scholarly journals Golgi-bound Rab34 Is a Novel Member of the Secretory Pathway

2007 ◽  
Vol 18 (12) ◽  
pp. 4762-4771 ◽  
Author(s):  
Neil M. Goldenberg ◽  
Sergio Grinstein ◽  
Mel Silverman
Keyword(s):  
Hela Cells ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Fluid Phase ◽  
Dominant Negative ◽  
Temperature Sensitive ◽  
Golgi Stack ◽  
Green Fluorescent ◽  
Intracellular Vesicle Transport

Golgi-localized Rab34 has been implicated in repositioning lysosomes and activation of macropinocytosis. Using HeLa cells, we undertook a detailed investigation of Rab34 involvement in intracellular vesicle transport. Immunoelectron microscopy and immunocytochemistry confirmed that Rab34 is localized to the Golgi stack and that active Rab34 shifts lysosomes to the cell center. Contrary to a previous report, we found that Rab34 is not concentrated at membrane ruffles and is not involved in fluid-phase uptake. Also, Rab34-induced repositioning of lysosomes does not affect mannose 6-phosphate receptor trafficking. Most strikingly, HeLa cells depleted of Rab34 by transfection with dominant-negative Rab34 or after RNA interference, failed to transport the temperature-sensitive vesicular stomatitis virus G-protein (VSVG) fused to green fluorescent protein (VSVG-GFP) from the Golgi to the plasma membrane. Transfection with mouse Rab34 rescued this defect. Using endogenous major histocompatibility complex class I (MHCI) as a marker, an endoglycosidase H resistance assay showed that endoplasmic reticulum (ER) to medial Golgi traffic remains intact in knockdown cells, indicating that Rab34 specifically functions downstream of the ER. Further, brefeldin A treatment revealed that Rab34 effects intra-Golgi transport, not exit from the trans-Golgi network. Collectively, these results define Rab34 as a novel member of the secretory pathway acting at the Golgi.

Golgi-bound Rab34 is a novel member of the secretory pathways

2007 ◽  
Vol 30 (4) ◽  
pp. 82
Author(s):  
Neil M. Goldenberg ◽  
Sergio Grinstein ◽  
Mel Silverman
Keyword(s):  
Hela Cells ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Dominant Negative ◽  
Temperature Sensitive ◽  
Class I Mhc ◽  
Golgi Stack ◽  
Vesicular Stomatitis ◽  
Golgi Network ◽  
Intracellular Vesicle Transport

Background: Golgi-localized Rab34 has been implicated in repositioning of lysosomes and activation of macropinocytosis. Methods: Using HeLa cells we undertook a detailed investigation of Rab34 involvement in intracellular vesicle transport. Results: Immunoelectron microscopy and immunocytochemistry confirmed that Rab34 is localized to the Golgi stack and that active Rab34 shifts lysosomes to the cell centre. Contrary to a previous report, we found that Rab34 is not concentrated at membrane ruffles and is not involved in macropinocytosis. Also, Rab34 induced repositioning of lysosomes does not affect transport of the mannose 6-phosphate receptor to endosomes. Most strikingly, HeLa cells depleted of Rab34 by transfection with dominant-negative Rab34, or following RNA interference, failed to transport the temperature-sensitive Vesicular Stomatitis Virus G-protein fused to GFP (VSVG-GFP) from the Golgi to the plasma membrane. Transfection with mouse Rab34 rescued this defect. Using endogenous MHC class I (MHC) as a marker, an endoglycosidase H resistance assay showed that ER to medial Golgi traffic remains intact in knock-down cells indicating that Rab34 specifically functions in post-Golgi transport. Further, brefeldin A treatment revealed that Rab34 acts at the Golgi, not the trans-Golgi network. Conclusion: Collectively, these results define Rab34 as a novel member of the secretory pathway acting at the Golgi.

Transport of virally expressed green fluorescent protein through the secretory pathway in tobacco leaves is inhibited by cold shock and brefeldin A

Planta ◽  
1999 ◽  
Vol 208 (3) ◽  
pp. 392-400 ◽  
Author(s):  
Petra Boevink ◽  
Barry Martin ◽  
Karl Oparka ◽  
Simon Santa Cruz ◽  
Chris Hawes
Keyword(s):  
Green Fluorescent Protein ◽  
Secretory Pathway ◽  
Cold Shock ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Tobacco Leaves ◽  
Green Fluorescent

scholarly journals Recycling of Golgi-resident Glycosyltransferases through the ER Reveals a Novel Pathway and Provides an Explanation for Nocodazole-induced Golgi Scattering

1998 ◽  
Vol 143 (6) ◽  
pp. 1505-1521 ◽  
Author(s):  
Brian Storrie ◽  
Jamie White ◽  
Sabine Röttger ◽  
Ernst H.K. Stelzer ◽  
Tatsuo Suganuma ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Time Lapse ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Protein Synthesis Inhibitors ◽  
Microtubule Depolymerization ◽  
Golgi Stack ◽  
Gradual Accumulation ◽  
Green Fluorescent ◽  
Er Export

During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway. To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11–amino acid epitope, VSV-G (VSV), and the trans/TGN enzyme β1,4-galactosyltransferase (GalT) fused to GFP. After nocodazole addition, time-lapse microscopy of GalNAc-T2–GFP and GalT–GFP revealed that scattered Golgi elements appeared abruptly and that no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fluorescence intensity for tens of minutes. During the entire process of dispersal, immunogold labeling for GalNAc-T2–VSV and GalT showed that these were continuously concentrated over stacked Golgi cisternae and tubulovesicular Golgi structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites. In fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident proteins with each other through what appeared to be an ER intermediate. That Golgi enzymes cycle through the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1pdn) blocking ER export. Sar1pdn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumulation of GalNAc-T2–VSV in the ER. Few to no peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1pdn. In conclusion, we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced Golgi scattering observed in interphase cells.

scholarly journals Intracellular Trafficking of a Palmitoylated Membrane-Associated Protein Component of Enveloped Vaccinia Virus

2003 ◽  
Vol 77 (16) ◽  
pp. 9008-9019 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Plasma Membrane ◽  
Vaccinia Virus ◽  
Intracellular Trafficking ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Coated Pits ◽  
Early Endosomes ◽  
Green Fluorescent

ABSTRACT The F13L protein of vaccinia virus, an essential and abundant palmitoylated peripheral membrane component of intra- and extracellular enveloped virions, associates with Golgi, endosomal, and plasma membranes in the presence or absence of other viral proteins. In the present study, the trafficking of a fully functional F13L-green fluorescent protein (GFP) chimera in transfected and productively infected cells was analyzed using specific markers and inhibitors. We found that Sar1H79G, a trans-dominant-negative protein inhibitor of cargo transport from the endoplasmic reticulum, had no apparent effect on the intracellular distribution of F13L-GFP, suggesting that the initial membrane localization occurs at a downstream compartment of the secretory pathway. Recycling of F13L-GFP from the plasma membrane was demonstrated by partial colocalization with FM4-64, a fluorescent membrane marker of endocytosis. Punctate F13L-GFP fluorescence overlapped with clathrin and Texas red-conjugated transferrin, suggesting that endocytosis occurred via clathrin-coated pits. The inhibitory effects of chlorpromazine and trans-dominant-negative forms of dynamin and Eps15 protein on the recycling of F13L-GFP provided further evidence for clathrin-mediated endocytosis. In addition, the F13L protein was specifically coimmunoprecipitated with α-adaptin, a component of the AP-2 complex that interacts with Eps15. Nocodazole and wortmannin perturbed the intracellular trafficking of F13L-GFP, consistent with its entry into late and early endosomes through the secretory and endocytic pathways, respectively. The recycling pathway described here provides a mechanism for the reutilization of the F13L protein following its deposition in the plasma membrane during the exocytosis of enveloped virions.

scholarly journals Heterogeneity of a Fluorescent Tegument Component in Single Pseudorabies Virus Virions and Enveloped Axonal Assemblies

2005 ◽  
Vol 79 (7) ◽  
pp. 3903-3919 ◽  
Author(s):  
T. del Rio ◽  
T. H. Ch'ng ◽  
E. A. Flood ◽  
S. P. Gross ◽  
L. W. Enquist
Keyword(s):  
Cell Body ◽  
Secretory Pathway ◽  
Pseudorabies Virus ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Cultured Neurons ◽  
Long Distance ◽  
Heterogeneous Distribution ◽  
Green Fluorescent

ABSTRACT The molecular mechanisms responsible for long-distance, directional spread of alphaherpesvirus infections via axons of infected neurons are poorly understood. We describe the use of red and green fluorescent protein (GFP) fusions to capsid and tegument components, respectively, to visualize purified, single extracellular virions and axonal assemblies after pseudorabies virus (PRV) infection of cultured neurons. We observed heterogeneity in GFP fluorescence when GFP was fused to the tegument component VP22 in both single extracellular virions and discrete puncta in infected axons. This heterogeneity was observed in the presence or absence of a capsid structure detected by a fusion of monomeric red fluorescent protein to VP26. The similarity of the heterogeneous distribution of these fluorescent protein fusions in both purified virions and in axons suggested that tegument-capsid assembly and axonal targeting of viral components are linked. One possibility was that the assembly of extracellular and axonal particles containing the dually fluorescent fusion proteins occurred by the same process in the cell body. We tested this hypothesis by treating infected cultured neurons with brefeldin A, a potent inhibitor of herpesvirus maturation and secretion. Brefeldin A treatment disrupted the neuronal secretory pathway, affected fluorescent capsid and tegument transport in the cell body, and blocked subsequent entry into axons of capsid and tegument proteins. Electron microscopy demonstrated that in the absence of brefeldin A treatment, enveloped capsids entered axons, but in the presence of the inhibitor, unenveloped capsids accumulated in the cell body. These results support an assembly process in which PRV capsids acquire a membrane in the cell body prior to axonal entry and subsequent transport.

Transport Through the Secretory Pathway: Observations of Cargo and Peripheral Coat Proteins

1998 ◽  
Vol 4 (S2) ◽  
pp. 1026-1027
Author(s):  
John F. Presley ◽  
Nelson B. Cole ◽  
Jennifer Lippincott-Schwartz
Keyword(s):  
Golgi Complex ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Temperature Sensitive ◽  
Coat Proteins ◽  
Viral Glycoprotein ◽  
Early Secretory Pathway ◽  
Green Fluorescent ◽  
Dynactin Complex

We have used green fluorescent protein (GFP) chimeras to examine the dynamics of the early secretory pathway and the role of the peripheral coat protein COP I. To describe the overall properties of ER to Golgi transport we used the temperature sensitive viral glycoprotein, ts045 VSVG, tagged with GFP at its cytoplasmic tail. VSVG-GFP retained the temperature sensitive phenotype of its parent: it reversibly misfolded and was retained in the ER at 40°C. Upon shift to 32°C it was rapidly exported from the ER, moving as a synchronous pool into the Golgi complex and then to the cell surface. Using time-lapse imaging of living cells expressing VSVG-GFP we found that the carriers for ER to Golgi traffic are tubulovesicular pre-Golgi intermediates that move centrosomally to the Golgi at speeds of>1 μM2/sec and then fuse with the cis face of the Golgi complex. These movements are dependant on microtubules and the dynein/dynactin complex.

Lumenal targeted GFP, used as a marker of soluble cargo, visualises rapid ERGIC to Golgi traffic by a tubulo-vesicular network

2000 ◽  
Vol 113 (18) ◽  
pp. 3151-3159 ◽  
Author(s):  
R. Blum ◽  
D.J. Stephens ◽  
I. Schulz
Keyword(s):  
Fluorescent Protein ◽  
Time Lapse ◽  
Temperature Sensitive ◽  
Long Distance ◽  
Anterograde Transport ◽  
Network Transport ◽  
Vesicular Stomatitis ◽  
Green Fluorescent ◽  
Time Lapse Imaging ◽  
Tubular Membranes

The mechanism by which soluble proteins without sorting motifs are transported to the cell surface is not clear. Here we show that soluble green fluorescent protein (GFP) targeted to the lumen of the endoplasmic reticulum but lacking any known retrieval, retention or targeting motifs, was accumulated in the lumen of the ERGIC if cells were kept at reduced temperature. Upon activation of anterograde transport by rewarming of cells, lumenal GFP stained a microtubule-dependent, pre-Golgi tubulo-vesicular network that served as transport structure between peripheral ERGIC-elements and the perinuclear Golgi complex. Individual examples of these tubular elements up to 20 microm in length were observed. Time lapse imaging indicated rapid anterograde flow of soluble lumenal GFP through this network. Transport tubules, stained by lumenal GFP, segregated rapidly from COPI-positive membranes after transport activation. A transmembrane cargo marker, the temperature sensitive glycoprotein of the vesicular stomatitis virus, ts-045 G, is also not present in tubules which contained the soluble cargo marker lum-GFP. These results suggest a role for pre-Golgi vesicular tubular membranes in long distance anterograde transport of soluble cargo. http://www.biologists.com/JCS/movies/jcs1334.html

Effects of cytokines on mycobacterial phagosome maturation

1998 ◽  
Vol 111 (7) ◽  
pp. 897-905 ◽  
Author(s):  
L.E. Via ◽  
R.A. Fratti ◽  
M. McFalone ◽  
E. Pagan-Ramos ◽  
D. Deretic ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fluorescent Protein ◽  
Fluid Phase ◽  
Transgenic Animals ◽  
Mononuclear Phagocytes ◽  
Phagosome Maturation ◽  
Ifn Gamma ◽  
The Status ◽  
Green Fluorescent ◽  
A Minor

One of the major mechanisms permitting intracellular pathogens to parasitize macrophages is their ability to alter maturation of the phagosome or affect its physical integrity. These processes are opposed by the host innate and adaptive immune defenses, and in many instances mononuclear phagocytes can be stimulated with appropriate cytokines to restrict the growth of the microorganisms within the phagosomal compartment. Very little is known about the effects that cytokines have on phagosome maturation. Here we have used green fluorescent protein (GFP)-labeled mycobacteria and a fixable acidotropic probe, LysoTracker Red DND-99, to monitor maturation of the mycobacterial phagosome. The macrophage compartments that stained with the LysoTracker probe were examined first. This dye was found to colocalize preferentially with the late endosomal and lysosomal markers rab7 and Lamp1, and with a fluid phase marker chased into the late endosomal compartments. In contrast, LysoTracker showed only a minor overlap with the early endosomal marker rab5. Pathogenic mycobacteria are believed to reside in nonacidified vacuoles sequestered away from late endosomal compartments as a part of their intracellular survival strategy. We examined the status of mycobacterial phagosomes in macrophages from IL-10 knockout mice, in quiescent cells, and in mononuclear phagocytes stimulated with the macrophage-activating cytokine IFN-(gamma). When macrophages were derived from the bone marrow of transgenic IL-10 mice lacking this major deactivating cytokine, colocalization of GFP-fluorescing mycobacteria with the LysoTracker staining appeared enhanced, suggestive of increased acidification of the mycobacterial phagosome relative to macrophages from normal mice. When bone marrow-derived macrophages from normal mice or a J774 murine macrophage cell line were stimulated with IFN-(gamma) and LPS, this resulted in increased colocalization of mycobacteria and LysoTracker, but no statistically significant enhancement was observed in IL-10 transgenic animals. These studies are consistent with the interpretation that proinflammatory and anti-inflammatory cytokines affect maturation of mycobacterial phagosomes. Although multiple mechanisms are likely to be at work, we propose the existence of a direct link between cytokine effects on the host cell and phagosome maturation in the macrophage.

Important role for the V-type H+-ATPase and the Golgi apparatus in the recycling of PTH/PTHrP receptor

2004 ◽  
Vol 286 (5) ◽  
pp. E704-E710 ◽  
Author(s):  
Hesham A. W. Tawfeek ◽  
Abdul B. Abou-Samra
Keyword(s):  
Golgi Apparatus ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Receptor Recycling ◽  
Bafilomycin A1 ◽  
Coated Pits ◽  
Concanamycin A ◽  
Hydrogen Atpase ◽  
Green Fluorescent ◽  
Pthrp Receptor

Our previous studies demonstrated that a green fluorescent protein-tagged parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor stably expressed in LLCPK-1 cells undergoes agonist-dependent internalization into clathrin-coated pits. The subcellular localization of the internalized PTH/PTHrP receptor is not known. In the present study, we explored the intracellular pathways of the internalized PTH/PTHrP receptor. Using immunofluorescence and confocal microscopy, we show that the internalized receptors localize at a juxtanuclear compartment identified as the Golgi apparatus. The receptors do not colocalize with lysosomes. Furthermore, whereas the internalized receptors exhibit rapid recycling, treatment with proton pump inhibitors (bafilomycin-A1 and concanamycin A) or brefeldin A, Golgi disrupting agents, reduces PTH/PTHrP receptor recycling. Together, these data indicate an important role for the vacuolar-type hydrogen-ATPase and the Golgi apparatus in postendocytic PTH/PTHrP receptor recovery.

Mammalian cell lines expressing functional RNA polymerase II tagged with the green fluorescent protein

2000 ◽  
Vol 113 (15) ◽  
pp. 2679-2683 ◽  
Author(s):  
K. Sugaya ◽  
M. Vigneron ◽  
P.R. Cook
Keyword(s):  
Green Fluorescent Protein ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Temperature Sensitive ◽  
Mammalian Cell Lines ◽  
Eukaryotic Genes ◽  
Green Fluorescent

RNA polymerase II is a multi-subunit enzyme responsible for transcription of most eukaryotic genes. It associates with other complexes to form enormous multifunctional ‘holoenzymes’ involved in splicing and polyadenylation. We wished to study these different complexes in living cells, so we generated cell lines expressing the largest, catalytic, subunit of the polymerase tagged with the green fluorescent protein. The tagged enzyme complements a deficiency in tsTM4 cells that have a temperature-sensitive mutation in the largest subunit. Some of the tagged subunit is incorporated into engaged transcription complexes like the wild-type protein; it both resists extraction with sarkosyl and is hyperphosphorylated at its C terminus. Remarkably, subunits bearing such a tag can be incorporated into the active enzyme, despite the size and complexity of the polymerizing complex. Therefore, these cells should prove useful in the analysis of the dynamics of transcription in living cells.

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