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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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Evidence for membrane flow from Golgi to ER: diffusive recovery from photobleach contrasted with the time course of Golgi  blinkout. (A) Three confocal images showing photobleaching and recovery of GFP-GalTase in the ER in cells treated with BFA for 1 h.  The left panel is the prebleach image, the middle panel is just after the bleach, and the right panel is after recovery. The bleached region  is outlined in dashed lines. The fluorescent intensities within the boxed ROIs labeled 1, 2, and 3 during recovery are plotted as symbols  to the right (diamonds, crosses, and pluses, respectively). The smooth curves are simulations with Deff of 2.5 × 10−9cm2/s as explained in  the text and A. The dashed curves are the same simulation assuming Deff of 5.0 × 10−9cm2/s. Note that even though each ROI  displayed a different recovery curve, a single Deff of 2.5 × 10−9cm2/s in the simulations could effectively account for all the experimental  data. (B) The same cell as in A was imaged during Golgi blinkout after addition of BFA. The two confocal images correspond to onset  of Golgi blinkout and after the redistribution into the ER is complete. The fluorescent intensities within the boxed ROIs labeled 1, 2,  and 3 during Golgi blinkout are plotted to the right as symbols (pluses, crosses, and diamonds, respectively). Numerical simulations began with the fluorescent density field shown in the left panel and assumed diffusive transport toward the field in the right panel. The  smooth curves in the graph show the simulated intensity assuming Deff of 2.5 × 10−9cm2/s, whereas the dashed curves show the simulated intensity assuming Deff of 5.0 × 10−9cm2/s. Note the latency period of the experimental data in relation to the simulated curves and  the more sigmoidal rise. (C) Three cooled CCD images of a GFP-KDELR–expressing cell at the beginning of Golgi blinkout (left), 7.4 s  later (middle), and the final time point 37 s later (right). Fluorescent intensity values of the four ROIs shown were plotted as a function  of time in BFA. Numerical simulations ran from the first panel toward the third as in B. Diffusion constants for each ROI that best fit  the data are shown. Bars (A and C), 10 μm.
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Figure 11: Evidence for membrane flow from Golgi to ER: diffusive recovery from photobleach contrasted with the time course of Golgi blinkout. (A) Three confocal images showing photobleaching and recovery of GFP-GalTase in the ER in cells treated with BFA for 1 h. The left panel is the prebleach image, the middle panel is just after the bleach, and the right panel is after recovery. The bleached region is outlined in dashed lines. The fluorescent intensities within the boxed ROIs labeled 1, 2, and 3 during recovery are plotted as symbols to the right (diamonds, crosses, and pluses, respectively). The smooth curves are simulations with Deff of 2.5 × 10−9cm2/s as explained in the text and A. The dashed curves are the same simulation assuming Deff of 5.0 × 10−9cm2/s. Note that even though each ROI displayed a different recovery curve, a single Deff of 2.5 × 10−9cm2/s in the simulations could effectively account for all the experimental data. (B) The same cell as in A was imaged during Golgi blinkout after addition of BFA. The two confocal images correspond to onset of Golgi blinkout and after the redistribution into the ER is complete. The fluorescent intensities within the boxed ROIs labeled 1, 2, and 3 during Golgi blinkout are plotted to the right as symbols (pluses, crosses, and diamonds, respectively). Numerical simulations began with the fluorescent density field shown in the left panel and assumed diffusive transport toward the field in the right panel. The smooth curves in the graph show the simulated intensity assuming Deff of 2.5 × 10−9cm2/s, whereas the dashed curves show the simulated intensity assuming Deff of 5.0 × 10−9cm2/s. Note the latency period of the experimental data in relation to the simulated curves and the more sigmoidal rise. (C) Three cooled CCD images of a GFP-KDELR–expressing cell at the beginning of Golgi blinkout (left), 7.4 s later (middle), and the final time point 37 s later (right). Fluorescent intensity values of the four ROIs shown were plotted as a function of time in BFA. Numerical simulations ran from the first panel toward the third as in B. Diffusion constants for each ROI that best fit the data are shown. Bars (A and C), 10 μm.

Mentions: HeLa cells grown on glass coverslips were sealed into a chamber fashioned out of silicon rubber (Ronsil; North American Reiss, Blackstone, VA) placed on a glass slide and containing buffered medium with Oxyrase (Oxyrase, Inc., Ashland, OH). The cells in Figs. 3 C and 4 were viewed with a scanning confocal attachment (model MRC 600; Bio-Rad Labs, Hercules, CA) attached to a microscope (model Axioplan; Carl Zeiss, Inc., Thornwood, NY) with a 63× planapochromat lens (NA 1.4; Carl Zeiss, Inc.). The 488-nm line of a krypton-argon laser was used with a 1 or 3% neutral density filter. Digital output was routed through a time and date generator (model WJ-810; Panasonic Corp., New York), and single frames were recorded on an optical memory disk recorder (model 3031F; Panasonic). Cells in Figs. 2; 3, A and B; and 11, A and B, were viewed on a confocal laser scanning microscope (model LSM 410; Carl Zeiss, Inc.) equipped with a Kr/Ar laser and a 100× 1.4 NA planapochromat oil immersion objective. The GFP molecule was excited with the 488 line of the laser and imaged with a 515–540 bandpass filter. The time-lapse sequence in Fig. 11 B was recorded using macros programmed with the Zeiss LSM software package. In all other experiments, cells were viewed with a custom built inverted wide field microscope (model Eikoscope; Yona Microscopes, Columbia, MD). This microscope was equipped with a 63×, 1.4 NA objective and a cooled charge-coupled device (Photometrics, Tucson, AZ) with a KAF 1400 pixel Kodak chip (Rochester, NY) for 12-bit image detection. A 100-W mercury lamp was used as the light source. Neutral density filters, excitation (485 nm band pass), emission (515 nm long pass), and dichroic filters (fluorescence set XF32; Omega Optical Inc., Brattleboro, VT) were used to select the appropriate spectra for imaging GFP and BODIPY-ceramide. Biological Detection Systems imaging software (version 1.6, now Oncor imaging, Oncor Instruments, San Diego, CA) or IPlab Spectrum was used to control image acquisition (Macintosh Quadra 800; Apple Computer Co., Cupertino, CA). Images were manipulated using IPlab Spectrum (Signal Analytics, Vienna, VA), NIH-Image software (Wayne Rasband, Research Services Branch, National Institutes of Health, Bethesda, MD), and Adobe Photoshop (San Jose, CA). Images were printed with a Fujix Pictrography 3000 Digital Printer (Fuji Photofil Co., Tokyo, Japan). None of the cooled CCD images collected and displayed had any saturated pixels. The dynamic range was 0–4,095 gray levels for the cooled CCD images. For the confocal images, the range was 0–255 gray levels. There was no overexposure in those confocal images (Fig. 11, A and B), which were used to analyze Golgi blinkout and to fit a diffusion constant since it clearly would have spoiled the quantitation.


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)

Evidence for membrane flow from Golgi to ER: diffusive recovery from photobleach contrasted with the time course of Golgi  blinkout. (A) Three confocal images showing photobleaching and recovery of GFP-GalTase in the ER in cells treated with BFA for 1 h.  The left panel is the prebleach image, the middle panel is just after the bleach, and the right panel is after recovery. The bleached region  is outlined in dashed lines. The fluorescent intensities within the boxed ROIs labeled 1, 2, and 3 during recovery are plotted as symbols  to the right (diamonds, crosses, and pluses, respectively). The smooth curves are simulations with Deff of 2.5 × 10−9cm2/s as explained in  the text and A. The dashed curves are the same simulation assuming Deff of 5.0 × 10−9cm2/s. Note that even though each ROI  displayed a different recovery curve, a single Deff of 2.5 × 10−9cm2/s in the simulations could effectively account for all the experimental  data. (B) The same cell as in A was imaged during Golgi blinkout after addition of BFA. The two confocal images correspond to onset  of Golgi blinkout and after the redistribution into the ER is complete. The fluorescent intensities within the boxed ROIs labeled 1, 2,  and 3 during Golgi blinkout are plotted to the right as symbols (pluses, crosses, and diamonds, respectively). Numerical simulations began with the fluorescent density field shown in the left panel and assumed diffusive transport toward the field in the right panel. The  smooth curves in the graph show the simulated intensity assuming Deff of 2.5 × 10−9cm2/s, whereas the dashed curves show the simulated intensity assuming Deff of 5.0 × 10−9cm2/s. Note the latency period of the experimental data in relation to the simulated curves and  the more sigmoidal rise. (C) Three cooled CCD images of a GFP-KDELR–expressing cell at the beginning of Golgi blinkout (left), 7.4 s  later (middle), and the final time point 37 s later (right). Fluorescent intensity values of the four ROIs shown were plotted as a function  of time in BFA. Numerical simulations ran from the first panel toward the third as in B. Diffusion constants for each ROI that best fit  the data are shown. Bars (A and C), 10 μm.
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Figure 11: Evidence for membrane flow from Golgi to ER: diffusive recovery from photobleach contrasted with the time course of Golgi blinkout. (A) Three confocal images showing photobleaching and recovery of GFP-GalTase in the ER in cells treated with BFA for 1 h. The left panel is the prebleach image, the middle panel is just after the bleach, and the right panel is after recovery. The bleached region is outlined in dashed lines. The fluorescent intensities within the boxed ROIs labeled 1, 2, and 3 during recovery are plotted as symbols to the right (diamonds, crosses, and pluses, respectively). The smooth curves are simulations with Deff of 2.5 × 10−9cm2/s as explained in the text and A. The dashed curves are the same simulation assuming Deff of 5.0 × 10−9cm2/s. Note that even though each ROI displayed a different recovery curve, a single Deff of 2.5 × 10−9cm2/s in the simulations could effectively account for all the experimental data. (B) The same cell as in A was imaged during Golgi blinkout after addition of BFA. The two confocal images correspond to onset of Golgi blinkout and after the redistribution into the ER is complete. The fluorescent intensities within the boxed ROIs labeled 1, 2, and 3 during Golgi blinkout are plotted to the right as symbols (pluses, crosses, and diamonds, respectively). Numerical simulations began with the fluorescent density field shown in the left panel and assumed diffusive transport toward the field in the right panel. The smooth curves in the graph show the simulated intensity assuming Deff of 2.5 × 10−9cm2/s, whereas the dashed curves show the simulated intensity assuming Deff of 5.0 × 10−9cm2/s. Note the latency period of the experimental data in relation to the simulated curves and the more sigmoidal rise. (C) Three cooled CCD images of a GFP-KDELR–expressing cell at the beginning of Golgi blinkout (left), 7.4 s later (middle), and the final time point 37 s later (right). Fluorescent intensity values of the four ROIs shown were plotted as a function of time in BFA. Numerical simulations ran from the first panel toward the third as in B. Diffusion constants for each ROI that best fit the data are shown. Bars (A and C), 10 μm.
Mentions: HeLa cells grown on glass coverslips were sealed into a chamber fashioned out of silicon rubber (Ronsil; North American Reiss, Blackstone, VA) placed on a glass slide and containing buffered medium with Oxyrase (Oxyrase, Inc., Ashland, OH). The cells in Figs. 3 C and 4 were viewed with a scanning confocal attachment (model MRC 600; Bio-Rad Labs, Hercules, CA) attached to a microscope (model Axioplan; Carl Zeiss, Inc., Thornwood, NY) with a 63× planapochromat lens (NA 1.4; Carl Zeiss, Inc.). The 488-nm line of a krypton-argon laser was used with a 1 or 3% neutral density filter. Digital output was routed through a time and date generator (model WJ-810; Panasonic Corp., New York), and single frames were recorded on an optical memory disk recorder (model 3031F; Panasonic). Cells in Figs. 2; 3, A and B; and 11, A and B, were viewed on a confocal laser scanning microscope (model LSM 410; Carl Zeiss, Inc.) equipped with a Kr/Ar laser and a 100× 1.4 NA planapochromat oil immersion objective. The GFP molecule was excited with the 488 line of the laser and imaged with a 515–540 bandpass filter. The time-lapse sequence in Fig. 11 B was recorded using macros programmed with the Zeiss LSM software package. In all other experiments, cells were viewed with a custom built inverted wide field microscope (model Eikoscope; Yona Microscopes, Columbia, MD). This microscope was equipped with a 63×, 1.4 NA objective and a cooled charge-coupled device (Photometrics, Tucson, AZ) with a KAF 1400 pixel Kodak chip (Rochester, NY) for 12-bit image detection. A 100-W mercury lamp was used as the light source. Neutral density filters, excitation (485 nm band pass), emission (515 nm long pass), and dichroic filters (fluorescence set XF32; Omega Optical Inc., Brattleboro, VT) were used to select the appropriate spectra for imaging GFP and BODIPY-ceramide. Biological Detection Systems imaging software (version 1.6, now Oncor imaging, Oncor Instruments, San Diego, CA) or IPlab Spectrum was used to control image acquisition (Macintosh Quadra 800; Apple Computer Co., Cupertino, CA). Images were manipulated using IPlab Spectrum (Signal Analytics, Vienna, VA), NIH-Image software (Wayne Rasband, Research Services Branch, National Institutes of Health, Bethesda, MD), and Adobe Photoshop (San Jose, CA). Images were printed with a Fujix Pictrography 3000 Digital Printer (Fuji Photofil Co., Tokyo, Japan). None of the cooled CCD images collected and displayed had any saturated pixels. The dynamic range was 0–4,095 gray levels for the cooled CCD images. For the confocal images, the range was 0–255 gray levels. There was no overexposure in those confocal images (Fig. 11, A and B), which were used to analyze Golgi blinkout and to fit a diffusion constant since it clearly would have spoiled the quantitation.

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