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Golgi cisternal unstacking stimulates COPI vesicle budding and protein transport.

Wang Y, Wei JH, Bisel B, Tang D, Seemann J - PLoS ONE (2008)

Bottom Line: We have used the Golgi stacking protein GRASP65 as a tool to modify the stacking state of Golgi cisternae.Cells with an unstacked Golgi showed a higher transport rate compared to cells with stacked Golgi membranes.The results further suggest that at the onset of mitosis, unstacking of cisternae allows extensive and rapid vesiculation of the Golgi in preparation for its subsequent partitioning.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA.

ABSTRACT
The Golgi apparatus in mammalian cells is composed of flattened cisternae that are densely packed to form stacks. We have used the Golgi stacking protein GRASP65 as a tool to modify the stacking state of Golgi cisternae. We established an assay to measure protein transport to the cell surface in post-mitotic cells in which the Golgi was unstacked. Cells with an unstacked Golgi showed a higher transport rate compared to cells with stacked Golgi membranes. Vesicle budding from unstacked cisternae in vitro was significantly increased compared to stacked membranes. These results suggest that Golgi cisternal stacking can directly regulate vesicle formation and thus the rate of protein transport through the Golgi. The results further suggest that at the onset of mitosis, unstacking of cisternae allows extensive and rapid vesiculation of the Golgi in preparation for its subsequent partitioning.

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CD8 transport assay.(A) A plasmid encoding the plasma membrane protein CD8 was microinjected together with Texas-Red dextran (top left panel, red) into NRK cells. After 45 min, a fluorescently labeled antibody against the luminal domain of CD8 was added to the medium. The recruitment of the antibody onto the cell surface was analyzed by time-lapse microscopy in 2 min intervals over 70 min (Supplemental Movie S1). The fluorescence intensity on the cell surface increased over time, representing the arrival of CD8 from the Golgi to the plasma membrane. Bar, 15 µm. (B) Quantitation. To validate the transport assay, a peptide (N73) that inhibits intra-Golgi transport, or a control peptide (wt), were co-injected into NRK cells along with the CD8 plasmid and Texas-Red dextran and assayed for CD8 transport as in (A). The mean fluorescence intensity of the cells in each frame was quantified and plotted (red curve: control, n = 10; black curve: N73 peptide, n = 6); it decreased from 9.7 per min for control-injected cells to 2.4 per min for N73-peptide injected cells. CD8 transport was therefore inhibited by 75%.
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pone-0001647-g002: CD8 transport assay.(A) A plasmid encoding the plasma membrane protein CD8 was microinjected together with Texas-Red dextran (top left panel, red) into NRK cells. After 45 min, a fluorescently labeled antibody against the luminal domain of CD8 was added to the medium. The recruitment of the antibody onto the cell surface was analyzed by time-lapse microscopy in 2 min intervals over 70 min (Supplemental Movie S1). The fluorescence intensity on the cell surface increased over time, representing the arrival of CD8 from the Golgi to the plasma membrane. Bar, 15 µm. (B) Quantitation. To validate the transport assay, a peptide (N73) that inhibits intra-Golgi transport, or a control peptide (wt), were co-injected into NRK cells along with the CD8 plasmid and Texas-Red dextran and assayed for CD8 transport as in (A). The mean fluorescence intensity of the cells in each frame was quantified and plotted (red curve: control, n = 10; black curve: N73 peptide, n = 6); it decreased from 9.7 per min for control-injected cells to 2.4 per min for N73-peptide injected cells. CD8 transport was therefore inhibited by 75%.

Mentions: This assay was validated by microinjection of a GM130 N-terminal peptide (N73), a known inhibitor of Golgi transport that disrupts the GM130-p115 tethering complex [21]. This peptide leads to an accumulation of COPI vesicles as well as a 65% reduction of protein transport to the cell surface [22]. Here we injected the N73 peptide or a control peptide together with the CD8 plasmid (Fig. 2 A). The N73 peptide displaced p115 from the Golgi, which is consistent with previous results [21], [22], but the control peptide had no effect (not shown). Injected cells were identified after 45 min by the injected fluorescent dextran, and recruitment of the fluorescent CD8 antibody to the plasma membrane was monitored at 2 min intervals over 70 min (Fig. 2 A, Movie S1). The increase in fluorescence intensity (Fig. 2 B) per minute was reduced from 9.7 (control) to 2.4 (N73 peptide), which corresponds to a 75% inhibition of CD8 transport. This is similar to the previously reported 65% inhibition in fixed cells where the viral plasma membrane protein VSV-G was used as cargo [22].


Golgi cisternal unstacking stimulates COPI vesicle budding and protein transport.

Wang Y, Wei JH, Bisel B, Tang D, Seemann J - PLoS ONE (2008)

CD8 transport assay.(A) A plasmid encoding the plasma membrane protein CD8 was microinjected together with Texas-Red dextran (top left panel, red) into NRK cells. After 45 min, a fluorescently labeled antibody against the luminal domain of CD8 was added to the medium. The recruitment of the antibody onto the cell surface was analyzed by time-lapse microscopy in 2 min intervals over 70 min (Supplemental Movie S1). The fluorescence intensity on the cell surface increased over time, representing the arrival of CD8 from the Golgi to the plasma membrane. Bar, 15 µm. (B) Quantitation. To validate the transport assay, a peptide (N73) that inhibits intra-Golgi transport, or a control peptide (wt), were co-injected into NRK cells along with the CD8 plasmid and Texas-Red dextran and assayed for CD8 transport as in (A). The mean fluorescence intensity of the cells in each frame was quantified and plotted (red curve: control, n = 10; black curve: N73 peptide, n = 6); it decreased from 9.7 per min for control-injected cells to 2.4 per min for N73-peptide injected cells. CD8 transport was therefore inhibited by 75%.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2249924&req=5

pone-0001647-g002: CD8 transport assay.(A) A plasmid encoding the plasma membrane protein CD8 was microinjected together with Texas-Red dextran (top left panel, red) into NRK cells. After 45 min, a fluorescently labeled antibody against the luminal domain of CD8 was added to the medium. The recruitment of the antibody onto the cell surface was analyzed by time-lapse microscopy in 2 min intervals over 70 min (Supplemental Movie S1). The fluorescence intensity on the cell surface increased over time, representing the arrival of CD8 from the Golgi to the plasma membrane. Bar, 15 µm. (B) Quantitation. To validate the transport assay, a peptide (N73) that inhibits intra-Golgi transport, or a control peptide (wt), were co-injected into NRK cells along with the CD8 plasmid and Texas-Red dextran and assayed for CD8 transport as in (A). The mean fluorescence intensity of the cells in each frame was quantified and plotted (red curve: control, n = 10; black curve: N73 peptide, n = 6); it decreased from 9.7 per min for control-injected cells to 2.4 per min for N73-peptide injected cells. CD8 transport was therefore inhibited by 75%.
Mentions: This assay was validated by microinjection of a GM130 N-terminal peptide (N73), a known inhibitor of Golgi transport that disrupts the GM130-p115 tethering complex [21]. This peptide leads to an accumulation of COPI vesicles as well as a 65% reduction of protein transport to the cell surface [22]. Here we injected the N73 peptide or a control peptide together with the CD8 plasmid (Fig. 2 A). The N73 peptide displaced p115 from the Golgi, which is consistent with previous results [21], [22], but the control peptide had no effect (not shown). Injected cells were identified after 45 min by the injected fluorescent dextran, and recruitment of the fluorescent CD8 antibody to the plasma membrane was monitored at 2 min intervals over 70 min (Fig. 2 A, Movie S1). The increase in fluorescence intensity (Fig. 2 B) per minute was reduced from 9.7 (control) to 2.4 (N73 peptide), which corresponds to a 75% inhibition of CD8 transport. This is similar to the previously reported 65% inhibition in fixed cells where the viral plasma membrane protein VSV-G was used as cargo [22].

Bottom Line: We have used the Golgi stacking protein GRASP65 as a tool to modify the stacking state of Golgi cisternae.Cells with an unstacked Golgi showed a higher transport rate compared to cells with stacked Golgi membranes.The results further suggest that at the onset of mitosis, unstacking of cisternae allows extensive and rapid vesiculation of the Golgi in preparation for its subsequent partitioning.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA.

ABSTRACT
The Golgi apparatus in mammalian cells is composed of flattened cisternae that are densely packed to form stacks. We have used the Golgi stacking protein GRASP65 as a tool to modify the stacking state of Golgi cisternae. We established an assay to measure protein transport to the cell surface in post-mitotic cells in which the Golgi was unstacked. Cells with an unstacked Golgi showed a higher transport rate compared to cells with stacked Golgi membranes. Vesicle budding from unstacked cisternae in vitro was significantly increased compared to stacked membranes. These results suggest that Golgi cisternal stacking can directly regulate vesicle formation and thus the rate of protein transport through the Golgi. The results further suggest that at the onset of mitosis, unstacking of cisternae allows extensive and rapid vesiculation of the Golgi in preparation for its subsequent partitioning.

Show MeSH
Related in: MedlinePlus