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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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Temporal Analysis of Lysosomal Exocytosis and Fusion Pore Opening Using 70 kDa FITC–Dextran as Lumenal Marker(A) Histogram of the release time (time taken for the vesicular fluorescence to drop from peak to postfusion resting value). Release time was less than 0.75 s for more than two-thirds of lysosomes in Syt VII KO MEF (n = 62 lysosomes), while most lysosomes (81%) in WT MEFs (n = 56 lysosomes) released their lumenal content for more than 0.75 s.(B) Analysis of release time of lysosomes using ionophore, bombesin, and thrombin to trigger lysosomal exocytosis. Irrespective of the means of calcium increase, lysosomes in Syt VII KO MEFs released their lumenal content significantly faster (p = 0.002, 0.01, and 0.03, respectively).(C) Histogram of the number of lysosomes exocytosing as a function of the time following calcium ionophore addition. Fluorescent dextran was used as a lumenal marker; the time axis indicates seconds elapsed following the addition of ionophore. Exocytosis initiated earlier in the Syt VII KO MEFs (white bars; n = 8 cells) compared to WT MEFs (gray bars; n = 6 cells).(D) No change was observed in the total number of lysosomes that exocytosed at the basal surface of WT or Syt VII KO MEFs when calcium was raised using ionophore or thrombin; however, compared to WT MEFs, bombesin triggered exocytosis of half as many lysosomes in the Syt VII KO MEFs (asterix represents p value < 0.02). The error bars represent SEM.
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pbio-0020233-g005: Temporal Analysis of Lysosomal Exocytosis and Fusion Pore Opening Using 70 kDa FITC–Dextran as Lumenal Marker(A) Histogram of the release time (time taken for the vesicular fluorescence to drop from peak to postfusion resting value). Release time was less than 0.75 s for more than two-thirds of lysosomes in Syt VII KO MEF (n = 62 lysosomes), while most lysosomes (81%) in WT MEFs (n = 56 lysosomes) released their lumenal content for more than 0.75 s.(B) Analysis of release time of lysosomes using ionophore, bombesin, and thrombin to trigger lysosomal exocytosis. Irrespective of the means of calcium increase, lysosomes in Syt VII KO MEFs released their lumenal content significantly faster (p = 0.002, 0.01, and 0.03, respectively).(C) Histogram of the number of lysosomes exocytosing as a function of the time following calcium ionophore addition. Fluorescent dextran was used as a lumenal marker; the time axis indicates seconds elapsed following the addition of ionophore. Exocytosis initiated earlier in the Syt VII KO MEFs (white bars; n = 8 cells) compared to WT MEFs (gray bars; n = 6 cells).(D) No change was observed in the total number of lysosomes that exocytosed at the basal surface of WT or Syt VII KO MEFs when calcium was raised using ionophore or thrombin; however, compared to WT MEFs, bombesin triggered exocytosis of half as many lysosomes in the Syt VII KO MEFs (asterix represents p value < 0.02). The error bars represent SEM.

Mentions: Since cargo of any size would be released more rapidly through a larger pore, we independently assayed for the size of the fusion pore by measuring the time taken by exocytosing lysosomes to release their lumenal cargo. Increase in FITC–dextran fluorescence was taken as the indicator for the time of opening of the fusion pore (see Figure 1C). We measured the time taken for the lumenal fluorescence of individual exocytic lysosomes (fusing partially or completely) to decrease to the post-fusion resting value in WT and Syt VII KO MEF (see Figure 1C). For most lysosomes (greater than 81%) in WT MEFs, it took longer than 0.75 s for the fluorescence of the lumenal cargo to reach their post-fusion resting value (gray bars in Figure 5A). In contrast, in Syt VII KO MEFs for most lysosomes (greater than 75%), this occurred in less than 0.75 s (white bars in Figure 5A). The increased propensity of lysosomes to rapidly release their lumenal content was also observed when Syt VII KO cells were treated with bombesin or thrombin (Figure 5B). This suggests that in the absence of Syt VII, the fusion pore either opens faster, opens to a larger size, or both. While we cannot distinguish among these possibilities, they are all consistent with Syt VII restricting the expansion of the fusion pore.


Synaptotagmin VII restricts fusion pore expansion during lysosomal exocytosis.

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

Temporal Analysis of Lysosomal Exocytosis and Fusion Pore Opening Using 70 kDa FITC–Dextran as Lumenal Marker(A) Histogram of the release time (time taken for the vesicular fluorescence to drop from peak to postfusion resting value). Release time was less than 0.75 s for more than two-thirds of lysosomes in Syt VII KO MEF (n = 62 lysosomes), while most lysosomes (81%) in WT MEFs (n = 56 lysosomes) released their lumenal content for more than 0.75 s.(B) Analysis of release time of lysosomes using ionophore, bombesin, and thrombin to trigger lysosomal exocytosis. Irrespective of the means of calcium increase, lysosomes in Syt VII KO MEFs released their lumenal content significantly faster (p = 0.002, 0.01, and 0.03, respectively).(C) Histogram of the number of lysosomes exocytosing as a function of the time following calcium ionophore addition. Fluorescent dextran was used as a lumenal marker; the time axis indicates seconds elapsed following the addition of ionophore. Exocytosis initiated earlier in the Syt VII KO MEFs (white bars; n = 8 cells) compared to WT MEFs (gray bars; n = 6 cells).(D) No change was observed in the total number of lysosomes that exocytosed at the basal surface of WT or Syt VII KO MEFs when calcium was raised using ionophore or thrombin; however, compared to WT MEFs, bombesin triggered exocytosis of half as many lysosomes in the Syt VII KO MEFs (asterix represents p value < 0.02). The error bars represent SEM.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC439782&req=5

pbio-0020233-g005: Temporal Analysis of Lysosomal Exocytosis and Fusion Pore Opening Using 70 kDa FITC–Dextran as Lumenal Marker(A) Histogram of the release time (time taken for the vesicular fluorescence to drop from peak to postfusion resting value). Release time was less than 0.75 s for more than two-thirds of lysosomes in Syt VII KO MEF (n = 62 lysosomes), while most lysosomes (81%) in WT MEFs (n = 56 lysosomes) released their lumenal content for more than 0.75 s.(B) Analysis of release time of lysosomes using ionophore, bombesin, and thrombin to trigger lysosomal exocytosis. Irrespective of the means of calcium increase, lysosomes in Syt VII KO MEFs released their lumenal content significantly faster (p = 0.002, 0.01, and 0.03, respectively).(C) Histogram of the number of lysosomes exocytosing as a function of the time following calcium ionophore addition. Fluorescent dextran was used as a lumenal marker; the time axis indicates seconds elapsed following the addition of ionophore. Exocytosis initiated earlier in the Syt VII KO MEFs (white bars; n = 8 cells) compared to WT MEFs (gray bars; n = 6 cells).(D) No change was observed in the total number of lysosomes that exocytosed at the basal surface of WT or Syt VII KO MEFs when calcium was raised using ionophore or thrombin; however, compared to WT MEFs, bombesin triggered exocytosis of half as many lysosomes in the Syt VII KO MEFs (asterix represents p value < 0.02). The error bars represent SEM.
Mentions: Since cargo of any size would be released more rapidly through a larger pore, we independently assayed for the size of the fusion pore by measuring the time taken by exocytosing lysosomes to release their lumenal cargo. Increase in FITC–dextran fluorescence was taken as the indicator for the time of opening of the fusion pore (see Figure 1C). We measured the time taken for the lumenal fluorescence of individual exocytic lysosomes (fusing partially or completely) to decrease to the post-fusion resting value in WT and Syt VII KO MEF (see Figure 1C). For most lysosomes (greater than 81%) in WT MEFs, it took longer than 0.75 s for the fluorescence of the lumenal cargo to reach their post-fusion resting value (gray bars in Figure 5A). In contrast, in Syt VII KO MEFs for most lysosomes (greater than 75%), this occurred in less than 0.75 s (white bars in Figure 5A). The increased propensity of lysosomes to rapidly release their lumenal content was also observed when Syt VII KO cells were treated with bombesin or thrombin (Figure 5B). This suggests that in the absence of Syt VII, the fusion pore either opens faster, opens to a larger size, or both. While we cannot distinguish among these possibilities, they are all consistent with Syt VII restricting the expansion of the fusion pore.

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