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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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Localization of  GFP-GalTase by immunogold microscopy. Gallery of  images from cryosections of  CHO cells expressing GFP-GalTase (A–C) or GFP-KDELR (D) that were immunostained with anti-GFP  antibody and 15-nm colloidal  gold protein A. Note that  multiple cisternae within  Golgi stacks are specifically  labeled with gold particles.  Nu, nucleus; Pm, plasma  membrane. Bars, 0.2 μm.
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Figure 1: Localization of GFP-GalTase by immunogold microscopy. Gallery of images from cryosections of CHO cells expressing GFP-GalTase (A–C) or GFP-KDELR (D) that were immunostained with anti-GFP antibody and 15-nm colloidal gold protein A. Note that multiple cisternae within Golgi stacks are specifically labeled with gold particles. Nu, nucleus; Pm, plasma membrane. Bars, 0.2 μm.

Mentions: Galactosyltransferase is a Golgi resident enzyme that functions in the remodeling of N-linked oligosaccharides attached to proteins moving through the secretory pathway. By contrast, KDEL receptor is an itinerant Golgi protein, which recycles KDEL-containing ligands passing through the Golgi back to the ER (Lewis and Pelham, 1992). Previous work has demonstrated that addition of the GFP tag to these proteins does not interfere with Golgi targeting of these proteins or with ligand-induced recycling of KDELR (Cole et al., 1996a). GFP-tagged galactosyltransferase (GFP-GalTase) and KDEL receptor (GFP-KDELR) were both shown to reside predominantly in juxtanuclear Golgi-like structures, with GFP-KDELR also found in small amounts in the ER (Cole et al., 1996a). To further analyze the Golgi location of these chimeras, we used immunogold electron microscopy, and confocal microscopy of double-labeled specimens. Immunoelectron microscopy using thin, frozen sections labeled with polyclonal antibodies to GFP followed by secondary antibodies coupled to 15-nm gold revealed the ultrastructural distribution of the chimeras. As shown in Fig. 1, A–C, for GFP-GalTase, nearly all of the gold particles were restricted to the Golgi complex and were mostly found associated with stacked cisternae. No significant gold labeling of plasma membrane or nuclear envelope was detected. In cells expressing GFP-KDELR (Fig. 1 D), a similar localization of gold particles to Golgi stacks was observed, but occasional gold particles were also found over the ER.


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)

Localization of  GFP-GalTase by immunogold microscopy. Gallery of  images from cryosections of  CHO cells expressing GFP-GalTase (A–C) or GFP-KDELR (D) that were immunostained with anti-GFP  antibody and 15-nm colloidal  gold protein A. Note that  multiple cisternae within  Golgi stacks are specifically  labeled with gold particles.  Nu, nucleus; Pm, plasma  membrane. Bars, 0.2 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140213&req=5

Figure 1: Localization of GFP-GalTase by immunogold microscopy. Gallery of images from cryosections of CHO cells expressing GFP-GalTase (A–C) or GFP-KDELR (D) that were immunostained with anti-GFP antibody and 15-nm colloidal gold protein A. Note that multiple cisternae within Golgi stacks are specifically labeled with gold particles. Nu, nucleus; Pm, plasma membrane. Bars, 0.2 μm.
Mentions: Galactosyltransferase is a Golgi resident enzyme that functions in the remodeling of N-linked oligosaccharides attached to proteins moving through the secretory pathway. By contrast, KDEL receptor is an itinerant Golgi protein, which recycles KDEL-containing ligands passing through the Golgi back to the ER (Lewis and Pelham, 1992). Previous work has demonstrated that addition of the GFP tag to these proteins does not interfere with Golgi targeting of these proteins or with ligand-induced recycling of KDELR (Cole et al., 1996a). GFP-tagged galactosyltransferase (GFP-GalTase) and KDEL receptor (GFP-KDELR) were both shown to reside predominantly in juxtanuclear Golgi-like structures, with GFP-KDELR also found in small amounts in the ER (Cole et al., 1996a). To further analyze the Golgi location of these chimeras, we used immunogold electron microscopy, and confocal microscopy of double-labeled specimens. Immunoelectron microscopy using thin, frozen sections labeled with polyclonal antibodies to GFP followed by secondary antibodies coupled to 15-nm gold revealed the ultrastructural distribution of the chimeras. As shown in Fig. 1, A–C, for GFP-GalTase, nearly all of the gold particles were restricted to the Golgi complex and were mostly found associated with stacked cisternae. No significant gold labeling of plasma membrane or nuclear envelope was detected. In cells expressing GFP-KDELR (Fig. 1 D), a similar localization of gold particles to Golgi stacks was observed, but occasional gold particles were also found over the ER.

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