“pore opening”). Following the pore opening, the dextran is released and it diffuses away from the site of the fusion, causing the lumenal fluorescence to decrease (“release”).(D) A histogram of the fraction of lumenal contents released by exocytosing lysosomes. Upon ionophore-triggered fusion, 21% of all lysosomes analyzed in WT MEFs (n = 47; gray bars) and 45% of all in Syt VII KO MEFs (n = 51; white bars) completely released their lumenal content.(E and F) To monitor the nature of lysosomal fusion in individual WT MEFs (E) and Syt VII KO MEFs (F), calcium was increased using ionophore (WT, n = 7 cells; KO, n = 9 cells) as well as the IP3 agonists bombesin (WT, n = 6 cells; KO, n = 9 cells) and thrombin (WT, n = 5 cells; KO, n = 7 cells). Irrespective of the means, increase in calcium led to most lysosomes to fuse partially in WT MEFs (E) and completely in Syt VII KO MEFs (F). The error bars represent SEM.
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pbio-0020233-g001: Fate of Lumenal Content during Lysosomal FusionMEFs were incubated for 2 h with 70 kDa FITC–dextran followed by more than 3 h in dextran-free media to chase the dextrans into the lysosomes. These cells were then treated with calcium ionophore (A23187) to trigger exocytosis of lysosomes.(A and B) The middle panels are images of lyosomes undergoing complete (A) and partial (B) exocytosis. Intensity plots for the regions in these images marked by dotted circles are shown in the lower panel. The top panel shows a schematic representation of these different stages.(C) Schematic fluorescence intensity plots for lysosomes undergoing partial (red) or complete (green) fusion. Owing to the exponential decay of the evanescent field (blue; top panels in [A] and [B]) away from the coverslip, a lysosome that is more than 150 nm from the cell membrane (black line in top panels in [A] and [B]) is not fluorescent. As this lysosome moves closer (labeled as “entry into evanescent field”), its fluorescence intensity increases. Since the lumen of lysosome is acidic, it quenches FITC fluorescence. As soon as the fusion pore is formed, the lysosomal lumen is rapidly alkalinized resulting in an increase of FITC–dextran fluorescence (“pore opening”). Following the pore opening, the dextran is released and it diffuses away from the site of the fusion, causing the lumenal fluorescence to decrease (“release”).(D) A histogram of the fraction of lumenal contents released by exocytosing lysosomes. Upon ionophore-triggered fusion, 21% of all lysosomes analyzed in WT MEFs (n = 47; gray bars) and 45% of all in Syt VII KO MEFs (n = 51; white bars) completely released their lumenal content.(E and F) To monitor the nature of lysosomal fusion in individual WT MEFs (E) and Syt VII KO MEFs (F), calcium was increased using ionophore (WT, n = 7 cells; KO, n = 9 cells) as well as the IP3 agonists bombesin (WT, n = 6 cells; KO, n = 9 cells) and thrombin (WT, n = 5 cells; KO, n = 7 cells). Irrespective of the means, increase in calcium led to most lysosomes to fuse partially in WT MEFs (E) and completely in Syt VII KO MEFs (F). The error bars represent SEM.

Mentions: To monitor the fate of exocytic lysosomes in MEFs, we labeled their lumen using fluorescent dextran (FITC–dextran). Treating MEFs with calcium ionophore A23187 or the IP3 agonist bombesin or thrombin caused lysosomal exocytosis (Figure 1A and 1B). Fusion of a FITC–dextran-loaded lysosome was indicated by a transient increase followed by a decrease in its fluorescence (Figure 1A–1C). The increase in fluorescence was due to a combination of two factors: (a) movement of the lysosome closer to the coverslip, which results in better excitation of its cargo by the evanescent wave; (b) opening of the fusion pore, which results in dissipation of the acidic pH of the lysosomes, resulting in dequenching of the fluorescence of FITC–dextran. The rapid decrease in fluorescence was due to the diffusion of lumenal cargo away from the site of fusion (Figure 1A–1C). In some of the exocytosing lysosomes, the lumenal fluorescence decreased down to baseline, indicating that they completely released their lumenal cargo (Figure 1A). The fluorescence of other lysosomes did not decrease down to baseline at the site of fusion (Figure 1B). Thus, these lysosomes only partially released their contents upon fusion. To resolve whether partial release represented a very slow diffusion of lumenal content or an opening of the fusion pore that was transient, we observed the lysosomes for longer periods. During partial release, the lumenal fluorescence decreased rapidly within the first second (Figure 1B), but remained relatively constant afterwards, decreasing only at the rate of photobleaching (t1/2 for FITC in our setup is 28.5 s). Absence of any subsequent decrease in its fluorescence, even over longer periods, indicated that cessation of release was the result of closure of the fusion pore prior to complete release of the lumenal cargo. Quantitation of the lumenal contents retained in all exocytosed lysosomes analyzed in the WT MEFs revealed that only 21% completely released their lumenal content (Figure 1D, gray bar). The percentage of lysosomes in individual WT MEFs that only partially released their lumenal cargo of 70 kDa dextran in response to A23187-induced increase in cellular Ca2+ was 65.3% (n = 7 cells) (Figure 1E, black bars; Table 1; Video S1). A comparable fraction of lysosomes, respectively, underwent partial release when calcium increase was triggered by the IP3 agonists thrombin (66.3) or bombesin (69.5), (Figure 1E; Table 1).


Synaptotagmin VII restricts fusion pore expansion during lysosomal exocytosis.

Jaiswal JK, Chakrabarti S, Andrews NW, Simon SM - PLoS Biol. (2004)

Fate of Lumenal Content during Lysosomal FusionMEFs were incubated for 2 h with 70 kDa FITC–dextran followed by more than 3 h in dextran-free media to chase the dextrans into the lysosomes. These cells were then treated with calcium ionophore (A23187) to trigger exocytosis of lysosomes.(A and B) The middle panels are images of lyosomes undergoing complete (A) and partial (B) exocytosis. Intensity plots for the regions in these images marked by dotted circles are shown in the lower panel. The top panel shows a schematic representation of these different stages.(C) Schematic fluorescence intensity plots for lysosomes undergoing partial (red) or complete (green) fusion. Owing to the exponential decay of the evanescent field (blue; top panels in [A] and [B]) away from the coverslip, a lysosome that is more than 150 nm from the cell membrane (black line in top panels in [A] and [B]) is not fluorescent. As this lysosome moves closer (labeled as “entry into evanescent field”), its fluorescence intensity increases. Since the lumen of lysosome is acidic, it quenches FITC fluorescence. As soon as the fusion pore is formed, the lysosomal lumen is rapidly alkalinized resulting in an increase of FITC–dextran fluorescence (“pore opening”). Following the pore opening, the dextran is released and it diffuses away from the site of the fusion, causing the lumenal fluorescence to decrease (“release”).(D) A histogram of the fraction of lumenal contents released by exocytosing lysosomes. Upon ionophore-triggered fusion, 21% of all lysosomes analyzed in WT MEFs (n = 47; gray bars) and 45% of all in Syt VII KO MEFs (n = 51; white bars) completely released their lumenal content.(E and F) To monitor the nature of lysosomal fusion in individual WT MEFs (E) and Syt VII KO MEFs (F), calcium was increased using ionophore (WT, n = 7 cells; KO, n = 9 cells) as well as the IP3 agonists bombesin (WT, n = 6 cells; KO, n = 9 cells) and thrombin (WT, n = 5 cells; KO, n = 7 cells). Irrespective of the means, increase in calcium led to most lysosomes to fuse partially in WT MEFs (E) and completely in Syt VII KO MEFs (F). The error bars represent SEM.
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pbio-0020233-g001: Fate of Lumenal Content during Lysosomal FusionMEFs were incubated for 2 h with 70 kDa FITC–dextran followed by more than 3 h in dextran-free media to chase the dextrans into the lysosomes. These cells were then treated with calcium ionophore (A23187) to trigger exocytosis of lysosomes.(A and B) The middle panels are images of lyosomes undergoing complete (A) and partial (B) exocytosis. Intensity plots for the regions in these images marked by dotted circles are shown in the lower panel. The top panel shows a schematic representation of these different stages.(C) Schematic fluorescence intensity plots for lysosomes undergoing partial (red) or complete (green) fusion. Owing to the exponential decay of the evanescent field (blue; top panels in [A] and [B]) away from the coverslip, a lysosome that is more than 150 nm from the cell membrane (black line in top panels in [A] and [B]) is not fluorescent. As this lysosome moves closer (labeled as “entry into evanescent field”), its fluorescence intensity increases. Since the lumen of lysosome is acidic, it quenches FITC fluorescence. As soon as the fusion pore is formed, the lysosomal lumen is rapidly alkalinized resulting in an increase of FITC–dextran fluorescence (“pore opening”). Following the pore opening, the dextran is released and it diffuses away from the site of the fusion, causing the lumenal fluorescence to decrease (“release”).(D) A histogram of the fraction of lumenal contents released by exocytosing lysosomes. Upon ionophore-triggered fusion, 21% of all lysosomes analyzed in WT MEFs (n = 47; gray bars) and 45% of all in Syt VII KO MEFs (n = 51; white bars) completely released their lumenal content.(E and F) To monitor the nature of lysosomal fusion in individual WT MEFs (E) and Syt VII KO MEFs (F), calcium was increased using ionophore (WT, n = 7 cells; KO, n = 9 cells) as well as the IP3 agonists bombesin (WT, n = 6 cells; KO, n = 9 cells) and thrombin (WT, n = 5 cells; KO, n = 7 cells). Irrespective of the means, increase in calcium led to most lysosomes to fuse partially in WT MEFs (E) and completely in Syt VII KO MEFs (F). The error bars represent SEM.
Mentions: To monitor the fate of exocytic lysosomes in MEFs, we labeled their lumen using fluorescent dextran (FITC–dextran). Treating MEFs with calcium ionophore A23187 or the IP3 agonist bombesin or thrombin caused lysosomal exocytosis (Figure 1A and 1B). Fusion of a FITC–dextran-loaded lysosome was indicated by a transient increase followed by a decrease in its fluorescence (Figure 1A–1C). The increase in fluorescence was due to a combination of two factors: (a) movement of the lysosome closer to the coverslip, which results in better excitation of its cargo by the evanescent wave; (b) opening of the fusion pore, which results in dissipation of the acidic pH of the lysosomes, resulting in dequenching of the fluorescence of FITC–dextran. The rapid decrease in fluorescence was due to the diffusion of lumenal cargo away from the site of fusion (Figure 1A–1C). In some of the exocytosing lysosomes, the lumenal fluorescence decreased down to baseline, indicating that they completely released their lumenal cargo (Figure 1A). The fluorescence of other lysosomes did not decrease down to baseline at the site of fusion (Figure 1B). Thus, these lysosomes only partially released their contents upon fusion. To resolve whether partial release represented a very slow diffusion of lumenal content or an opening of the fusion pore that was transient, we observed the lysosomes for longer periods. During partial release, the lumenal fluorescence decreased rapidly within the first second (Figure 1B), but remained relatively constant afterwards, decreasing only at the rate of photobleaching (t1/2 for FITC in our setup is 28.5 s). Absence of any subsequent decrease in its fluorescence, even over longer periods, indicated that cessation of release was the result of closure of the fusion pore prior to complete release of the lumenal cargo. Quantitation of the lumenal contents retained in all exocytosed lysosomes analyzed in the WT MEFs revealed that only 21% completely released their lumenal content (Figure 1D, gray bar). The percentage of lysosomes in individual WT MEFs that only partially released their lumenal cargo of 70 kDa dextran in response to A23187-induced increase in cellular Ca2+ was 65.3% (n = 7 cells) (Figure 1E, black bars; Table 1; Video S1). A comparable fraction of lysosomes, respectively, underwent partial release when calcium increase was triggered by the IP3 agonists thrombin (66.3) or bombesin (69.5), (Figure 1E; Table 1).

Bottom Line: Synaptotagmin is considered a calcium-dependent trigger for regulated exocytosis.These observations indicate that Syt VII does not function as the calcium-dependent trigger for lysosomal exocytosis.Instead, it restricts the kinetics and extent of calcium-dependent lysosomal fusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biophysics, Rockefeller University, New York, New York, USA.

ABSTRACT
Synaptotagmin is considered a calcium-dependent trigger for regulated exocytosis. We examined the role of synaptotagmin VII (Syt VII) in the calcium-dependent exocytosis of individual lysosomes in wild-type (WT) and Syt VII knockout (KO) mouse embryonic fibroblasts (MEFs) using total internal reflection fluorescence microscopy. In WT MEFs, most lysosomes only partially released their contents, their membrane proteins did not diffuse into the plasma membrane, and inner diameters of their fusion pores were smaller than 30 nm. In Syt VII KO MEFs, not only was lysosomal exocytosis triggered by calcium, but all of these restrictions on fusion were also removed. These observations indicate that Syt VII does not function as the calcium-dependent trigger for lysosomal exocytosis. Instead, it restricts the kinetics and extent of calcium-dependent lysosomal fusion.

Show MeSH
Related in: MedlinePlus