scholarly journals alpha 2-macroglobulin adsorbed to colloidal gold: a new probe in the study of receptor-mediated endocytosis.

1981 ◽  
Vol 89 (1) ◽  
pp. 29-34 ◽  
Author(s):  
R B Dickson ◽  
M C Willingham ◽  
I Pastan
Keyword(s):  
Electron Microscope ◽  
Cell Surface ◽  
Vesicular Stomatitis Virus ◽  
Golgi Complex ◽  
Colloidal Gold ◽  
Microscope Level ◽  
Coated Pits ◽  
Electron Microscope Level ◽  
Receptor Mediated Endocytosis ◽  
Vesicular Stomatitis

alpha 2-Macroglobulin (alpha 2 M) was adsorbed to colloidal gold and used as a new tool in the study of receptor-mediated endocytosis. alpha 2 M-gold is easy to prepare and is clearly visualized at the electron microscope level. When cells were incubated with alpha 2 M-gold at 0 degrees C, gold was visualized both diffusely over the cell surface and concentrated in coated pits. After cells to which alpha 2 M-gold had been bound at 0 degrees C were warmed, the gold was rapidly internalized into uncoated vesicles, previously termed receptosomes. After 30 min of incubation or longer, gold was found in small lysosomes and, later, in large lysosomes and very small vesicles in the region of the Golgi complex. This pattern of localization is similar to that previously described, using peroxidase-labeled anti-alpha 2 M antibodies. By incubating cells with both alpha 2 M-gold and vesicular stomatitis virus (VSV), we studied the internalization of these two markers simultaneously. VSV and alpha 2 M-gold rapidly clustered in the same coated pits and were internalized in the same receptosomes. Proteins and hormones adsorbed to gold may be useful in the study of receptor-mediated endocytosis.

scholarly journals Intracellular transport of transferrin- and asialoorosomucoid-colloidal gold conjugates to lysosomes after receptor-mediated endocytosis.

1985 ◽  
Vol 33 (11) ◽  
pp. 1134-1144 ◽  
Author(s):  
M R Neutra ◽  
A Ciechanover ◽  
L S Owen ◽  
H F Lodish
Keyword(s):  
Acid Phosphatase ◽  
Cell Surface ◽  
Colloidal Gold ◽  
Intracellular Transport ◽  
Ruthenium Red ◽  
Endocytic Pathway ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Gold Probe ◽  
20 Nm

Proteins coupled to colloidal gold particles have been widely used to visualize the uptake and intracellular transport of specific ligands by receptor-mediated endocytosis. The intracellular route of lysosome-directed ligands such as asialoglycoproteins (ASGP) are apparently unaltered by conjugation to gold, but the pathway of transferrin, a ligand that normally recycles to the cell surface, was reported to be altered by conjugation to 15-20 nm gold. In this study, we sought to determine whether a smaller transferrin-gold probe would recycle, and whether it might enter the same endosomal and lysosomal compartments as does a larger, lysosome-directed ASGP gold probe by visualizing their simultaneous uptake in human hepatoma (HepG2) cells. In the same cells, endocytosis of fluid-phase protein was followed using the soluble tracer native ferritin; lysosomal compartments were identified by acid phosphatase cytochemistry; and cell surfaces were labeled with ruthenium red or cationized ferritin. During the first 10 min of uptake at 37 degrees C, specific receptor-bound ferrotransferrin (FeTf)-8 nm gold and asialoorosomucoid (ASOR)-20 nm gold were clustered together in coated pits and entered the same coated vesicles, smooth vesicles, and tubules in the peripheral cytoplasm. At later times, however, transferrin-gold did not return to the cell surface; unlike native transferrin, this gold probe accompanied ASOR-gold into multivesicular bodies (MVB). The MVBs that contained probes were at first devoid of acid phosphatase activity, but at 30 min, enzyme activity was detected in a few MVBs. Native ferritin was present, along with gold probes, in all compartments of the endocytic pathway. We conclude that the normal intracellular pathway of transferrin is altered by its association with a colloidal gold particle.

scholarly journals Kinesin-5/Eg5 is important for transport of CARTS from the trans-Golgi network to the cell surface

2013 ◽  
Vol 202 (2) ◽  
pp. 241-250 ◽  
Author(s):  
Yuichi Wakana ◽  
Julien Villeneuve ◽  
Josse van Galen ◽  
David Cruz-Garcia ◽  
Mitsuo Tagaya ◽  
...  
Keyword(s):  
Cell Surface ◽  
Vesicular Stomatitis Virus ◽  
Golgi Complex ◽  
Protein Transport ◽  
S2 Cells ◽  
Trans Golgi Network ◽  
Drosophila S2 Cells ◽  
Vesicular Stomatitis ◽  
Golgi Network

Here we report that the kinesin-5 motor Klp61F, which is known for its role in bipolar spindle formation in mitosis, is required for protein transport from the Golgi complex to the cell surface in Drosophila S2 cells. Disrupting the function of its mammalian orthologue, Eg5, in HeLa cells inhibited secretion of a protein called pancreatic adenocarcinoma up-regulated factor (PAUF) but, surprisingly, not the trafficking of vesicular stomatitis virus G protein (VSV-G) to the cell surface. We have previously reported that PAUF is transported from the trans-Golgi network (TGN) to the cell surface in specific carriers called CARTS that exclude VSV-G. Inhibition of Eg5 function did not affect the biogenesis of CARTS; however, their migration was delayed and they accumulated near the Golgi complex. Altogether, our findings reveal a surprising new role of Eg5 in nonmitotic cells in the facilitation of the transport of specific carriers, CARTS, from the TGN to the cell surface.

scholarly journals Three distinct steps in transport of vesicular stomatitis virus glycoprotein from the ER to the cell surface in vivo with differential sensitivities to GTP gamma S

1998 ◽  
Vol 111 (13) ◽  
pp. 1877-1888 ◽  
Author(s):  
R. Pepperkok ◽  
M. Lowe ◽  
B. Burke ◽  
T.E. Kreis
Keyword(s):  
Cell Surface ◽  
Vesicular Stomatitis Virus ◽  
Golgi Complex ◽  
Mammalian Cells ◽  
Coat Proteins ◽  
Gtp Hydrolysis ◽  
Dominant Mutant ◽  
Vesicular Stomatitis ◽  
Gamma S

Microinjected GTP gamma S revealed three distinct steps in the exocytic transport of the temperature sensitive glycoprotein of vesicular stomatitis virus (ts-O45-G) from the ER to the cell surface in intact Vero cells. While COPII dependent export of ts-O45-G from the ER is blocked in cells injected with recombinant protein of a dominant mutant of SAR1a (SAR1a[H79G]) inhibited in GTP hydrolysis, neither injected GTP gamma S nor antibodies against beta-COP (anti-EAGE) interfere with this transport step significantly. In contrast, transport to the Golgi complex is blocked by 50 microM GTP gamma S, a dominant mutant of ARF1 (ARF1[Q71L]) inhibited in GTP hydrolysis, or microinjected anti-EAGE, but injected Sar1a[H79G]p has no effect. Microinjection of GTP gamma S or expression of ARF[Q71L] rapidly induces accumulation of COPI coated vesicular structures lacking ts-O45-G. Finally, transport of ts-O45-G from the trans-Golgi network (TGN) to the cell surface is inhibited only by high concentrations of GTP gamma S (500 microM). Interestingly, this step is only partially brefeldin A sensitive, and injected antibodies against beta-COP and p200/myosin II, a TGN membrane associated protein, have no effect. These data provide first strong in vivo evidence for at least three distinct steps in the exocytic pathway of mammalian cells regulated by different sets of GTPases and coat proteins. COPII, but not COPI, is required for ER export of ts-O45-G. COPI plays a role in subsequent transport to the Golgi complex, and a so far unidentified GTP gamma S sensitive coat appears to be involved in transport from the TGN to the cell surface.

scholarly journals Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis

2005 ◽  
Vol 169 (2) ◽  
pp. 285-295 ◽  
Author(s):  
Daniela A. Sahlender ◽  
Rhys C. Roberts ◽  
Susan D. Arden ◽  
Giulietta Spudich ◽  
Marcus J. Taylor ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rna Interference ◽  
Vesicular Stomatitis Virus ◽  
Protein Interaction ◽  
Golgi Complex ◽  
Myosin Vi ◽  
Binding Partner ◽  
Binding Partners ◽  
Vesicular Stomatitis ◽  
Golgi Organization

Myosin VI plays a role in the maintenance of Golgi morphology and in exocytosis. In a yeast 2-hybrid screen we identified optineurin as a binding partner for myosin VI at the Golgi complex and confirmed this interaction in a range of protein interaction studies. Both proteins colocalize at the Golgi complex and in vesicles at the plasma membrane. When optineurin is depleted from cells using RNA interference, myosin VI is lost from the Golgi complex, the Golgi is fragmented and exocytosis of vesicular stomatitis virus G-protein to the plasma membrane is dramatically reduced. Two further binding partners for optineurin have been identified: huntingtin and Rab8. We show that myosin VI and Rab8 colocalize around the Golgi complex and in vesicles at the plasma membrane and overexpression of constitutively active Rab8-Q67L recruits myosin VI onto Rab8-positive structures. These results show that optineurin links myosin VI to the Golgi complex and plays a central role in Golgi ribbon formation and exocytosis.

scholarly journals Receptor-mediated endocytosis of alpha 2-macroglobulin in cultured fibroblasts.

1980 ◽  
Vol 28 (8) ◽  
pp. 818-823 ◽  
Author(s):  
M C Willingham ◽  
F R Maxfield ◽  
I Pastan
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Coated Pits ◽  
Cultured Fibroblasts ◽  
Receptor Mediated Endocytosis ◽  
Cytoplasmic Vesicles

Alpha 2-macroglobulin is internalized into cultured fibroblasts by receptor-mediated endocytosis. This ligand binds initially to diffusely distributed receptors on the cell surface which cluster rapidly into bristle-coated pits. Within a few minutes at 37 degrees C, these complexes are internalized into uncoated cytoplasmic vesicles, called receptosomes, which move about in the cell by saltatory motion. These vesicles interact with the Golgi-endoplasmic reticulum-lysosome system in the cell to deliver the ligand to newly formed lysosomes within 30--60 min.

Intracellular transport of the glycoprotein of VSV is inhibited by CCCP at a late stage of post-translational processing

1989 ◽  
Vol 92 (4) ◽  
pp. 633-642
Author(s):  
J.K. Burkhardt ◽  
Y. Argon
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Late Stage ◽  
Intracellular Transport ◽  
Post Translational Modifications ◽  
Glycoprotein G ◽  
Golgi Compartment ◽  
Vesicular Stomatitis

The appearance of newly synthesized glycoprotein (G) of vesicular stomatitis virus at the surface of infected BHK cells is inhibited reversibly by treatment with carbonylcyanide m-chlorophenylhydrazone (CCCP). Under the conditions used, CCCP treatment depleted the cellular ATP levels by 40–60%, consistent with inhibition of transport at energy-requiring stages. The G protein that accumulates in cells treated with CCCP is heterogeneous. Most of it is larger than the newly synthesized G protein, is acylated with palmitic acid, and is resistant to endoglycosidase H (Endo H). Most of the arrested G protein is also sensitive to digestion with neuraminidase, indicating that it has undergone at least partial sialylation. A minority of G protein accumulates under these conditions in a less-mature form, suggesting its inability to reach the mid-Golgi compartment. The oligosaccharides of this G protein are Endo-H-sensitive and seem to be partly trimmed. Whereas sialylated G protein was arrested intracellularly, fucose-labelled G protein was able to complete its transport to the cell surface, indicating that a late CCCP-sensitive step separates sialylation from fucosylation. These post-translational modifications indicate that G protein can be transported as far as the trans-Golgi in the presence of CCCP and is not merely arrested in the endoplasmic reticulum.

Sign in / Sign up

Export Citation Format

Share Document