scholarly journals In exocrine pancreas, the basolateral endocytic pathway converges with the autophagic pathway immediately after the early endosome.

1990 ◽  
Vol 111 (2) ◽  
pp. 329-345 ◽  
Author(s):  
J Tooze ◽  
M Hollinshead ◽  
T Ludwig ◽  
K Howell ◽  
B Hoflack ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Lysosomal Enzymes ◽  
Early Endosome ◽  
Endocytic Pathway ◽  
Cell Periphery ◽  
Type I ◽  
Type Ii ◽  
Autophagic Vacuoles ◽  
Early Endosomes ◽  
Mannose 6 Phosphate

Intracisternal granules (ICGs) are insoluble aggregates of pancreatic digestive enzymes and proenzymes that develop within the lumen of the rough endoplasmic reticulum of exocrine pancreatic cells, especially in guinea pigs. These ICGs are eliminated by autophagy. By morphological criteria, we identified three distinct and sequential classes of autophagic compartments, which we refer to as phagophores, Type I autophagic vacuoles, and Type II autophagic vacuoles. Lobules of guinea pig pancreas were incubated in media containing HRP for periods of 5-120 min to determine the relationship between the endocytic and autophagic pathways. Incubations with HRP of 15 min or less labeled early endosomes at the cell periphery that were not involved in autophagy of ICGs, but after these short incubations none of the autophagic compartments were HRP positive. After 30-min incubation with HRP, early endosomes at the cell periphery, late endosomes in the pericentriolar region, and, in addition, Type I autophagic vacuoles containing ICGs were all labeled by the tracer. Type II autophagic vacuoles were not labeled after 30-min incubation with HRP but were labeled after incubations of 60-120 min. Phagophores did not receive HRP even after 120 min incubations. We concluded that the autophagic and endocytic pathways converge immediately after the early endosome level and that Type I autophagic vacuoles precede Type II autophagic vacuoles on the endocytic pathway. We studied the distribution of acid phosphatase, lysosomal proteases and cation-independent-mannose-6-phosphate receptor (CI-M6PR) in the three classes of autophagic compartments by histochemical and immunocytochemical methods. Phagophores, the earliest autophagic compartment, contained none of these markers. Type I autophagic vacuoles contained acid phosphatase but, at most, only very low levels of cathepsin D and CI-M6PR. Type II autophagic vacuoles, by contrast, are enriched for acid phosphatase, cathepsin D, and other lysosomal enzymes, and they are also enriched for CI-M6PR. Moreover, soluble fragments of bovine CI-M6PR conjugated to colloidal gold particles heavily labeled Type II but not Type I autophagic vacuoles, and this labeling was specifically blocked by mannose-6-phosphate. This indicates that the lysosomal enzymes present in Type II autophagic vacuoles carry mannose-6-phosphate monoester residues. Using 3-C2, 4-dinitroanilino-3'-amino-N-methyldipropylamine (DAMP), we showed that Type II autophagic vacuoles are acidic. We interpret these findings as indicating that Type II autophagic vacuoles are a prelysosomal compartment in which the already combined endocytic and autophagic pathways meet the delivery pathway of lysosomal enzymes.

Characterization of the cation-independent mannose 6-phosphate receptor-enriched prelysosomal compartment in NRK cells

1990 ◽  
Vol 95 (3) ◽  
pp. 441-461
Author(s):  
G. Griffiths ◽  
R. Matteoni ◽  
R. Back ◽  
B. Hoflack
Keyword(s):  
Acid Phosphatase ◽  
Membrane Surface ◽  
Three Dimensional ◽  
Endocytic Pathway ◽  
Dimensional Structure ◽  
Surface Areas ◽  
Early Endosomes ◽  
Mannose 6 Phosphate ◽  
Golgi Network

The structure of a late endosomal compartment, which contains the bulk of the cation-independent mannose 6-phosphate receptor (MPR) in NRK cells, is documented using immunocytochemistry and cryo-sections, as well as conventional Epon sections. This compartment, which we refer to as the prelysosomal compartment (PLC), has a complex three-dimensional structure consisting of tubuloreticular domains in continuity with vesicular parts. The latter are characterized by a high density of internal membranes, which may be either tubular or sheet-like, that label extensively for the MPR. This structural organization was also maintained after fractionation in sucrose gradients. The amount of MPR immunolabelling was then quantitated with respect to the membrane surface areas of the four compartments where it is found: namely, the plasma membrane, early endosomes, the trans Golgi network and the PLC. The results showed that in NRK cells 90% of the labelling for the receptor was found in the PLC, with the rest distributed over the other three compartments. Cytochemical studies indicated that the PLC is the first structure along the endocytic pathway that gives a significant reaction for acid phosphatase. However, the PLC is clearly distinct from the MPR-negative lysosomes, which are also acid phosphatase-positive, since the two organelles could be physically separated from each other after fractionation on Percoll gradients.

scholarly journals QUANTITATIVE CHARACTERIZATION OF DENSE BODY, AUTOPHAGIC VACUOLE, AND ACID PHOSPHATASE-BEARING PARTICLE POPULATIONS DURING THE EARLY PHASES OF GLUCAGON-INDUCED AUTOPHAGY IN RAT LIVER

1971 ◽  
Vol 48 (3) ◽  
pp. 473-489 ◽  
Author(s):  
Russell L. Deter
Keyword(s):  
Acid Phosphatase ◽  
Dense Body ◽  
Autophagic Vacuole ◽  
Type I ◽  
Type Ii ◽  
Average Volume ◽  
Quantitative Characterization ◽  
Autophagic Vacuoles ◽  
Dense Bodies

Quantitative characterization of dense body, autophagic vacuole and acid phosphatase-bearing particle populations of rat liver have been made at 10 min intervals during the first 50 min following the intraperitoneal administration of glucagon. Beginning 10 to 20 min postinjection, increases in the number of autophagic vacuoles and in the osmotic sensitivity of acid phosphatase-bearing particles were observed, associated with a progressive disappearance of dense bodies. These changes appeared to reach a maximum 50 min after treatment. The average volume of autophagic vacuoles was found to be 440–870% greater than that of normal dense bodies during this time period. No consistent change in total acid phosphatase activity was noted. A detailed study of autophagic vacuole profile populations revealed the presence of five different types of profiles, two of which, types I and II, accounted for 76.3–94.4% of the profiles examined. Type I profiles primarily contained elements of the endoplasmic reticulum, free ribosomes, and ground cytoplasm. Type II profiles had mitochondrial profiles as their principal constituent, but endoplasmic reticulum and free ribosomes were also seen. At all time points type I profiles predominated, comprising 55–69% of the profiles found. Both profile types were bounded by single and double limiting membranes, the former being predominate. A time-dependent change in the ratio of single to double membrane-limited profiles could not be demonstrated. Morphometric parameters derived from profile size distributions indicated that the number of types I and II autophagic vacuoles increased with time, the rate being greater for the type II particle, except between 40 and 50 min. The average volume of the type II autophagic vacuole was consistently greater than that of the type I.

scholarly journals Endocytic Pathway of Feline Coronavirus for Cell Entry: Differences in Serotype-Dependent Viral Entry Pathway

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 300 ◽  
Author(s):  
Tomomi Takano ◽  
Yumeho Wakayama ◽  
Tomoyoshi Doki
Keyword(s):  
Late Endosome ◽  
Endocytic Pathway ◽  
Type I ◽  
Type Ii ◽  
Cholesterol Accumulation ◽  
Endosome Trafficking ◽  
Early Endosomes ◽  
Trafficking Inhibitor ◽  
Intracellular Cholesterol ◽  
Feline Coronavirus

Feline coronavirus (FCoV) is a pathogen causing a lethal infectious disease in cats, feline infectious peritonitis. It has two serotypes (type I FCoV and type II FCoV). According to our previous study, type I FCoV infection is inhibited by compounds inducing intracellular cholesterol accumulation, whereas type II FCoV infection is not inhibited. Intracellular cholesterol accumulation was reported to disrupt late endosome function. Based on these findings, types I and II FCoV are considered to enter the cytosol through late and early endosomes, respectively. We investigated whether the antiviral activities of a late endosome trafficking inhibitor and cholesterol-accumulating agents are different between the FCoV serotypes. The late endosome trafficking inhibitor did not inhibit type II FCoV infection, but it inhibited type I FCoV infection. Type I FCoV infection was inhibited by cholesterol-accumulating triazoles, but not by non-cholesterol-accumulating triazoles. These phenomena were observed in both feline cell lines and feline primary macrophages. This study provides additional information on the differences in intracellular reproductive cycle between type I and type II FCoV.

scholarly journals Clathrin Hub Expression Affects Early Endosome Distribution with Minimal Impact on Receptor Sorting and Recycling

2001 ◽  
Vol 12 (9) ◽  
pp. 2790-2799 ◽  
Author(s):  
Elizabeth M. Bennett ◽  
Sharron X. Lin ◽  
Mhairi C. Towler ◽  
Frederick R. Maxfield ◽  
Frances M. Brodsky
Keyword(s):  
Plasma Membrane ◽  
Early Endosome ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Endocytic Trafficking ◽  
Minimal Impact ◽  
Recycling Endosomes ◽  
Receptor Mediated Endocytosis ◽  
Early Endosomes ◽  
Endosomal Membranes

Clathrin-coated vesicles execute receptor-mediated endocytosis at the plasma membrane. However, a role for clathrin in later endocytic trafficking processes, such as receptor sorting and recycling or maintaining the organization of the endocytic pathway, has not been thoroughly characterized. The existence of clathrin-coated buds on endosomes suggests that clathrin might mediate later endocytic trafficking events. To investigate the function of clathrin-coated buds on endosomal membranes, endosome function and distribution were analyzed in a HeLa cell line that expresses the dominant-negative clathrin inhibitor Hub in an inducible manner. As expected, Hub expression reduced receptor-mediated endocytosis at the plasma membrane. Hub expression also induced a perinuclear aggregation of early endosome antigen 1-positive early endosomes, such that sorting and recycling endosomes were found tightly concentrated in the perinuclear region. Despite the dramatic redistribution of endosomes, Hub expression did not affect the overall kinetics of receptor sorting or recycling. These data show that clathrin function is necessary to maintain proper cellular distribution of early endosomes but does not play a prominent role in sorting and recycling events. Thus, clathrin's role on endosomal membranes is to influence organelle localization and is distinct from its role in trafficking pathways at the plasma membrane and trans-Golgi network.

scholarly journals Role of LAMP-2 in Lysosome Biogenesis and Autophagy

2002 ◽  
Vol 13 (9) ◽  
pp. 3355-3368 ◽  
Author(s):  
Eeva-Liisa Eskelinen ◽  
Anna Lena Illert ◽  
Yoshitaka Tanaka ◽  
Günter Schwarzmann ◽  
Judith Blanz ◽  
...  
Keyword(s):  
Half Life ◽  
Lysosomal Enzymes ◽  
State Level ◽  
Autophagic Vacuoles ◽  
Lysosome Biogenesis ◽  
Golgi Region ◽  
Activity Measurements ◽  
Phenotype Analysis ◽  
Mannose 6 Phosphate

In LAMP-2–deficient mice autophagic vacuoles accumulate in many tissues, including liver, pancreas, muscle, and heart. Here we extend the phenotype analysis using cultured hepatocytes. In LAMP-2–deficient hepatocytes the half-life of both early and late autophagic vacuoles was prolonged as evaluated by quantitative electron microscopy. However, an endocytic tracer reached the autophagic vacuoles, indicating delivery of endo/lysosomal constituents to autophagic vacuoles. Enzyme activity measurements showed that the trafficking of some lysosomal enzymes to lysosomes was impaired. Immunoprecipitation of metabolically labeled cathepsin D indicated reduced intracellular retention and processing in the knockout cells. The steady-state level of 300-kDa mannose 6-phosphate receptor was slightly lower in LAMP-2–deficient hepatocytes, whereas that of 46-kDa mannose 6-phosphate receptor was decreased to 30% of controls due to a shorter half-life. Less receptor was found in the Golgi region and in vesicles and tubules surrounding multivesicular endosomes, suggesting impaired recycling from endosomes to the Golgi. More receptor was found in autophagic vacuoles, which may explain its shorter half-life. Our data indicate that in hepatocytes LAMP-2 deficiency either directly or indirectly leads to impaired recycling of 46-kDa mannose 6-phosphate receptors and partial mistargeting of a subset of lysosomal enzymes. Autophagic vacuoles may accumulate due to impaired capacity for lysosomal degradation.

scholarly journals Identification of a membrane glycoprotein found primarily in the prelysosomal endosome compartment.

1991 ◽  
Vol 112 (2) ◽  
pp. 245-255 ◽  
Author(s):  
J E Park ◽  
J M Lopez ◽  
E B Cluett ◽  
W J Brown
Keyword(s):  
Lysosomal Enzymes ◽  
Endocytic Pathway ◽  
Membrane Glycoprotein ◽  
Cell Fractionation ◽  
Density Fractions ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Acidic Compartment ◽  
Intracellular Vesicles ◽  
Secondary Lysosomes

Cells contain an intracellular compartment that serves as both the "prelysosomal" delivery site for newly synthesized lysosomal enzymes by the mannose 6-phosphate (Man6P) receptor and as a station along the endocytic pathway to lysosomes. We have obtained mAbs to a approximately 57-kD membrane glycoprotein, (called here plgp57), found predominantly in this prelysosomal endosome compartment. This conclusion is supported by the following results: (a) plgp57 was primarily found in a population of late endosomes that were located just distal to the 20 degrees C block site in the endocytic pathway to lysosomes (approximately 83% of the prelysosomes were positive for plgp57 but less than 5% of the early endosomes had detectable amounts of this marker); (b) plgp57 and the cation-independent (CI) Man6P receptor were located in many of the same intracellular vesicles; (c) plgp57 was found in the membranes of an acidic compartment; (d) immunoelectron microscopy showed that plgp57 was located in characteristic multilamellar- and multivesicular-type vacuoles believed to be prelysosomal endosomes; and (e) cell fractionation studies demonstrated that plgp57 was predominantly found in low density organelles which comigrated with late endosomes and CI Man6P receptors, and only approximately 10-15% of the antigen was found in high density fractions containing the majority of secondary lysosomes. These results indicate that plgp57 is a novel marker for a unique prelysosomal endosome compartment that is the site of confluence of the endocytic and biosynthetic pathways to lysosomes.

scholarly journals The disintegrin and metalloproteinase ADAM12 contributes to TGF-β signaling through interaction with the type II receptor

2007 ◽  
Vol 178 (2) ◽  
pp. 201-208 ◽  
Author(s):  
Azeddine Atfi ◽  
Emmanuelle Dumont ◽  
Frédéric Colland ◽  
Dominique Bonnier ◽  
Annie L'Helgoualc'h ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Type I ◽  
Type Ii ◽  
Transmembrane Receptors ◽  
Smad Proteins ◽  
Early Endosomes ◽  
A Disintegrin And Metalloproteinase

Transforming growth factor-β (TGF-β) regulates a wide variety of biological processes through two types of Ser/Thr transmembrane receptors: the TGF-β type I receptor and the TGF-β type II receptor (TβRII). Upon ligand binding, TGF-β type I receptor activated by TβRII propagates signals to Smad proteins, which mediate the activation of TGF-β target genes. In this study, we identify ADAM12 (a disintegrin and metalloproteinase 12) as a component of the TGF-β signaling pathway that acts through association with TβRII. We found that ADAM12 functions by a mechanism independent of its protease activity to facilitate the activation of TGF-β signaling, including the phosphorylation of Smad2, association of Smad2 with Smad4, and transcriptional activation. Furthermore, ADAM12 induces the accumulation of TβRII in early endosomal vesicles and stabilizes the TβRII protein presumably by suppressing the association of TβRII with Smad7. These results define ADAM12 as a new partner of TβRII that facilitates its trafficking to early endosomes in which activation of the Smad pathway is initiated.

scholarly journals Rab4 Regulates Formation of Synaptic-like Microvesicles from Early Endosomes in PC12 Cells

2001 ◽  
Vol 12 (11) ◽  
pp. 3703-3715 ◽  
Author(s):  
Heidi de Wit ◽  
Yael Lichtenstein ◽  
Regis B. Kelly ◽  
Hans J. Geuze ◽  
Judith Klumperman ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Pc12 Cells ◽  
Early Endosome ◽  
Small Gtpase ◽  
Immunogold Electron Microscopy ◽  
Cell Periphery ◽  
Early Endosomes ◽  
Cytoplasmic Vesicles ◽  
Important Site

Early endosomes in PC12 cells are an important site for the formation of synaptic-like microvesicles and constitutive recycling vesicles. By immunogold electron microscopy, the small GTPase rab4 was localized to early endosomes and numerous small vesicles in the cell periphery and Golgi area of PC12 cells. Overexpression of GTPase-deficient Q67Lrab4 increased the number of early endosome-associated and cytoplasmic vesicles, whereas expression of GDP-bound S22Nrab4 significantly increased the length of early endosomal tubules. In parallel, Q67Lrab4 induced a shift in rab4, VAMP2, and TfR label from early endosomes to peripheral vesicles, whereas S22Nrab4 increased early endosome labeling of all three proteins. These observations were corroborated by early endosome budding assays. Together, our data document a thus far unrecognized role for rab4 in the formation of synaptic-like microvesicles and add to our understanding of the formation of constitutive recycling vesicles from early endosomes.

The endocytic pathway in animal cells

1990 ◽  
Vol 48 (3) ◽  
pp. 41-41
Author(s):  
Gareth Griffiths
Keyword(s):  
Early Endosome ◽  
Endocytic Pathway ◽  
Three Dimensions ◽  
Cell Periphery ◽  
Animal Cells ◽  
The Third ◽  
Negative Stain ◽  
Late Endosomes ◽  
Work Done

Our recent data have provided evidence that the endocytic pathway comprises four structurally and functionally distinct compartments in addition to the clathrin coated vesicles. The most proximal of these compartments, the early endosome, is structurally complex, consists of cisternal, tubular, as well as vesicular domains and is predominantly localized to the cell periphery. We have proposed that the vesicular domains bud off the early endosome to form the spherical endosome (carrier) vesicles that transport material from early to late endosomes, a process that requires microtubules. These endosome vesicles are spherical (0.2-0.4μm diameter) structures whose lumen is packed with thin tubules. The structure of the latter has recently become more evident in negative stain preparations. These vesicles can also be shown to bind microtubules in vitro (work done in collaboration with Jean Gruenberg's group, EMBL). The third compartment, the late endosome or prelysosomal compartment (PLC) is, like the early endosome, complex in three-dimensions.

The effect of wortmannin on the localisation of lysosomal type I integral membrane glycoproteins suggests a role for phosphoinositide 3-kinase activity in regulating membrane traffic late in the endocytic pathway

1996 ◽  
Vol 109 (4) ◽  
pp. 749-762 ◽  
Author(s):  
B.J. Reaves ◽  
N.A. Bright ◽  
B.M. Mullock ◽  
J.P. Luzio
Keyword(s):  
Rat Kidney ◽  
Membrane Traffic ◽  
Endocytic Pathway ◽  
Type I ◽  
Morphological Effect ◽  
Trans Golgi Network ◽  
Endosomal Compartment ◽  
Vacuolar Compartment ◽  
Mannose 6 Phosphate ◽  
Golgi Network

Addition of wortmannin to normal rat kidney cells caused a redistribution of the lysosomal type I integral membrane proteins Igp110 and Igp120 to a swollen vacuolar compartment. This compartment did not contain the cation independent mannose 6-phosphate receptor and was depleted in acid hydrolases. It was distinct from another swollen vacuolar compartment containing the cation independent mannose 6-phosphate receptor. The swollen Igp110-positive compartment was accessible to a monoclonal antibody against Igp120 added extracellularly, showing that it had the characteristics of an endosomal compartment. Wortmannin had no gross morphological effect on the trans-Golgi network or lysosomes nor any effect on the delivery to the trans-Golgi network of endocytosed antibodies against the type I membrane protein TGN38. We propose that the observed effects of wortmannin were due to inhibition of membrane traffic between cation independent mannose 6-phosphate receptor-positive late endosomes and the trans-Golgi network and to inhibition of membrane traffic between a novel Igp120-positive, cation independent mannose 6-phosphate receptor-negative late endosomal compartment and lysosomes. The effects of wortmannin suggest a function for a phosphatidylinositol 3-kinase(s) in regulating membrane traffic in the late endocytic pathway.

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