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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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Time-course and  kinetics of Golgi disassembly in a population of cells  treated with BFA. Cells expressing GFP-GalTase were  treated with BFA and imaged with a cooled CCD  microscope system at 37°C.  (A) A field of cells showing  redistribution of GFP-GalTase into the ER. Arrows  point to Golgi structures undergoing blinkout. Note that  Golgi blinkout occurred at  different times in different  cells. Images shown begin at  4 min 16 s (4:16) of BFA  treatment and extend until 8  min 54 s (8:54). (B) Time-lapse sequences were taken  as described above with images taken at 13.5-s intervals. Bars show duration  (start to finish) of Golgi  blinkout. Start of blinkout  was the first frame showing  spreading of fluorescence.  The end was the last frame to  show change. (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 7: Time-course and kinetics of Golgi disassembly in a population of cells treated with BFA. Cells expressing GFP-GalTase were treated with BFA and imaged with a cooled CCD microscope system at 37°C. (A) A field of cells showing redistribution of GFP-GalTase into the ER. Arrows point to Golgi structures undergoing blinkout. Note that Golgi blinkout occurred at different times in different cells. Images shown begin at 4 min 16 s (4:16) of BFA treatment and extend until 8 min 54 s (8:54). (B) Time-lapse sequences were taken as described above with images taken at 13.5-s intervals. Bars show duration (start to finish) of Golgi blinkout. Start of blinkout was the first frame showing spreading of fluorescence. The end was the last frame to show change. (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 redistribution into the ER occurred at different times in different cells (Fig. 7 A) but was always a sudden, explosive event that we called Golgi “blinkout.” Within a population of over one hundred cells, Golgi blinkout usually occurred 4 to 8 min after BFA was added and had a duration of 15–60 s during which Golgi-localized fluorescence dispersed into the ER (Fig. 7 B). Significantly, the number of Golgi structures in a population of cells treated with BFA decreased exponentially in time (after a latency period of about 4 min) (Fig. 7 C), resembling a first-order process (i.e., radioactive decay). This suggested that entry of BFA-treated Golgi membranes into the ER system is likely to involve a unique fusion event or entry site rather than targeting and fusion of a mass of tubules or vesicles.


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)

Time-course and  kinetics of Golgi disassembly in a population of cells  treated with BFA. Cells expressing GFP-GalTase were  treated with BFA and imaged with a cooled CCD  microscope system at 37°C.  (A) A field of cells showing  redistribution of GFP-GalTase into the ER. Arrows  point to Golgi structures undergoing blinkout. Note that  Golgi blinkout occurred at  different times in different  cells. Images shown begin at  4 min 16 s (4:16) of BFA  treatment and extend until 8  min 54 s (8:54). (B) Time-lapse sequences were taken  as described above with images taken at 13.5-s intervals. Bars show duration  (start to finish) of Golgi  blinkout. Start of blinkout  was the first frame showing  spreading of fluorescence.  The end was the last frame to  show change. (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 7: Time-course and kinetics of Golgi disassembly in a population of cells treated with BFA. Cells expressing GFP-GalTase were treated with BFA and imaged with a cooled CCD microscope system at 37°C. (A) A field of cells showing redistribution of GFP-GalTase into the ER. Arrows point to Golgi structures undergoing blinkout. Note that Golgi blinkout occurred at different times in different cells. Images shown begin at 4 min 16 s (4:16) of BFA treatment and extend until 8 min 54 s (8:54). (B) Time-lapse sequences were taken as described above with images taken at 13.5-s intervals. Bars show duration (start to finish) of Golgi blinkout. Start of blinkout was the first frame showing spreading of fluorescence. The end was the last frame to show change. (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 redistribution into the ER occurred at different times in different cells (Fig. 7 A) but was always a sudden, explosive event that we called Golgi “blinkout.” Within a population of over one hundred cells, Golgi blinkout usually occurred 4 to 8 min after BFA was added and had a duration of 15–60 s during which Golgi-localized fluorescence dispersed into the ER (Fig. 7 B). Significantly, the number of Golgi structures in a population of cells treated with BFA decreased exponentially in time (after a latency period of about 4 min) (Fig. 7 C), resembling a first-order process (i.e., radioactive decay). This suggested that entry of BFA-treated Golgi membranes into the ER system is likely to involve a unique fusion event or entry site rather than targeting and fusion of a mass of tubules or vesicles.

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