scholarly journals High-resolution mapping reveals topologically distinct cellular pools of phosphatidylserine

2011 ◽  
Vol 194 (2) ◽  
pp. 257-275 ◽  
Author(s):  
Gregory D. Fairn ◽  
Nicole L. Schieber ◽  
Nicholas Ariotti ◽  
Samantha Murphy ◽  
Lars Kuerschner ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Light And Electron Microscopy ◽  
Transmembrane Topology ◽  
Trans Golgi Network ◽  
High Resolution Mapping ◽  
Section Technique ◽  
Resolution Mapping ◽  
Golgi Network ◽  
Multivesicular Endosomes ◽  
Labeling Pattern

Phosphatidylserine (PS) plays a central role in cell signaling and in the biosynthesis of other lipids. To date, however, the subcellular distribution and transmembrane topology of this crucial phospholipid remain ill-defined. We transfected cells with a GFP-tagged C2 domain of lactadherin to detect by light and electron microscopy PS exposed on the cytosolic leaflet of the plasmalemma and organellar membranes. Cytoplasmically exposed PS was found to be clustered on the plasma membrane, and to be associated with caveolae, the trans-Golgi network, and endocytic organelles including intraluminal vesicles of multivesicular endosomes. This labeling pattern was compared with the total cellular distribution of PS as visualized using a novel on-section technique. These complementary methods revealed PS in the interior of the ER, Golgi complex, and mitochondria. These results indicate that PS in the lumenal monolayer of the ER and Golgi complex becomes exposed cytosolically at the trans-Golgi network. Transmembrane flipping of PS may contribute to the exit of cargo from the Golgi complex.

scholarly journals Oligomerization of atrans-Golgi/trans-Golgi Network Retained Protein Occurs in the Golgi Complex and May Be Part of Its Retention

1995 ◽  
Vol 270 (15) ◽  
pp. 8815-8821 ◽  
Author(s):  
Jacomine Krijnse Locker ◽  
Dirk-Jan E. Opstelten ◽  
Maria Ericsson ◽  
Marian C. Horzinek ◽  
Peter J. M. Rottier
Keyword(s):  
Golgi Complex ◽  
Trans Golgi Network ◽  
Golgi Network

Nucleotide depletion increases trafficking of gentamicin to the Golgi complex in LLC-PK1 cells

2002 ◽  
Vol 283 (6) ◽  
pp. F1422-F1429 ◽  
Author(s):  
Ruben M. Sandoval ◽  
Robert L. Bacallao ◽  
Kenneth W. Dunn ◽  
Jeffrey D. Leiser ◽  
Bruce A. Molitoris
Keyword(s):  
Cell Culture ◽  
Golgi Complex ◽  
Lens Culinaris ◽  
Ricinus Communis ◽  
Physiological State ◽  
Complex Mechanism ◽  
Trans Golgi Network ◽  
Golgi Network ◽  
Texas Red ◽  
Number Of Cells

Having shown rapid trafficking of aminoglycosides to the Golgi complex in cell culture, we focused on the injurious interaction that occurs when gentamicin administration is preceded by renal ischemia. Using Texas red-labeled gentamicin as a tracer, we determined that 15 min of cellular nucleotide depletion did not significantly increase subsequent uptake. However, cells previously depleted of nucleotides accumulated significantly more Texas red-labeled gentamicin within a dispersed Golgi complex. Using Ricinus communis and Lens culinaris lectins, which label specific compartments of the Golgi complex ( trans-Golgi network/ trans and medial/ cis compartments, respectively), we determined that the medial/ cis compartment dispersed after 15 min of nucleotide depletion but the trans-Golgi network/ trans compartment remained unaffected. An increase in the number of cells exhibiting disrupted medial/ cis-Golgi morphology after repletion in physiological media containing gentamicin was also seen. In summary, the increase in nephrotoxicity seen when ischemia precedes aminoglycoside uptake may be part of a complex mechanism initially involving increased Golgi accumulation and prolonged Golgi dispersion. The Golgi complex must then endure the effects of gentamicin accumulated in larger quantities in an aberrant physiological state.

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 Immunocytochemical Localization of the Menkes Copper Transport Protein (ATP7A) to the Trans-Golgi Network

1997 ◽  
Vol 6 (3) ◽  
pp. 409-416 ◽  
Author(s):  
Herman A. Dierick ◽  
Ayla N. Adam ◽  
June F. Escara-Wilke ◽  
Thomas W. Glover
Keyword(s):  
Golgi Complex ◽  
Cho Cells ◽  
Cultured Cells ◽  
Human Fibroblasts ◽  
Western Blots ◽  
Fusion Construct ◽  
Copper Trafficking ◽  
Trans Golgi Network ◽  
Amino Terminal ◽  
Golgi Network

Abstract We have generated polyclonal antibodies against the amino-terminal third of the Menkes protein (ATP7A; MNK) by immunizing rabbits with a histidine-tagged MNK fusion construct containing metal-binding domains 1–4. The purified antibodies were used in Western analysis of cell lysates and in indirect immunofluorescence experiments on cultured cells. On Western blots, the antibodies recognized the ∼165 kDa MNK protein in CHO cells and human fibroblasts. No MNK signal could be detected in fibroblasts from a patient with Menkes disease or in Hep3B hepatocellular carcinoma cells, confirming the specificity of the antibodies. Immunocytochemical analysis of CHO cells and human fibroblasts showed a distinct perinuclear signal corresponding to the pattern of the Golgi complex. This staining pattern was similar to that of α-mannosidase II which is a known resident enzyme of the Golgi complex. Using brefeldin A, a fungal inhibitor of protein secretion, we further demonstrated that the MNK protein is localized to the trans-Golgi network. This data provides direct evidence for a subcellular localization of the MNK protein which is similar to the proposed vacuolar localization of Ccc2p, the yeast homolog of MNK and WND (ATP7B), the Wilson disease gene product. In light of the proposed role of MNK both in subcellular copper trafficking and in copper efflux, these data suggest a model for how these two processes are linked and represent an important step in the functional analysis of the MNK protein.

GLUT4 in cultured skeletal myotubes is segregated from the transferrin receptor and stored in vesicles associated with TGN

1996 ◽  
Vol 109 (13) ◽  
pp. 2967-2978 ◽  
Author(s):  
E. Ralston ◽  
T. Ploug
Keyword(s):  
Immunoelectron Microscopy ◽  
Golgi Complex ◽  
Transferrin Receptor ◽  
Glucose Transporter ◽  
Brefeldin A ◽  
Immunogold Electron Microscopy ◽  
Trans Golgi Network ◽  
Storage Vesicles ◽  
Skeletal Myotubes ◽  
Golgi Network

There is little consensus on the nature of the storage compartment of the glucose transporter GLUT4, in non-stimulated cells of muscle and fat. More specifically, it is not known whether GLUT4 is localized to unique, specialized intracellular storage vesicles, or to vesicles that are part of the constitutive endosomal-lysosomal pathway. To address this question, we have investigated the localization of the endogenous GLUT4 in non-stimulated skeletal myotubes from the cell line C2, by immunofluorescence and immunoelectron microscopy. We have used a panel of antibodies to markers of the Golgi complex (alpha mannosidase II and giantin), of the trans-Golgi network (TGN38), of lysosomes (lgp110), and of early and late endosomes (transferrin receptor and mannose-6-phosphate receptor, respectively), to define the position of their subcellular compartments. By immunofluorescence, GLUT4 appears concentrated in the core of the myotubes. It is primarily found around the nuclei, in a pattern suggesting an association with the Golgi complex, which is further supported by colocalization with giantin and by immunogold electron microscopy. GLUT4 appears to be in the trans-most cisternae of the Golgi complex and in vesicles just beyond, i.e. in the structures that constitute the trans-Golgi network (TGN). In myotubes treated with brefeldin A, the immunofluorescence pattern of GLUT4 is modified, but it differs from both Golgi complex markers and TGN38. Instead, it resembles the pattern of the transferrin receptor, which forms long tubules. In untreated cells, double staining for GLUT4 and transferrin receptor by immunofluorescence shows similar but distinct patterns. Immunoelectron microscopy localizes transferrin receptor, detected by immunoperoxidase, to large vesicles, presumably endosomes, very close to the GLUT4-containing tubulo-vesicular elements. In brefeldin A-treated cells, a network of tubules of approximately 70 nm diameter, studded with varicosities, stains for both GLUT4 and transferrin receptor, suggesting that brefeldin A has caused fusion of the transferrin receptor and GLUT4-containing compartments. The results suggest that GLUT4 storage vesicles constitute a specialized compartment that is either a subset of the TGN, or is very closely linked to it. The link between GLUT4 vesicles and transferrin receptor containing endosomes, as revealed by brefeldin A, may be important for GLUT4 translocation in response to muscle stimulation.

scholarly journals Compartmentation of the Golgi complex: brefeldin-A distinguishes trans-Golgi cisternae from the trans-Golgi network.

1990 ◽  
Vol 111 (3) ◽  
pp. 893-899 ◽  
Author(s):  
N W Chege ◽  
S R Pfeffer
Keyword(s):  
Sialic Acid ◽  
Golgi Complex ◽  
Brefeldin A ◽  
Fungal Metabolite ◽  
Trans Golgi Network ◽  
Lectin Affinity ◽  
Mannose 6 Phosphate ◽  
Lectin Chromatography ◽  
Golgi Network ◽  
Lines Of Evidence

The Golgi complex is composed of at least four distinct compartments, termed the cis-, medial, and trans-Golgi cisternae and the trans-Golgi network (TGN). It has recently been reported that the organization of the Golgi complex is disrupted in cells treated with the fungal metabolite, brefeldin-A. Under these conditions, it was shown that resident enzymes of the cis-, medial, and trans-Golgi return to the ER. We report here that 300-kD mannose 6-phosphate receptors, when pulse-labeled within the ER of brefeldin-A-treated cells, acquired numerous N-linked galactose residues with a half time of approximately 2 h, as measured by their ability to bind to RCA-I lectin affinity columns. In contrast, Limax flavus lectin chromatography revealed that less than 10% of these receptors acquired sialic acid after 8 h in brefeldin-A. Two lines of evidence suggested that proteins within and beyond the TGN did not return to the ER in the presence of brefeldin-A. First, the majority of 300-kD mannose 6-phosphate receptors present in the TGN and endosomes did not return to the ER after up to 6 h in brefeldin-A, as determined by their failure to contact galactosyltransferase that had relocated there. Moreover, although mannose 6-phosphate receptors did not acquire sialic acid when present in the ER of brefeldin-A-treated cells, they were readily sialylated when labeled at the cell surface and transported to the TGN. These experiments indicate that galactosyltransferase, a trans-Golgi enzyme, returns to the endoplasmic reticulum in the presence of brefeldin-A, while the bulk of sialyltransferase, a resident of the TGN, does not. Our findings support the proposal that the TGN is a distinct, fourth compartment of the Golgi apparatus that is insensitive to brefeldin-A.

Structural and histochemical studies of Golgi complex differentiation in salivary gland cells during Drosophila development

1992 ◽  
Vol 102 (1) ◽  
pp. 169-184 ◽  
Author(s):  
G.N. Thomopoulos ◽  
E.P. Neophytou ◽  
M. Alexiou ◽  
A. Vadolas ◽  
S. Limberi-Thomopoulos ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Golgi Complex ◽  
Secretory Granules ◽  
Differential Staining ◽  
Reaction Products ◽  
Trans Golgi Network ◽  
Gland Cells ◽  
Salivary Gland Cells ◽  
Complex Carbohydrates ◽  
Golgi Network

Morphological alterations in the Golgi complex (GC) and changes in the distribution of acid phosphatase (AcPase), thiamine pyrophosphatase (TPPase), complex carbohydrates and reduced osmium tetroxide compounds in this organelle were studied in the salivary gland cells of Drosophila during larval and prepupal development. The morphology and the AcPase, TPPase and complex carbohydrates cytochemical patterns of the Golgi complex varied characteristically during cell differentiation. At the early 3rd instar period the Golgi complex consisted mainly of vesiculated cisternae, and AcPase activity was observed in all cisternae but not in the secretory granules. As development proceeded to the late 3rd instar the Golgi complex displayed its typical appearance, consisting of four to six cisternae, and only the two to three cisternae towards the trans-face as well as the trans-Golgi network and some of the immature secretory granules exhibited AcPase reactivity. In the course of a ‘wave’ of production of the ‘glue’ secretory granules proceeding proximally through the gland, the number of AcPase positive cisternae changed correspondingly. After secretion of the ‘glue’ secretory granules, the size of the Golgi complex decreased and almost all cisternae displayed AcPase reactivity. The detection of TPPase activity presented some specificity problems, since staining was observed not only in the GC cisternae but in the endoplasmic reticulum (ER) and microvilli. The reaction products were seen in a few GC vesicles during the early 3rd instar and in the trans side of the organelle at the end of the 3rd instar. During production of the secretory granules, every GC cisterna was intensely stained. These results agree with previous findings suggesting that AcPase and TPPase in secretory cells may be primarily involved in the processing of exportable proteins. The vicinal (vic)-glycol groups of the complex carbohydrates were detected using the periodic acid/thiocarbohydrazide/silver proteinate (PA-TCH-SP) technique. During synthesis of the ‘glue’ secretory granules, the reaction products were observed over the GC cisternae and the trans-Golgi network, with increasing intensity from the cis to the trans side of the organelle. No PA-TCH-SP staining was observed over the GC cisternae during the early 3rd instar. Following discharge of the ‘glue’ secretory granules, all GC cisternae displayed uniform PA-TCH-SP staining. After OsO4 impregnation, the reaction products were observed mainly in ER and mitochondria and rarely in the GC. In numerous cells, only the mitochondria were stained, while in many cases the ER of neighboring cells exhibited differential staining.(ABSTRACT TRUNCATED AT 400 WORDS)

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 Apiconuclear Organization of Microtubules Does Not Specify Protein Delivery from the Trans-Golgi Network to Different Membrane Domains in Polarized Epithelial Cells

1998 ◽  
Vol 9 (3) ◽  
pp. 685-699 ◽  
Author(s):  
Kent K. Grindstaff ◽  
Robert L. Bacallao ◽  
W. James Nelson
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Golgi Complex ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Membrane Domains ◽  
Trans Golgi Network ◽  
G Cells ◽  
Polarized Epithelial Cells ◽  
Golgi Network

In nonpolarized epithelial cells, microtubules originate from a broad perinuclear region coincident with the distribution of the Golgi complex and extend outward to the cell periphery (perinuclear [PN] organization). During development of epithelial cell polarity, microtubules reorganize to form long cortical filaments parallel to the lateral membrane, a meshwork of randomly oriented short filaments beneath the apical membrane, and short filaments at the base of the cell; the Golgi becomes localized above the nucleus in the subapical membrane cytoplasm (apiconuclear [AN] organization). The AN-type organization of microtubules is thought to be specialized in polarized epithelial cells to facilitate vesicle trafficking between the trans-Golgi Network (TGN) and the plasma membrane. We describe two clones of MDCK cells, which have different microtubule distributions: clone II/G cells, which gradually reorganize a PN-type distribution of microtubules and the Golgi complex to an AN-type during development of polarity, and clone II/J cells which maintain a PN-type organization. Both cell clones, however, exhibit identical steady-state polarity of apical and basolateral proteins. During development of cell surface polarity, both clones rapidly establish direct targeting pathways for newly synthesized gp80 and gp135/170, and E-cadherin between the TGN and apical and basolateral membrane, respectively; this occurs before development of the AN-type microtubule/Golgi organization in clone II/G cells. Exposure of both clone II/G and II/J cells to low temperature and nocodazole disrupts >99% of microtubules, resulting in: 1) 25–50% decrease in delivery of newly synthesized gp135/170 and E-cadherin to the apical and basolateral membrane, respectively, in both clone II/G and II/J cells, but with little or no missorting to the opposite membrane domain during all stages of polarity development; 2) ∼40% decrease in delivery of newly synthesized gp80 to the apical membrane with significant missorting to the basolateral membrane in newly established cultures of clone II/G and II/J cells; and 3) variable and nonspecific delivery of newly synthesized gp80 to both membrane domains in fully polarized cultures. These results define several classes of proteins that differ in their dependence on intact microtubules for efficient and specific targeting between the Golgi and plasma membrane domains.

Three-dimensional reconstruction of the golgi complex and trans-golgi network: A HVEM tomographic study

1993 ◽  
Vol 51 ◽  
pp. 184-185
Author(s):  
Mark S. Ladinsky ◽  
James R. Kremer ◽  
Paul S. Furcinitti ◽  
J. Richard McIntosh ◽  
Kathryn E. Howell
Keyword(s):  
Golgi Complex ◽  
Secretory Pathway ◽  
Rat Kidney ◽  
Three Dimensional ◽  
Specific Staining ◽  
Axial Tomography ◽  
High Voltage Electron Microscopy ◽  
Trans Golgi Network ◽  
Voltage Electron ◽  
Golgi Network

The Golgi complex is the central organelle in the secretory pathway of eukaryotic cells. It is composed of multiple cisternae that contain various enzymes involved in posttranslational modification of newly synthesized proteins. The cisternae are connected to a reticular lattice, the trans-Golgi Network (TGN), which is responsible for sorting and targeting proteins and lipids to other cellular locations. Transport between the Golgi cisternae is thought to occur largely via carrier vesicles, but recent studies suggest that some transport may be via tubules which link the cisternae and TGN. Although the Golgi complex has been well characterized biochemically, only a few studies have addressed its 3-D fine structure.Specific staining, high voltage electron microscopy (HVEM), and computer axial tomography were used to visualize the structure of the Golgi cisternae and the TGN in 3-D. A technique developed by R. E. Pagano was used to facilitate the visualization of the TGN and trans-most cisternae: Normal rat kidney (NRK) cells are treated with a fluorescent analog of ceramide (BODIPY-cer) which is internalized and metabolized in the Golgi complex (Fig. 1a).

Sign in / Sign up

Export Citation Format

Share Document