scholarly journals Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells.

1991 ◽  
Vol 115 (6) ◽  
pp. 1561-1572 ◽  
Author(s):  
T Ludwig ◽  
G Griffiths ◽  
B Hoflack
Keyword(s):  
Lysosomal Enzymes ◽  
Rat Kidney ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Early Endosomes ◽  
Fluid Phase Endocytosis ◽  
Normal Rat ◽  
Endocytic Compartments ◽  
Endocytic Pathways

We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6-P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome.

scholarly journals Endocytosis in filter-grown Madin-Darby canine kidney cells.

1989 ◽  
Vol 109 (6) ◽  
pp. 3243-3258 ◽  
Author(s):  
M Bomsel ◽  
K Prydz ◽  
R G Parton ◽  
J Gruenberg ◽  
K Simons
Keyword(s):  
Cell Layer ◽  
Mdck Cells ◽  
Fluid Phase ◽  
Perinuclear Region ◽  
Apical Side ◽  
Early Endosomes ◽  
Basolateral Side ◽  
Endocytic Compartments ◽  
Kinetics Of ◽  
Endocytic Pathways

In this paper, we have characterized the apical and basolateral endocytic pathways of epithelial MDCK cells grown on filters. The three-dimensional organization of the endocytic compartments was analyzed by confocal microscopy after internalization of a fluorescent fluid-phase marker from either side of the cell layer. After 5 min of internalization, distinct sets of apical and basolateral early endosomes were observed lining the plasma membrane domain from which internalization had occurred. At later time points, the apical and the basolateral endocytic pathways were shown to converge in the perinuclear region. Mixing of two different fluorescent markers could be detected after their simultaneous internalization from opposite sides of the cell layer. The extent of the meeting was quantitated by measuring the amount of complex formed intracellularly between avidin internalized from the apical side and biotinylated horseradish peroxidase (HRP) from the basolateral side. After 15 min, 14% of the avidin marker was complexed with the biotinylated HRP and this value increased to 50% during a subsequent chase of 60 min in avidin-free medium. We also determined the kinetics of fluid internalization, recycling, transcytosis, and intracellular retention using HRP as a marker. Fluid was internalized with the same rates from either surface domain (1.2 x 10(-4) microns 3/min per microns 2 of surface area). However, significant differences were observed for each pathway in the amounts and kinetics of marker recycled and transcytosed. The content of apical early endosomes was primarily recycled and transcytosed (45% along Bach route after 1 h internalization), whereas delivery to late endocytic compartments was favored from the basolateral early endosome (77% after 1 h). Our results demonstrate that early apical and basolateral endosomes are functionally and topologically distinct, but that the endocytic pathways converge at later stages in the perinuclear region of the cell.

scholarly journals Targeting of an Intestinal Apical Endosomal Protein to Endosomes in Nonpolarized Cells

1997 ◽  
Vol 136 (2) ◽  
pp. 319-330 ◽  
Author(s):  
Jean M. Wilson ◽  
Tamara L. Colton
Keyword(s):  
Epithelial Cells ◽  
Rat Kidney ◽  
Neuronal Cell ◽  
Brefeldin A ◽  
Fluid Phase ◽  
Neuronal Cell Body ◽  
Intestinal Epithelial ◽  
Endosomal Compartment ◽  
Early Endosomes ◽  
Normal Rat

Polarized cells such as epithelial cells and neurons have distinct endosomal compartments associated with different plasma membrane domains. The endosomes of the neuronal cell body and the basolateral cytoplasm of epithelial cells are thought to perform cellular “housekeeping” functions such as the uptake of nutrients and metabolites, while the endosomes in the apical cytoplasm or axons are thought to be specialized for the sorting and transcytosis of cell type–specific ligands and receptors. However, it is not known if nonpolarized cells such as fibroblasts contain a specialized endosomal compartment analogous to the specialized endosomes found in neurons and epithelia. We have expressed a protein that is normally found in the apical early endosomes of developing intestinal epithelial cells in normal rat kidney fibroblasts. This apical endosomal marker, called endotubin, is targeted to early endosomes in transfected fibroblasts, and is present in peripheral as well as perinuclear endosomes. The peripheral endosomes that contain endotubin appear to exclude transferrin, fluid phase markers, and the mannose-6-phosphate receptor, although in the perinuclear region colocalization of endotubin and these markers is present. In addition, endotubin positive structures do not tubulate in response to brefeldin A and instead redistribute to a diffuse perinuclear location. Since this endosomal compartment has many of the characteristics of an apical or axonal endosomal compartment, our results indicate that nonpolarized cells also contain a specialized early endosomal compartment.

scholarly journals Relationship between invariant chain expression and major histocompatibility complex class II transport into early and late endocytic compartments.

1993 ◽  
Vol 177 (3) ◽  
pp. 583-596 ◽  
Author(s):  
P Romagnoli ◽  
C Layet ◽  
J Yewdell ◽  
O Bakke ◽  
R N Germain
Keyword(s):  
Major Histocompatibility Complex ◽  
Mhc Class Ii ◽  
Class Ii ◽  
Endocytic Pathway ◽  
Invariant Chain ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endocytic Compartments

Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, contains a targeting signal for transport to intracellular vesicles in the endocytic pathway. The characteristics of the target vesicles and the relationship between Ii structure and class II localization in distinct endosomal subcompartments have not been well defined. We demonstrate here that in transiently transfected COS cells expressing high levels of the p31 or p41 forms of Ii, uncleaved Ii is transported to and accumulates in transferrin-accessible (early) endosomes. Coexpressed MHC class II is also found in this same compartment. These early endosomes show altered morphology and a slower rate of content movement to later parts of the endocytic pathway. At more moderate levels of Ii expression, or after removal of a highly conserved region in the cytoplasmic tail of Ii, coexpressed class II molecules are found primarily in vesicles with the characteristics of late endosomes/prelysosomes. The Ii chains in these late endocytic vesicles have undergone proteolytic cleavage in the lumenal region postulated to control MHC class II peptide binding. These data indicate that the association of class II with Ii results in initial movement to early endosomes. At high levels of Ii expression, egress to later endocytic compartments is delayed and class II-Ii complexes accumulate together with endocytosed material. At lower levels of Ii expression, class II-Ii complexes are found primarily in late endosomes/prelysosomes. These data provide evidence that the route of class II transport to the site of antigen processing and loading involves movement through early endosomes to late endosomes/prelysosomes. Our results also reveal an unexpected ability of intact Ii to modify the structure and function of the early endosomal compartment, which may play a role in regulating this processing pathway.

scholarly journals Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes

1992 ◽  
Vol 103 (4) ◽  
pp. 1139-1152
Author(s):  
J.W. Kok ◽  
K. Hoekstra ◽  
S. Eskelinen ◽  
D. Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Intracellular Ph ◽  
Brefeldin A ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Recycling Pathway ◽  
Golgi Network ◽  
Extensive Involvement

Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's trafficking from peripheral early endosomes to centrally located late endosomes. When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway. Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.

Ligand-stimulated beta 2-adrenergic receptor internalization via the constitutive endocytic pathway into rab5-containing endosomes

1995 ◽  
Vol 108 (9) ◽  
pp. 2983-2991 ◽  
Author(s):  
R.H. Moore ◽  
N. Sadovnikoff ◽  
S. Hoffenberg ◽  
S. Liu ◽  
P. Woodford ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Adrenergic Receptor ◽  
Fluid Phase ◽  
Small Gtpase ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Beta Agonist ◽  
Hek293 Cells ◽  
Fluid Phase Endocytosis ◽  
Beta 2

The small GTPase rab5 appears to be rate-limiting for the constitutive internalization of transferrin receptor and for fluid-phase endocytosis. However, it is unknown whether rab5 regulates receptors whose internalization is stimulated by the binding of ligand, and whether such receptors change the underlying rate of the endocytic pathways they utilize. As a model for ligand-stimulated endocytosis, we used transfected HEK293 cells expressing high levels of an epitope-tagged human beta 2-adrenergic receptor. Nearly all receptors were on the cell surface in the absence of agonist, but within ten minutes of agonist addition > 50% of receptors internalized and colocalized extensively with rab5. Hypertonic sucrose blocked beta 2-adrenergic receptor internalization, as well as that of transferrin receptor, suggesting a clathrin-mediated process. In contrast, an inhibitor of potocytosis had little effect upon beta 2-adrenergic receptor internalization, suggesting that this process did not require active caveolae. Consistent with this finding, caveolin was not detectable in the 12 beta 6 line, as assessed by western blotting with a polyclonal anti-caveolin antibody. Stimulated receptor internalization did not affect the rate or capacity of the constitutive endocytic pathway since there was no detectable increase in fluid-phase endocytosis after addition of beta-agonist, nor was there a significant change in the amount of surface transferrin receptor. Altogether, these data suggest that beta 2-adrenergic receptors internalize by a clathrin-mediated and rab5-regulated constitutive endocytic pathway. Further, agonist-stimulated receptor internalization has no detectable effect upon the function of this pathway.

Characterization of Endocytic Pathways by Quantitative Fluorescence Microscopy

1998 ◽  
Vol 4 (S2) ◽  
pp. 1024-1025
Author(s):  
Frederick R. Maxfield ◽  
Richik N. Ghosh ◽  
William G. Mallet ◽  
Thwe Thwe Soe ◽  
Philip L. Leopold ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
Light And Electron Microscopy ◽  
Endocytic Pathway ◽  
Early Endosomes ◽  
Pass Through ◽  
Golgi Network ◽  
Quantitative Fluorescence ◽  
Endocytic Pathways

We have used light and electron microscopy to analyze endocytic trafficking pathways. In one set of studies, we have used fluorescently labeled antibodies to trace an endocytic pathway from the cell surface to the trans- Golgi network (TGN). Cells were transfected with a construct consisting of the transmembrane and cytoplasmic domains of TGN38 and the extracellular domain of Tac. TGN38 is predominantly in the TGN, but a small fraction is found on the cell surface. We used FITC-labeled anti-Tac monoclonal IgG to analyze the pathway from the surface to the TGN. We compared the distribution of internalized Tac-TGN38 to internalized transferrin. We found that most Tac-TGN38 enters the same early endosomes as transferrin. Furthermore, most Tac-TGN38 returns to the cell surface from the endocytic recycling compartment (ERC) at the same rate as transferrin. However, on each pass through the cell approximately 18% of Tac-TGN is retained, and this Tac-TGN38 is delivered to the TGN.

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 Fluid phase endocytosis and galactosyl receptor-mediated endocytosis employ different early endosomes

1999 ◽  
Vol 1421 (2) ◽  
pp. 317-328 ◽  
Author(s):  
Marianne Synnes ◽  
Kristian Prydz ◽  
Torunn Løvdal ◽  
Andreas Brech ◽  
Trond Berg
Keyword(s):  
Fluid Phase ◽  
Receptor Mediated Endocytosis ◽  
Early Endosomes ◽  
Fluid Phase Endocytosis

scholarly journals Low-Dose Fluvoxamine Modulates Endocytic Trafficking of SARS-CoV-2 Spike Protein: A Potential Mechanism for Anti-COVID-19 Protection by Antidepressants

2021 ◽  
Vol 12 ◽  
Author(s):  
Oleg O. Glebov
Keyword(s):  
Membrane Trafficking ◽  
Low Dose ◽  
Protein A ◽  
Antidepressant Drug ◽  
Fluid Phase ◽  
Spike Protein ◽  
Endosomal Trafficking ◽  
Therapeutic Concentration ◽  
Early Endosomes ◽  
Fluid Phase Endocytosis

Commonly prescribed antidepressants may be associated with protection against severe COVID-19. The mechanism of their action in this context, however, remains unknown. Here, I investigated the effect of an antidepressant drug fluvoxamine on membrane trafficking of the SARS-CoV-2 spike protein and its cell host receptor ACE2 in HEK293T cells. A sub-therapeutic concentration (80 nM) of fluvoxamine rapidly upregulated fluid-phase endocytosis, resulting in enhanced accumulation of the spike-ACE2 complex in enlarged early endosomes. Diversion of endosomal trafficking provides a simple cell biological mechanism consistent with the protective effect of antidepressants against COVID-19, highlighting their therapeutic and prophylactic potential.

scholarly journals Effect of Starvation on the Endocytic Pathway in Dictyostelium Cells

2010 ◽  
Vol 9 (3) ◽  
pp. 387-392 ◽  
Author(s):  
Ewan W. Smith ◽  
Wanessa C. Lima ◽  
Steve J. Charette ◽  
Pierre Cosson
Keyword(s):  
Cell Surface ◽  
Functional Organization ◽  
General Structure ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Rich Medium ◽  
Content Type ◽  
Structure And Dynamics ◽  
Endocytic Compartments ◽  
Mutant Cells

ABSTRACT Dictyostelium discoideum amoebae have been used extensively to study the structure and dynamics of the endocytic pathway. Here, we show that while the general structure of the endocytic pathway is maintained in starved cells, its dynamics rapidly slow down. In addition, analysis of apm3 and lvsB mutants reveals that the functional organization of the endocytic pathway is profoundly modified upon starvation. Indeed, in these mutant cells, some of the defects observed in rich medium persist in starved cells, notably an abnormally slow transfer of endocytosed material between endocytic compartments. Other parameters, such as endocytosis of the fluid phase or the rate of fusion of postlysosomes to the cell surface, vary dramatically upon starvation. Studying the endocytic pathway in starved cells can provide a different perspective, allowing the primary (invariant) defects resulting from specific mutations to be distinguished from their secondary (conditional) consequences.

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