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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 Membrane Protein during Lysosomal FusionLysosomal membranes in MEFs were labeled by transfecting cells with a vector encoding a CD63–GFP fusion protein, and expression was allowed for 48 h. For simultaneous labeling of lysosomal membrane and lumen, the CD63–GFP transfected cells were labeled with 70 kDa TRITC–dextran as described in Figure 1.(A) Following ionophore-induced calcium increase in WT MEFs, when the TRITC–dextran was released completely (left), CD63–GFP (right) was delivered to the plasma membrane, but it remained in multiple puncta near the site of fusion rather than diffuse away. The panels are pseudocolor surface plots, with the x and y axis representing the coordinates and the z axis representing the fluorescence intensity of individual pixels.(B) In the event of partial release of TRITC–dextran (top row), the CD63–GFP (bottom row) did not appear to be delivered to the plasma membrane. The lower panel shows the plot of fluorescent intensity of lumenal and membrane label (within the dotted circle) of the lysosome shown in (B).(C and D) Analysis of CD63–GFP-labeled lysosomes in WT MEFs (C) and in Syt VII KO MEFs (D) indicates that while CD63–GFP is retained in puncta in the WT MEFs, it diffuses freely in the plasma membrane in the SytVII KO MEFs. The lower panel shows the total and peak intensity plots of CD63–GFP-labeled lysosome in (D).
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pbio-0020233-g002: Fate of Membrane Protein during Lysosomal FusionLysosomal membranes in MEFs were labeled by transfecting cells with a vector encoding a CD63–GFP fusion protein, and expression was allowed for 48 h. For simultaneous labeling of lysosomal membrane and lumen, the CD63–GFP transfected cells were labeled with 70 kDa TRITC–dextran as described in Figure 1.(A) Following ionophore-induced calcium increase in WT MEFs, when the TRITC–dextran was released completely (left), CD63–GFP (right) was delivered to the plasma membrane, but it remained in multiple puncta near the site of fusion rather than diffuse away. The panels are pseudocolor surface plots, with the x and y axis representing the coordinates and the z axis representing the fluorescence intensity of individual pixels.(B) In the event of partial release of TRITC–dextran (top row), the CD63–GFP (bottom row) did not appear to be delivered to the plasma membrane. The lower panel shows the plot of fluorescent intensity of lumenal and membrane label (within the dotted circle) of the lysosome shown in (B).(C and D) Analysis of CD63–GFP-labeled lysosomes in WT MEFs (C) and in Syt VII KO MEFs (D) indicates that while CD63–GFP is retained in puncta in the WT MEFs, it diffuses freely in the plasma membrane in the SytVII KO MEFs. The lower panel shows the total and peak intensity plots of CD63–GFP-labeled lysosome in (D).

Mentions: In order to determine the fate of the membrane proteins during Ca2+-triggered lysosomal exocytosis, we simultaneously imaged the lysosomal lumenal (TRITC–dextran) and membrane (CD63–GFP) cargoes in WT MEFs. We observed that the membrane proteins were delivered to the plasma membrane during complete release of lumenal content (Figure 2A), but not when the lumenal contents were released partially (Figure 2B). Even when the lysosomal membrane proteins were delivered to the plasma membrane, their diffusion into plasma membrane was restricted (Figure 2A and 2C; Table 2). This is unlike the fate of membrane proteins during exocytosis of biosynthetic vesicles (Schmoranzer et al. 2000; Kreitzer et al. 2003; Schmoranzer and Simon 2003) or recycling endosomes (Lampson et al. 2001), where following its delivery to plasma membrane, the vesicular membrane protein diffuses away completely from the site of fusion. Thus, exocytosis of lysosomes in the WT MEF is different from other exocytic events in two manners. First, the majority of exocytic lysosomes only partially release their lumenal cargo with no release of membrane proteins. Second, even when the lumenal cargo is completely released, the membrane proteins delivered to the plasma membrane do not diffuse freely, but are retained into punctae at the site of fusion.


Synaptotagmin VII restricts fusion pore expansion during lysosomal exocytosis.

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

Fate of Membrane Protein during Lysosomal FusionLysosomal membranes in MEFs were labeled by transfecting cells with a vector encoding a CD63–GFP fusion protein, and expression was allowed for 48 h. For simultaneous labeling of lysosomal membrane and lumen, the CD63–GFP transfected cells were labeled with 70 kDa TRITC–dextran as described in Figure 1.(A) Following ionophore-induced calcium increase in WT MEFs, when the TRITC–dextran was released completely (left), CD63–GFP (right) was delivered to the plasma membrane, but it remained in multiple puncta near the site of fusion rather than diffuse away. The panels are pseudocolor surface plots, with the x and y axis representing the coordinates and the z axis representing the fluorescence intensity of individual pixels.(B) In the event of partial release of TRITC–dextran (top row), the CD63–GFP (bottom row) did not appear to be delivered to the plasma membrane. The lower panel shows the plot of fluorescent intensity of lumenal and membrane label (within the dotted circle) of the lysosome shown in (B).(C and D) Analysis of CD63–GFP-labeled lysosomes in WT MEFs (C) and in Syt VII KO MEFs (D) indicates that while CD63–GFP is retained in puncta in the WT MEFs, it diffuses freely in the plasma membrane in the SytVII KO MEFs. The lower panel shows the total and peak intensity plots of CD63–GFP-labeled lysosome in (D).
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Related In: Results  -  Collection

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

pbio-0020233-g002: Fate of Membrane Protein during Lysosomal FusionLysosomal membranes in MEFs were labeled by transfecting cells with a vector encoding a CD63–GFP fusion protein, and expression was allowed for 48 h. For simultaneous labeling of lysosomal membrane and lumen, the CD63–GFP transfected cells were labeled with 70 kDa TRITC–dextran as described in Figure 1.(A) Following ionophore-induced calcium increase in WT MEFs, when the TRITC–dextran was released completely (left), CD63–GFP (right) was delivered to the plasma membrane, but it remained in multiple puncta near the site of fusion rather than diffuse away. The panels are pseudocolor surface plots, with the x and y axis representing the coordinates and the z axis representing the fluorescence intensity of individual pixels.(B) In the event of partial release of TRITC–dextran (top row), the CD63–GFP (bottom row) did not appear to be delivered to the plasma membrane. The lower panel shows the plot of fluorescent intensity of lumenal and membrane label (within the dotted circle) of the lysosome shown in (B).(C and D) Analysis of CD63–GFP-labeled lysosomes in WT MEFs (C) and in Syt VII KO MEFs (D) indicates that while CD63–GFP is retained in puncta in the WT MEFs, it diffuses freely in the plasma membrane in the SytVII KO MEFs. The lower panel shows the total and peak intensity plots of CD63–GFP-labeled lysosome in (D).
Mentions: In order to determine the fate of the membrane proteins during Ca2+-triggered lysosomal exocytosis, we simultaneously imaged the lysosomal lumenal (TRITC–dextran) and membrane (CD63–GFP) cargoes in WT MEFs. We observed that the membrane proteins were delivered to the plasma membrane during complete release of lumenal content (Figure 2A), but not when the lumenal contents were released partially (Figure 2B). Even when the lysosomal membrane proteins were delivered to the plasma membrane, their diffusion into plasma membrane was restricted (Figure 2A and 2C; Table 2). This is unlike the fate of membrane proteins during exocytosis of biosynthetic vesicles (Schmoranzer et al. 2000; Kreitzer et al. 2003; Schmoranzer and Simon 2003) or recycling endosomes (Lampson et al. 2001), where following its delivery to plasma membrane, the vesicular membrane protein diffuses away completely from the site of fusion. Thus, exocytosis of lysosomes in the WT MEF is different from other exocytic events in two manners. First, the majority of exocytic lysosomes only partially release their lumenal cargo with no release of membrane proteins. Second, even when the lumenal cargo is completely released, the membrane proteins delivered to the plasma membrane do not diffuse freely, but are retained into punctae at the site of fusion.

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