scholarly journals Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase.

1994 ◽  
Vol 127 (3) ◽  
pp. 641-652 ◽  
Author(s):  
C N Connolly ◽  
C E Futter ◽  
A Gibson ◽  
C R Hopkins ◽  
D F Cutler
Keyword(s):  
Golgi Complex ◽  
Secretory Pathway ◽  
High Sensitivity ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Golgi Stack ◽  
Er Retention ◽  
Peroxidase Cytochemistry ◽  
Golgi Network

We have developed a novel technique with which to investigate the morphological basis of exocytotic traffic. We have used expression of HRP from cDNA in a variety of cells in combination with peroxidase cytochemistry to outline traffic into and out of the Golgi apparatus at the electron microscopic level with very high sensitivity. A secretory form of the peroxidase (ssHRP) is active from the beginning of the secretory pathway and the activity is efficiently cleared from cells. Investigation of the morphological elements involved in the itinerary of soluble ER proteins using ssHRP tagged with the ER retention motif (ssHRPKDEL) shows that it progresses through the Golgi stack no further than the cis-most element. Traffic between the RER and the Golgi stack as outlined by ssHRPKDEL occurs via vesicular carriers as well as by tubular elements. ssHRP has also been used to investigate the trans side of the Golgi complex, where incubation at reduced temperatures outlines the trans-Golgi network with HRP reaction product. Tracing the endosomal compartment with transferrin receptor in double-labeling experiments with ssHRP fails to show any overlap between these two compartments.

Retrograde trafficking of both Golgi complex and TGN markers to the ER induced by nordihydroguaiaretic acid and cyclofenil diphenol

1998 ◽  
Vol 111 (7) ◽  
pp. 951-965 ◽  
Author(s):  
D. Drecktrah ◽  
P. de Figueiredo ◽  
R.M. Mason ◽  
W.J. Brown
Keyword(s):  
Golgi Complex ◽  
Membrane Trafficking ◽  
Molecular Mechanisms ◽  
Nordihydroguaiaretic Acid ◽  
Retrograde Transport ◽  
Lipoxygenase Inhibitor ◽  
Golgi Stack ◽  
Golgi Network ◽  
In Vivo Effects

Previous studies have shown that the Golgi stack and the trans-Golgi network (TGN) may play a role in capturing escaped resident endoplasmic reticulum (ER) proteins, and directing their retrograde transport back to that organelle. Whether this retrograde movement represents a highly specific or more generalized membrane trafficking pathway is unclear. To better understand both the retrograde and anterograde trafficking pathways of the secretory apparatus, we examined more closely the in vivo effects of two structurally unrelated compounds, the potent lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), and the non-steroidal estrogen cyclofenil diphenol (CFD), both of which are known to inhibit secretion. In the presence of these compounds, transport of vesicular stomatitis virus G membrane glycoprotein from the ER to the Golgi complex, and from the TGN to the cell surface, was inhibited potently and rapidly. Surprisingly, we found that NDGA and CFD stimulated the rapid, but not concomitant, retrograde movement of both Golgi stack and TGN membrane proteins back to the ER until both organelles were morphologically absent from cells. Both NDGA- and CFD-stimulated TGN and Golgi retrograde membrane trafficking were inhibited by microtubule depolymerizing agents and energy poisons. Removal of NDGA and CFD resulted in the complete, but not concomitant, reformation of both Golgi stacks and their closely associated TGN compartments. These studies suggest that NDGA and CFD unmask a generalized bulk recycling pathway to the ER for both Golgi and TGN membranes and, further, that NDGA and CFD are useful for investigating the molecular mechanisms that control the formation and maintenance of both the Golgi stack proper and the TGN.

scholarly journals The dynamic nature of the Golgi complex.

1989 ◽  
Vol 108 (2) ◽  
pp. 277-297 ◽  
Author(s):  
G Griffiths ◽  
S D Fuller ◽  
R Back ◽  
M Hollinshead ◽  
S Pfeiffer ◽  
...  
Keyword(s):  
Surface Area ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Semliki Forest Virus ◽  
Total Surface Area ◽  
Morphological Evidence ◽  
Golgi Stack ◽  
Golgi Compartment ◽  
Vesicular Stomatitis ◽  
Golgi Network

The intracellular transport of newly synthesized G protein of vesicular stomatitis virus is blocked at 20 degrees C and this spanning membrane glycoprotein accumulates in the last Golgi compartment, the trans Golgi-network (TGN). Previous morphological evidence suggested that the TGN enlarged significantly under this condition. In the present study we have used stereological procedures to estimate the volume and surface area of the Golgi stack and the TGN of baby hamster kidney cells under different conditions. The results indicate that the increase in the size of the TGN at 20 degrees C is accompanied by a significant decrease in the surface area and volume of the preceding Golgi compartments. A similar effect is also seen in uninfected cells at 20 degrees C, as well as during normal (37 degrees C) infection with Semliki Forest virus. In the latter case, however, the decrease in the size of the Golgi stack and the increase in that of the TGN is not accompanied by inhibition of transport from the Golgi complex to the cell surface. The results indicate that the Golgi stack and the TGN are dynamic and interrelated structures that are capable of rapid alteration in total surface area in response to changes in the rates of membrane transport.

scholarly journals ER to Golgi Transport

1999 ◽  
Vol 147 (6) ◽  
pp. 1205-1222 ◽  
Author(s):  
Cecilia Alvarez ◽  
Hideaki Fujita ◽  
Ann Hubbard ◽  
Elizabeth Sztul
Keyword(s):  
G Protein ◽  
Golgi Complex ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Intact Cell ◽  
Cell Transport ◽  
Golgi Stack ◽  
Golgi Transport ◽  
Transport Assay ◽  
Golgi Structure

The membrane transport factor p115 functions in the secretory pathway of mammalian cells. Using biochemical and morphological approaches, we show that p115 participates in the assembly and maintenance of normal Golgi structure and is required for ER to Golgi traffic at a pre-Golgi stage. Injection of antibodies against p115 into intact WIF-B cells caused Golgi disruption and inhibited Golgi complex reassembly after BFA treatment and wash-out. Addition of anti–p115 antibodies or depletion of p115 from a VSVtsO45 based semi-intact cell transport assay inhibited transport. The inhibition occurred after VSV glycoprotein (VSV-G) exit from the ER but before its delivery to the Golgi complex, and resulted in VSV-G protein accumulating in peripheral vesicular tubular clusters (VTCs). The p115-requiring step of transport followed the rab1-requiring step and preceded the Ca2+-requiring step. Unexpectedly, mannosidase I redistributed from the Golgi complex to colocalize with VSV-G protein arrested in pre-Golgi VTCs by p115 depletion. Redistribution of mannosidase I was also observed in cells incubated at 15°C. Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack. This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.

scholarly journals Electron microscopic immunocytochemical evidence for the involvement of the convertases PC1 and PC2 in the processing of proinsulin in pancreatic beta-cells.

1995 ◽  
Vol 43 (1) ◽  
pp. 11-19 ◽  
Author(s):  
D Malide ◽  
N G Seidah ◽  
M Chrétien ◽  
M Bendayan
Keyword(s):  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Protein A ◽  
Gold Complex ◽  
Immunocytochemical Study ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Endoproteolytic Cleavage

Endoproteolytic cleavage of pairs of basic amino acids is the key mechanism in the specific processing of precursor hormone molecules. Two endoproteases, PC1 (or PC3) and PC2, have recently been implicated in the conversion of proinsulin. Using antibodies against these proteases and proinsulin, followed by protein A-gold complex, we performed an immunocytochemical study for precise identification of the subcellular compartments involved in the processing of insulin. Both PC1 and PC2 immunoreactivities followed a pattern of gradually increasing density along the secretory pathway, being higher in the immature granules. Proinsulin labeling was detected in the Golgi apparatus and in the coated immature secretory granules located mainly in the Golgi area. Using double labeling, we demonstrated the presence of PC1 and/or PC2 in the majority of proinsulin-rich granules. In addition, we provided evidence that PC1 and PC2 are co-localized within the same granules. Co-expression of PC1 and PC2 with proinsulin in islet beta-cells indicates that these proteases are actively involved, probably in a sequential manner, in the conversion of proinsulin into insulin.

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.

scholarly journals Estimation of the amount of internalized ricin that reaches the trans-Golgi network

1988 ◽  
Vol 106 (2) ◽  
pp. 253-267 ◽  
Author(s):  
B van Deurs ◽  
K Sandvig ◽  
OW Petersen ◽  
S Olsnes ◽  
K Simons ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Golgi Complex ◽  
Endocytic Pathway ◽  
Membrane Glycoproteins ◽  
Double Labeling ◽  
Trans Golgi Network ◽  
A Chain ◽  
Golgi Network ◽  
Ricin A

We have used a protocol for internalization of ricin, a ligand binding to plasma membrane glycoproteins and glycolipids with terminal galactosyl residues, and infection with the vesicular stomatitis virus ts 045 mutant in BHK-21 cells to determine whether internalized plasma membrane molecules tagged by ricin reach distinct compartments of the biosynthetic-exocytic pathway. At 39.5 degrees C newly synthesized G protein of ts 045 was largely prevented from leaving the endoplasmic reticulum. At the same temperature ricin was endocytosed and reached, in addition to endosomes and lysosomes, elements of the Golgi complex. When the temperature was lowered to 19.5 degrees C, no more ricin was delivered to the Golgi complex, but now G protein accumulated in the Golgi stacks and the trans-Golgi network (TGN). Double-labeling immunogold cytochemistry on ultracryosections was used to detect G protein and ricin simultaneously. These data, combined with stereological and biochemical methods, showed that approximately 5% of the total amount of ricin within the cells, corresponding to 6-8 X 10(4) molecules per cell, colocalized with G protein in the Golgi complex after 60 min at 39.5 degrees C. Of this amount approximately 70-80% was present in the TGN. Since most of the ricin molecules remain bound to their binding sites at the low pH prevailing in compartments of the endocytic pathway, the results indicate that a fraction of the internalized plasma membrane molecules with terminal galactose are not recycled directly from endosomes or delivered to lysosomes, but are routed to the Golgi complex. Also, the results presented here, in combination with other recent studies on ricin internalization, suggest that translocation of the toxic ricin A-chain to the cytosol occurs in the TGN.

scholarly journals Inter- and intracellular relationship of substance P-containing neurons with serotonin and GABA in the dorsal raphe nucleus: combination of autoradiographic and immunocytochemical techniques.

1986 ◽  
Vol 34 (6) ◽  
pp. 735-742 ◽  
Author(s):  
R Mâgoul ◽  
B Onteniente ◽  
A Oblin ◽  
A Calas
Keyword(s):  
Substance P ◽  
Dorsal Raphe Nucleus ◽  
Dorsal Raphe ◽  
Raphe Nucleus ◽  
Electron Microscopic Level ◽  
Gamma Aminobutyric Acid ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Relationship Of ◽  
Dorsal Raphé

Double-labeling experiments were performed at the electron microscopic level in the dorsal raphe nucleus of rat, in order to study the inter- and intracellular relationship of substance P with gamma-aminobutyric acid (GABA) and serotonin. Autoradiography for either [3H]serotonin or [3H]GABA was coupled, on the same tissue section, with peroxidase-antiperoxidase immunocytochemistry for substance P in colchicine-treated animals. Intercellular relationships were represented by synaptic contacts made by [3H]serotonin-labeled terminals on substance P-containing somata and dendrites, and by substance P-containing terminals on [3H]GABA-labeled cells. Intracellular relationships were suggested by the occurrence of the peptide within [3H]serotonin-containing and [3H]GABA-containing cell bodies and fibers. Doubly labeled varicosities of the two kinds were also observed in the supraependymal plexus adjacent to the dorsal raphe nucleus. The results demonstrated that, in addition to reciprocal synaptic interactions made by substance P with serotonin and GABA, the dorsal raphe nucleus is the site of intracellular relationships between the peptide and either the amine or the amino acid.

scholarly journals Exclusion of Golgi Residents from Transport Vesicles Budding from Golgi Cisternae in Intact Cells

2000 ◽  
Vol 150 (6) ◽  
pp. 1263-1270 ◽  
Author(s):  
Lelio Orci ◽  
Mylène Amherdt ◽  
Mariella Ravazzola ◽  
Alain Perrelet ◽  
James E. Rothman
Keyword(s):  
Steady State ◽  
Protein Transport ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Central Feature ◽  
Electron Microscopic ◽  
Anterograde Transport ◽  
Intact Cells ◽  
Golgi Stack ◽  
Transport Vesicles

A central feature of cisternal progression/maturation models for anterograde transport across the Golgi stack is the requirement that the entire population of steady-state residents of this organelle be continuously transported backward to earlier cisternae to avoid loss of these residents as the membrane of the oldest (trans-most) cisterna departs the stack. For this to occur, resident proteins must be packaged into retrograde-directed transport vesicles, and to occur at the rate of anterograde transport, resident proteins must be present in vesicles at a higher concentration than in cisternal membranes. We have tested this prediction by localizing two steady-state residents of medial Golgi cisternae (mannosidase II and N-acetylglucosaminyl transferase I) at the electron microscopic level in intact cells. In both cases, these abundant cisternal constituents were strongly excluded from buds and vesicles. This result suggests that cisternal progression takes place substantially more slowly than most protein transport and therefore is unlikely to be the predominant mechanism of anterograde movement.

scholarly journals Membrane-cytoskeleton dynamics in rat parietal cells: mobilization of actin and spectrin upon stimulation of gastric acid secretion.

1989 ◽  
Vol 108 (2) ◽  
pp. 441-453 ◽  
Author(s):  
F Mercier ◽  
H Reggio ◽  
G Devilliers ◽  
D Bataille ◽  
P Mangeat
Keyword(s):  
Gastric Acid ◽  
Dynamic Equilibrium ◽  
Gastric Gland ◽  
Cytoskeletal Proteins ◽  
Parietal Cells ◽  
Electron Microscopic Level ◽  
Apical Surface ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Acid Secreting

The gastric parietal (oxyntic) cell is presented as a model for studying the dynamic assembly of the skeletal infrastructure of cell membranes. A monoclonal antibody directed to a 95-kD antigen of acid-secreting membranes of rat parietal cells was characterized as a tracer of the membrane movement occurring under physiological stimuli. The membrane rearrangement was followed by immunocytochemistry both at the light and electron microscopic level on semithin and thin frozen sections from resting and stimulated rat gastric mucosa. Double labeling experiments demonstrated that a specific and massive mobilization of actin, and to a lesser extent of spectrin (fodrin), was involved in this process. In the resting state, actin and spectrin were mostly localized beneath the membranes of all cells of the gastric gland, whereas the bulk of acid-secreting membranes appeared diffusely distributed in the cytoplasmic space of parietal cells without any apparent connection with cytoskeletal proteins. In stimulated cells, both acid-secreting material and actin (or spectrin) extensively colocalized at the secretory apical surface of parietal cells, reflecting that acid-secreting membranes were now exposed at the lumen of the secretory canaliculus and that this insertion was stabilized by cortical proteins. The data are compatible with a model depicting the membrane movement occurring in parietal cells as an apically oriented insertion of activated secretory membranes from an intracellular storage pool. The observed redistribution of actin and spectrin argues for a direct control by gastric acid secretagogues of the dynamic equilibrium existing between nonassembled (or preassembled) and assembled forms of cytoskeletal proteins.

scholarly journals Enhanced sensitivity for light and electron microscopic in situ hybridization with multiple simultaneous non-radioactive oligodeoxynucleotide probes.

1995 ◽  
Vol 43 (8) ◽  
pp. 829-841 ◽  
Author(s):  
A Trembleau ◽  
F E Bloom
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent Dyes ◽  
General Procedure ◽  
Simultaneous Detection ◽  
High Sensitivity ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Enhanced Sensitivity ◽  
Low Sensitivity

Although oligonucleotide probes are useful for in situ hybridization, their low sensitivity compared to riboprobes and cDNA remains a problem. We have systematically examined the protocols to provide a general procedure that increases the sensitivity of oligoprobes for light and electron in situ hybridizations by using mixtures of multiple non-overlapping oligonucleotides (multi-oligoprobes). The protocol achieves these improvements with both radioactive and non-radioactive oligoprobes. With 33P-labeled probes in a semiquantitative assay, we found that mixtures of up to six vasopressin-directed multi-oligoprobes, each employed at saturating concentration, led to an additive signal with no significant increase of the background. Using this approach with non-radioactive oligoprobes, we were able to detect in the hypothalamus several low or moderately abundant mRNAs, such as vasopressin heterogeneous nuclear RNA and the galanin, dynorphin, and tyrosine hydroxylase mRNAs. Moreover, we showed that multi-oligoprobes used in a pre-embedding procedure were suitable for studying the ultrastructural compartmentalization of moderately abundant mRNAs. Finally, with the same basic approach we demonstrated that two sets of multi-oligoprobes can be combined for simultaneous detection of two different mRNAs using fluorescent dyes, making this approach suitable for high-resolution confocal analyses. Overall, our data demonstrate that multi-oligoprobes provide a sensitive tool of choice for various applications in which both well-preserved morphology and high sensitivity are needed. In particular, these probes appear ideal for study of the comparative subcellular localization of mRNAs at both the light and the electron microscopic level.

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