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Golgi tubule traffic and the effects of brefeldin A visualized in living cells.

Sciaky N, Presley J, Smith C, Zaal KJ, Cole N, Moreira JE, Terasaki M, Siggia E, Lippincott-Schwartz J - J. Cell Biol. (1997)

Bottom Line: Both lipid and protein emptied from the Golgi at similar rapid rates, leaving no Golgi structure behind, indicating that Golgi membranes do not simply mix but are absorbed into the ER in BFA-treated cells.Analysis of its kinetics suggested a mechanism that is analogous to wetting or adsorptive phenomena in which a tension-driven membrane flow supplements diffusive transfer of Golgi membrane into the ER.Such nonselective, flow-assisted transport of Golgi membranes into ER suggests that mechanisms that regulate retrograde tubule formation and detachment from the Golgi complex are integral to the existence and maintenance of this organelle.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The Golgi complex is a dynamic organelle engaged in both secretory and retrograde membrane traffic. Here, we use green fluorescent protein-Golgi protein chimeras to study Golgi morphology in vivo. In untreated cells, membrane tubules were a ubiquitous, prominent feature of the Golgi complex, serving both to interconnect adjacent Golgi elements and to carry membrane outward along microtubules after detaching from stable Golgi structures. Brefeldin A treatment, which reversibly disassembles the Golgi complex, accentuated tubule formation without tubule detachment. A tubule network extending throughout the cytoplasm was quickly generated and persisted for 5-10 min until rapidly emptying Golgi contents into the ER within 15-30 s. Both lipid and protein emptied from the Golgi at similar rapid rates, leaving no Golgi structure behind, indicating that Golgi membranes do not simply mix but are absorbed into the ER in BFA-treated cells. The directionality of redistribution implied Golgi membranes are at a higher free energy state than ER membranes. Analysis of its kinetics suggested a mechanism that is analogous to wetting or adsorptive phenomena in which a tension-driven membrane flow supplements diffusive transfer of Golgi membrane into the ER. Such nonselective, flow-assisted transport of Golgi membranes into ER suggests that mechanisms that regulate retrograde tubule formation and detachment from the Golgi complex are integral to the existence and maintenance of this organelle.

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Kinetics of disassembly of BODIPY-ceramide–labeled Golgi structures  in BFA-treated cells. Cells  were labeled with BODIPY-ceramide and treated with  BFA. They were then imaged at 37°C using a cooled  CCD microscope system. (A)  Image sequences of a field of  cells from 3 min (3:00) to 19  min 45 s (19:45) of BFA  treatment showing redistribution of BODIPY-ceramide  from juxtanuclear Golgi  structures into the ER. Arrows point to cells that have  just undergone blink out. (B)  Time-lapse sequences were  taken as described above  with images collected at 13.5-s  intervals. Bars show duration  (start to finish) of Golgi  blinkout for 110 cells. Start of  blinkout was the first frame  showing spreading of fluorescence. The end was the last  frame to show change. Onset  and duration of Golgi blinkout in BODIPY-ceramide– labeled Golgi membranes  was indistinguishable from  GFP-GalTase–labeled Golgi  membranes. (C) The data set  in B was replotted to show  the number of Golgi structures remaining as a function  of time in BFA. The best fit  exponential function to the  numbers is the line shown on  the graph, indicating that the  kinetics of Golgi blinkout in  a cell population is a first order process. Bar, 10 μm.
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Figure 8: Kinetics of disassembly of BODIPY-ceramide–labeled Golgi structures in BFA-treated cells. Cells were labeled with BODIPY-ceramide and treated with BFA. They were then imaged at 37°C using a cooled CCD microscope system. (A) Image sequences of a field of cells from 3 min (3:00) to 19 min 45 s (19:45) of BFA treatment showing redistribution of BODIPY-ceramide from juxtanuclear Golgi structures into the ER. Arrows point to cells that have just undergone blink out. (B) Time-lapse sequences were taken as described above with images collected at 13.5-s intervals. Bars show duration (start to finish) of Golgi blinkout for 110 cells. Start of blinkout was the first frame showing spreading of fluorescence. The end was the last frame to show change. Onset and duration of Golgi blinkout in BODIPY-ceramide– labeled Golgi membranes was indistinguishable from GFP-GalTase–labeled Golgi membranes. (C) The data set in B was replotted to show the number of Golgi structures remaining as a function of time in BFA. The best fit exponential function to the numbers is the line shown on the graph, indicating that the kinetics of Golgi blinkout in a cell population is a first order process. Bar, 10 μm.

Mentions: Golgi membranes labeled with BODIPY-ceramide also redistributed into the ER in BFA-treated cells by a rapid and discrete process (Fig. 8 A). The onset and duration of Golgi blinkout in these cells was indistinguishable from that of cells expressing the GFP-Golgi protein chimeras. For both, blinkout usually occurred between 4 and 8 min after adding BFA and lasted only 15–60 s (Fig. 8 B). In addition, Golgi structures in the population of cells disappeared exponentially in time, consistent with a unique fusion event triggering the redistribution process (Fig. 8 C). As found for the GFP-Golgi protein chimeras, very little BODIPY label was left concentrated in Golgi structures after this redistribution (Fig. 8 A), with the label instead found distributed throughout the ER system. In cells stained with higher concentrations of BODIPY-ceramide, however, a Golgi remnant sometimes remained and presumably corresponded to the trans-Golgi network, which does not redistribute its membranes into the ER with BFA (Lippincott-Schwartz, 1991). These results suggest that both Golgi lipid and protein are delivered at equivalent rapid rates into the ER upon initiation of a distinct event (possibly fusion of a single Golgi tubule with the ER) in BFA-treated cells.


Golgi tubule traffic and the effects of brefeldin A visualized in living cells.

Sciaky N, Presley J, Smith C, Zaal KJ, Cole N, Moreira JE, Terasaki M, Siggia E, Lippincott-Schwartz J - J. Cell Biol. (1997)

Kinetics of disassembly of BODIPY-ceramide–labeled Golgi structures  in BFA-treated cells. Cells  were labeled with BODIPY-ceramide and treated with  BFA. They were then imaged at 37°C using a cooled  CCD microscope system. (A)  Image sequences of a field of  cells from 3 min (3:00) to 19  min 45 s (19:45) of BFA  treatment showing redistribution of BODIPY-ceramide  from juxtanuclear Golgi  structures into the ER. Arrows point to cells that have  just undergone blink out. (B)  Time-lapse sequences were  taken as described above  with images collected at 13.5-s  intervals. Bars show duration  (start to finish) of Golgi  blinkout for 110 cells. Start of  blinkout was the first frame  showing spreading of fluorescence. The end was the last  frame to show change. Onset  and duration of Golgi blinkout in BODIPY-ceramide– labeled Golgi membranes  was indistinguishable from  GFP-GalTase–labeled Golgi  membranes. (C) The data set  in B was replotted to show  the number of Golgi structures remaining as a function  of time in BFA. The best fit  exponential function to the  numbers is the line shown on  the graph, indicating that the  kinetics of Golgi blinkout in  a cell population is a first order process. Bar, 10 μm.
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Figure 8: Kinetics of disassembly of BODIPY-ceramide–labeled Golgi structures in BFA-treated cells. Cells were labeled with BODIPY-ceramide and treated with BFA. They were then imaged at 37°C using a cooled CCD microscope system. (A) Image sequences of a field of cells from 3 min (3:00) to 19 min 45 s (19:45) of BFA treatment showing redistribution of BODIPY-ceramide from juxtanuclear Golgi structures into the ER. Arrows point to cells that have just undergone blink out. (B) Time-lapse sequences were taken as described above with images collected at 13.5-s intervals. Bars show duration (start to finish) of Golgi blinkout for 110 cells. Start of blinkout was the first frame showing spreading of fluorescence. The end was the last frame to show change. Onset and duration of Golgi blinkout in BODIPY-ceramide– labeled Golgi membranes was indistinguishable from GFP-GalTase–labeled Golgi membranes. (C) The data set in B was replotted to show the number of Golgi structures remaining as a function of time in BFA. The best fit exponential function to the numbers is the line shown on the graph, indicating that the kinetics of Golgi blinkout in a cell population is a first order process. Bar, 10 μm.
Mentions: Golgi membranes labeled with BODIPY-ceramide also redistributed into the ER in BFA-treated cells by a rapid and discrete process (Fig. 8 A). The onset and duration of Golgi blinkout in these cells was indistinguishable from that of cells expressing the GFP-Golgi protein chimeras. For both, blinkout usually occurred between 4 and 8 min after adding BFA and lasted only 15–60 s (Fig. 8 B). In addition, Golgi structures in the population of cells disappeared exponentially in time, consistent with a unique fusion event triggering the redistribution process (Fig. 8 C). As found for the GFP-Golgi protein chimeras, very little BODIPY label was left concentrated in Golgi structures after this redistribution (Fig. 8 A), with the label instead found distributed throughout the ER system. In cells stained with higher concentrations of BODIPY-ceramide, however, a Golgi remnant sometimes remained and presumably corresponded to the trans-Golgi network, which does not redistribute its membranes into the ER with BFA (Lippincott-Schwartz, 1991). These results suggest that both Golgi lipid and protein are delivered at equivalent rapid rates into the ER upon initiation of a distinct event (possibly fusion of a single Golgi tubule with the ER) in BFA-treated cells.

Bottom Line: Both lipid and protein emptied from the Golgi at similar rapid rates, leaving no Golgi structure behind, indicating that Golgi membranes do not simply mix but are absorbed into the ER in BFA-treated cells.Analysis of its kinetics suggested a mechanism that is analogous to wetting or adsorptive phenomena in which a tension-driven membrane flow supplements diffusive transfer of Golgi membrane into the ER.Such nonselective, flow-assisted transport of Golgi membranes into ER suggests that mechanisms that regulate retrograde tubule formation and detachment from the Golgi complex are integral to the existence and maintenance of this organelle.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The Golgi complex is a dynamic organelle engaged in both secretory and retrograde membrane traffic. Here, we use green fluorescent protein-Golgi protein chimeras to study Golgi morphology in vivo. In untreated cells, membrane tubules were a ubiquitous, prominent feature of the Golgi complex, serving both to interconnect adjacent Golgi elements and to carry membrane outward along microtubules after detaching from stable Golgi structures. Brefeldin A treatment, which reversibly disassembles the Golgi complex, accentuated tubule formation without tubule detachment. A tubule network extending throughout the cytoplasm was quickly generated and persisted for 5-10 min until rapidly emptying Golgi contents into the ER within 15-30 s. Both lipid and protein emptied from the Golgi at similar rapid rates, leaving no Golgi structure behind, indicating that Golgi membranes do not simply mix but are absorbed into the ER in BFA-treated cells. The directionality of redistribution implied Golgi membranes are at a higher free energy state than ER membranes. Analysis of its kinetics suggested a mechanism that is analogous to wetting or adsorptive phenomena in which a tension-driven membrane flow supplements diffusive transfer of Golgi membrane into the ER. Such nonselective, flow-assisted transport of Golgi membranes into ER suggests that mechanisms that regulate retrograde tubule formation and detachment from the Golgi complex are integral to the existence and maintenance of this organelle.

Show MeSH
Related in: MedlinePlus