scholarly journals The Autophagic and Endocytic Pathways Converge at the Nascent Autophagic Vacuoles

1997 ◽  
Vol 136 (1) ◽  
pp. 61-70 ◽  
Author(s):  
Willisa Liou ◽  
Hans J. Geuze ◽  
Math. J.H. Geelen ◽  
Jan W. Slot
Keyword(s):  
Rat Hepatocytes ◽  
Endocytic Pathway ◽  
Fusion Partner ◽  
Isolated Rat Hepatocytes ◽  
Autophagic Vacuoles ◽  
Exponential Increase ◽  
Morphological Criteria ◽  
Endosomal Vesicles ◽  
Multivesicular Endosomes ◽  
Endocytic Pathways

We used an improved cryosectioning technique in combination with immunogold cytochemistry and morphometric analysis to study the convergence of the autophagic and endocytic pathways in isolated rat hepatocytes. The endocytic pathway was traced by continuous uptake of gold tracer for various time periods, up to 45 min, while the cells were incubated in serum-free medium to induce autophagy. Endocytic structures involved in fusion with autophagic vacuoles (AV) were categorized into multivesicular endosomes (MVE) and vesicular endosomes (VE). Three types of AV—initial (AVi), intermediate (AVi/d), and degradative (AVd)—were defined by morphological criteria and immunogold labeling characteristics of marker enzymes. The entry of tracer into AV, manifested as either tracer-containing AV profiles (AV+) or fusion profiles (FP+) between AV and tracer-positive endosomal vesicles/vacuoles, was detected as early as 10 min after endocytosis. The number of AV+ exhibited an exponential increase with time. FP+ between MVE or VE and all three types of AV were observed. Among the 112 FP+ scored, 36% involved VE. Of the AV types, AVi and AVi/d were found five to six times more likely to be involved in fusions than AVd. These fusion patterns did not significantly change during the period of endocytosis (15–45 min). We conclude that the autophagic and endocytic pathways converge in a multistage fashion starting within 10 min of endocytosis. The nascent AV is the most upstream and preferred fusion partner for endosomes.

scholarly journals Use of glycyl-l-phenylalanine 2-naphthylamide, a lysosome-disrupting cathepsin C substrate, to distinguish between lysosomes and prelysosomal endocytic vacuoles

1994 ◽  
Vol 300 (1) ◽  
pp. 229-236 ◽  
Author(s):  
T O Berg ◽  
P E Strømhaug ◽  
T Løvdal ◽  
P O Seglen ◽  
T Berg
Keyword(s):  
Acid Phosphatase ◽  
Degradation Products ◽  
Rat Hepatocytes ◽  
Endocytic Pathway ◽  
Marker Enzyme ◽  
Major Peak ◽  
Isolated Rat Hepatocytes ◽  
Cathepsin C ◽  
Minor Peak ◽  
A Minor

Lysosome-disrupting enzyme substrates have been used to distinguish between lysosomal and prelysosomal compartments along the endocytic pathway in isolated rat hepatocytes. The cells were incubated for various periods of time with 125I-labelled tyramine cellobiose (125I-TC) covalently coupled to asialoorosomucoid (AOM) (125I-TC-AOM); this molecule is internalized by receptor-mediated endocytosis and degraded in lysosomes, where the degradation products (acid-soluble, radio-labelled short peptides) accumulate, Glycyl-L-phenylalanine 2-naphthylamide (GPN) and methionine O-methyl ester (MOM), which are hydrolysed by lysosomal cathepsin C and a lysosomal esterase respectively, both diffused into hepatocytic lysosomes after electrodisruption of the cells. Intralysosomal accumulation of the hydrolysis products (amino acids) of these substrates caused osmotic lysis of more than 90% of the lysosomes, as measured by the release of acid-soluble radioactivity derived from 125I-TC-AOM degradation. The acid-soluble radioactivity coincided in sucrose-density gradients with a major peak of the lysosomal marker enzyme acid phosphatase at 1.18 g/ml; in addition a minor, presumably endosomal, acid phosphatase peak was observed around 1.14 g/ml. The major peak of acid phosphatase was almost completely released by GPN (and by MOM), while the minor peak seemed unaffected by GPN. Acid-insoluble radioactivity, presumably in endosomes, banded (after 1 h of 125I-TC-AOM uptake) as a major peak at 1.14 and a minor peak at 1.18 g/ml in sucrose gradients, and was not significantly released by GPN. GPN thus appears to be an excellent tool by which to distinguish between endosomes and lysosomes. MOM, on the other hand, released some radioactivity and acid phosphatase from endosomes as well as from lysosomes.

scholarly journals Inhibition of asialoglycoprotein endocytosis and degradation in rat hepatocytes by protein phosphatase inhibitors

1995 ◽  
Vol 311 (1) ◽  
pp. 317-326 ◽  
Author(s):  
I Holen ◽  
P B Gordon ◽  
P E Strømhaug ◽  
T O Berg ◽  
M Fengsrud ◽  
...  
Keyword(s):  
Cell Surface ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Rat Hepatocytes ◽  
Maximal Effect ◽  
Isolated Rat Hepatocytes ◽  
Phosphatase Inhibitors ◽  
Protein Phosphatase Inhibitors ◽  
Endocytic Vesicles ◽  
Multivesicular Endosomes

In isolated rat hepatocytes, a radiolabelled tyramine-cellobiose conjugate of asialo-orosomucoid, 125I-TC-AOM, was rapidly taken up by receptor-mediated endocytosis and proteolytically degraded in the lysosomes, where radioactive degradation products accumulated. Okadaic acid and other protein phosphatase inhibitors (microcystin-LR, calyculin A) strongly reduced the fraction of asialoglycoprotein (ASGP) receptors localized to the cell surface, and correspondingly inhibited the uptake of 125I-TC-AOM. In addition, the inhibitors suppressed 125I-TC-AOM degradation strongly (90% at 150 nM) and potently (half-maximal effect at 20 nM okadaic acid), indicating an involvement of protein phosphorylation, and of a protein phosphatase of type 2A, in the regulation of intracellular endocytic flux. The effects of okadaic acid on 125I-TC-AOM accumulation, as well as on degradation, could be eliminated by the protein kinase inhibitor genistein. Okadaic acid prevented the transfer of 125I-TC-AOM to a non-recycling endocytic compartment, causing its retention in a recycling compartment from which about one-third of the endocytosed 125I-TC-AOM could be returned to the cell surface and detached from its receptor in the presence of EGTA. ASGP receptors recycled extensively both in the presence and absence of okadaic acid, as indicated by a sustained uptake of 125I-TC-AOM. Sucrose density gradient analysis and sedimentation studies indicated that okadaic acid caused accumulation of 125I-TC-AOM in light endosomes (1.11 g/ml), preventing its transfer to dense endosomes (1.14 g/ml) and lysosomes (1.18 g/ml). The lysosomes could be identified in density gradients by their contents of lysosomal marker enzymes and acid-soluble radioactivity, and by their sensitivity towards the lysosome-disrupting agent glycyl-L-phenylalanine-2-naphthylamide. By using endocytosed AOM-gold particles as an ultrastructural endocytic marker, it could be shown that the light endosomes accumulating ASGP in the presence of okadaic acid had the morphological appearance of small endocytic vesicles/tubules and multivesicular endosomes. Whereas in control cells 4% of the AOM-gold was in small vesicles/tubules, 55% in multivesicular endosomes and 41% in lysosomes, the corresponding figures for okadaic acid-treated cells were 17%, 73% and 11%. Our results thus indicate that protein phosphatase inhibitors have two effects on ASGP endocytosis: (1) an early inhibition of ligand uptake, due to a reduction in the fraction of ASGP receptors at the cell surface, and (2) an inhibition of ASGP transfer from a recycling compartment consisting of light, small endocytic vesicles and multivesicular endosomes, to a non-recycling compartment consisting of dense multivesicular endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Role of Ca2+ for protein turnover in isolated rat hepatocytes

1983 ◽  
Vol 216 (3) ◽  
pp. 529-536 ◽  
Author(s):  
B Grinde
Keyword(s):  
Proteinase Inhibitors ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Inhibitory Effect ◽  
Ionophore A23187 ◽  
Lysosomal Degradation ◽  
Bivalent Cation ◽  
Isolated Rat Hepatocytes ◽  
Autophagic Vacuoles ◽  
Endogenous Protein

Experiments with bivalent-cation chelators (EGTA and EDTA), a Ca2+ ionophore (A23187) and a Ca2+-channel blocker (verapamil) indicate that Ca2+ is required for the lysosomal degradation of endogenous protein in hepatocytes. A distinction is made between lysosomal and non-lysosomal degradation by using the lysosomotropic agent methylamine. As Ca2+ does not appear to be required for the lysosomal degradation of endocytosed asialo-fetuin, the Ca2+-dependence for the degradation of endogenous protein is probably connected with the formation of autophagic vacuoles or the fusion of autophagic vacuoles with lysosomes. EGTA and EDTA had a slight inhibitory effect on the non-lysosomal degradation. This effect could be due to the activity of non-lysosomal Ca2+-dependent thiol proteinases. Together with previous experiments with thiol-proteinase inhibitors, the present experiments indicate that these proteinases have a very limited impact on the bulk protein degradation in the isolated hepatocytes.

scholarly journals Prelysosomal and lysosomal connections between autophagy and endocytosis

1992 ◽  
Vol 283 (2) ◽  
pp. 361-369 ◽  
Author(s):  
P B Gordon ◽  
H Høyvik ◽  
P O Seglen
Keyword(s):  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Autophagic Vacuoles ◽  
Complete Degradation ◽  
Isolated Rat

In isolated rat hepatocytes electroloaded with [14C]sucrose, autophaged sugar accumulated in lysosomes under control conditions, and in prelysosomal autophagic vacuoles (amphisomes) in the presence of asparagine, an inhibitor of autophagic-lysosomal fusion. Endocytic uptake of the sucrose-cleaving enzyme invertase resulted in rapid and complete degradation of autophaged sucrose in both amphisomes and lysosomes. Pre-accumulated sucrose was degraded equally well in both compartments, regardless of amphisomal-lysosomal flux inhibition by asparagine, suggesting that endocytic entry into the autophagic pathway can take place both at the lysosomal and at the amphisomal level. The completeness of sucrose degradation by endocytosed invertase furthermore indicates that all lysosomes involved in autophagy can also engage in endocytosis. Endocytosed invertase reached the amphisomes even when autophagy was blocked by 3-methyladenine, and autophaged sucrose reached this compartment even when endocytic influx was blocked by vinblastine, suggesting that amphisomes may exhibit some degree of permanence independently of either pathway.

scholarly journals Lysosomal triacylglycerol lipase and lipolysis in isolated rat hepatocytes.

1979 ◽  
Vol 254 (18) ◽  
pp. 8841-8846
Author(s):  
L.J. Debeer ◽  
J. Thomas ◽  
P.J. De Schepper ◽  
G.P. Mannaerts
Keyword(s):  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Triacylglycerol Lipase ◽  
Isolated Rat
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