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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 to the plasma membrane is accelerated through unstacked Golgi membranes.(A) Metaphase NRK cells were microinjected with affinity-purified antibodies against GRASP65 together with a plasmid encoding the plasma membrane protein CD8. After cell division, CD8 was expressed in the daughter cells and transported to the plasma membrane. 45 min before fixation, cycloheximide was added to inhibit protein translation. Cells were double labeled with a monoclonal antibody against CD8 and the microinjected polyclonal antibodies against GRASP65. Note that CD8 was transported to the cell surface in cells where Golgi stack formation was inhibited by the injected GRASP65 antibodies. Bar, 15 µm. (B) Metaphase NRK cells were injected with G2A GRASP65 protein together with a CD8 plasmid and Texas-Red dextran as a marker. At the end of mitosis, a fluorescently labeled antibody against CD8 was added to the medium. The fluorescence increase was followed by time-lapse microscopy in 2 min intervals over 70 min (Movie S2). Bar, 15 µm. (C) Quantitation. The mean fluorescence intensity of the cells in each frame was quantified and plotted (red curve: G2A GRASP65 injection, blue curve: control injection). The data shown were obtained from one pair of divided cells for each condition. Note the increased rate of transport in cells injected with G2A GRASP65. (D) The transport rate of CD8 to the plasma membrane increased two-fold in cells with unstacked Golgi cisternae. Quantitation from three independent experiments (mean±SD; p<0.05). Appearance of CD8 per minute at the cell surface was stimulated by 90% in G2A GRASP65 injected cells (n = 34) compared to control cells (n = 44).
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pone-0001647-g003: CD8 transport to the plasma membrane is accelerated through unstacked Golgi membranes.(A) Metaphase NRK cells were microinjected with affinity-purified antibodies against GRASP65 together with a plasmid encoding the plasma membrane protein CD8. After cell division, CD8 was expressed in the daughter cells and transported to the plasma membrane. 45 min before fixation, cycloheximide was added to inhibit protein translation. Cells were double labeled with a monoclonal antibody against CD8 and the microinjected polyclonal antibodies against GRASP65. Note that CD8 was transported to the cell surface in cells where Golgi stack formation was inhibited by the injected GRASP65 antibodies. Bar, 15 µm. (B) Metaphase NRK cells were injected with G2A GRASP65 protein together with a CD8 plasmid and Texas-Red dextran as a marker. At the end of mitosis, a fluorescently labeled antibody against CD8 was added to the medium. The fluorescence increase was followed by time-lapse microscopy in 2 min intervals over 70 min (Movie S2). Bar, 15 µm. (C) Quantitation. The mean fluorescence intensity of the cells in each frame was quantified and plotted (red curve: G2A GRASP65 injection, blue curve: control injection). The data shown were obtained from one pair of divided cells for each condition. Note the increased rate of transport in cells injected with G2A GRASP65. (D) The transport rate of CD8 to the plasma membrane increased two-fold in cells with unstacked Golgi cisternae. Quantitation from three independent experiments (mean±SD; p<0.05). Appearance of CD8 per minute at the cell surface was stimulated by 90% in G2A GRASP65 injected cells (n = 34) compared to control cells (n = 44).

Mentions: To test whether the unstacked Golgi was capable of transporting proteins, prometaphase cells were injected with GRASP65 antibodies together with a plasmid encoding CD8. Two hours later, immunofluorescence analysis showed that CD8 was transported to the cell surface in both daughter cells, as shown by a typical staining pattern for plasma membrane proteins (Fig. 3 A). This showed that unstacked Golgi membranes were still transporting cargo proteins. We then measured the rate of transport in post-mitotic cells with disrupted Golgi stacks. Unsynchronized NRK cells in metaphase were injected with G2A GRASP65 protein to inhibit stacking, together with the CD8 plasmid and Texas Red dextran as a marker. After mitosis, CD8 was synthesized and its arrival on the plasma membrane was monitored by time-lapse microscopy (Fig. 3 B, Movie S2). The mean fluorescence of a representative cell pair from each condition showed that the CD8 signal increased linearly within the first 30 min and that CD8 accumulated faster on the plasma membrane in cells with an unstacked Golgi (Fig. 3 C). The results from three independent experiments showed that transport is nearly doubled in cells injected with G2A GRASP65 compared to control cells (Fig. 3 D). The signal was increased to 5.7±0.9 fluorescence units/min compared to 3.0±0.5, which corresponds to an increase of 90% in CD8 transport. This indicates that unstacking of the Golgi increases the transport rate to the cell surface, perhaps by accelerating vesicle budding (Fig. 1 E–G).


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 to the plasma membrane is accelerated through unstacked Golgi membranes.(A) Metaphase NRK cells were microinjected with affinity-purified antibodies against GRASP65 together with a plasmid encoding the plasma membrane protein CD8. After cell division, CD8 was expressed in the daughter cells and transported to the plasma membrane. 45 min before fixation, cycloheximide was added to inhibit protein translation. Cells were double labeled with a monoclonal antibody against CD8 and the microinjected polyclonal antibodies against GRASP65. Note that CD8 was transported to the cell surface in cells where Golgi stack formation was inhibited by the injected GRASP65 antibodies. Bar, 15 µm. (B) Metaphase NRK cells were injected with G2A GRASP65 protein together with a CD8 plasmid and Texas-Red dextran as a marker. At the end of mitosis, a fluorescently labeled antibody against CD8 was added to the medium. The fluorescence increase was followed by time-lapse microscopy in 2 min intervals over 70 min (Movie S2). Bar, 15 µm. (C) Quantitation. The mean fluorescence intensity of the cells in each frame was quantified and plotted (red curve: G2A GRASP65 injection, blue curve: control injection). The data shown were obtained from one pair of divided cells for each condition. Note the increased rate of transport in cells injected with G2A GRASP65. (D) The transport rate of CD8 to the plasma membrane increased two-fold in cells with unstacked Golgi cisternae. Quantitation from three independent experiments (mean±SD; p<0.05). Appearance of CD8 per minute at the cell surface was stimulated by 90% in G2A GRASP65 injected cells (n = 34) compared to control cells (n = 44).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2249924&req=5

pone-0001647-g003: CD8 transport to the plasma membrane is accelerated through unstacked Golgi membranes.(A) Metaphase NRK cells were microinjected with affinity-purified antibodies against GRASP65 together with a plasmid encoding the plasma membrane protein CD8. After cell division, CD8 was expressed in the daughter cells and transported to the plasma membrane. 45 min before fixation, cycloheximide was added to inhibit protein translation. Cells were double labeled with a monoclonal antibody against CD8 and the microinjected polyclonal antibodies against GRASP65. Note that CD8 was transported to the cell surface in cells where Golgi stack formation was inhibited by the injected GRASP65 antibodies. Bar, 15 µm. (B) Metaphase NRK cells were injected with G2A GRASP65 protein together with a CD8 plasmid and Texas-Red dextran as a marker. At the end of mitosis, a fluorescently labeled antibody against CD8 was added to the medium. The fluorescence increase was followed by time-lapse microscopy in 2 min intervals over 70 min (Movie S2). Bar, 15 µm. (C) Quantitation. The mean fluorescence intensity of the cells in each frame was quantified and plotted (red curve: G2A GRASP65 injection, blue curve: control injection). The data shown were obtained from one pair of divided cells for each condition. Note the increased rate of transport in cells injected with G2A GRASP65. (D) The transport rate of CD8 to the plasma membrane increased two-fold in cells with unstacked Golgi cisternae. Quantitation from three independent experiments (mean±SD; p<0.05). Appearance of CD8 per minute at the cell surface was stimulated by 90% in G2A GRASP65 injected cells (n = 34) compared to control cells (n = 44).
Mentions: To test whether the unstacked Golgi was capable of transporting proteins, prometaphase cells were injected with GRASP65 antibodies together with a plasmid encoding CD8. Two hours later, immunofluorescence analysis showed that CD8 was transported to the cell surface in both daughter cells, as shown by a typical staining pattern for plasma membrane proteins (Fig. 3 A). This showed that unstacked Golgi membranes were still transporting cargo proteins. We then measured the rate of transport in post-mitotic cells with disrupted Golgi stacks. Unsynchronized NRK cells in metaphase were injected with G2A GRASP65 protein to inhibit stacking, together with the CD8 plasmid and Texas Red dextran as a marker. After mitosis, CD8 was synthesized and its arrival on the plasma membrane was monitored by time-lapse microscopy (Fig. 3 B, Movie S2). The mean fluorescence of a representative cell pair from each condition showed that the CD8 signal increased linearly within the first 30 min and that CD8 accumulated faster on the plasma membrane in cells with an unstacked Golgi (Fig. 3 C). The results from three independent experiments showed that transport is nearly doubled in cells injected with G2A GRASP65 compared to control cells (Fig. 3 D). The signal was increased to 5.7±0.9 fluorescence units/min compared to 3.0±0.5, which corresponds to an increase of 90% in CD8 transport. This indicates that unstacking of the Golgi increases the transport rate to the cell surface, perhaps by accelerating vesicle budding (Fig. 1 E–G).

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