scholarly journals Arf6-independent GPI-anchored Protein-enriched Early Endosomal Compartments Fuse with Sorting Endosomes via a Rab5/Phosphatidylinositol-3′-Kinase–dependent Machinery

2006 ◽  
Vol 17 (8) ◽  
pp. 3689-3704 ◽  
Author(s):  
Manjula Kalia ◽  
Sudha Kumari ◽  
Rahul Chadda ◽  
Michelle M. Hill ◽  
Robert G. Parton ◽  
...  
Keyword(s):  
Fluid Phase ◽  
Endocytic Pathway ◽  
Phosphatidylinositol 3 Kinase ◽  
Extracellular Milieu ◽  
Pi3k Inhibition ◽  
Independent Mechanism ◽  
A Cell ◽  
Endocytic Vesicles ◽  
Endocytic Pathways ◽  
Phosphatidylinositol 3

In the process of internalization of molecules from the extracellular milieu, a cell uses multiple endocytic pathways, consequently generating different endocytic vesicles. These primary endocytic vesicles are targeted to specific destinations inside the cell. Here, we show that GPI-anchored proteins are internalized by an Arf6-independent mechanism into GPI-anchored protein-enriched early endosomal compartments (GEECs). Internalized GPI-anchored proteins and the fluid phase are first visualized in GEECs that are acidic, primary endocytic structures, negative for early endosomal markers, Rab4, Rab5, and early endosome antigen (EEA)1. They subsequently acquire Rab5 and EEA1 before homotypic fusion with other GEECs, and heterotypic fusion with endosomes containing cargo from the clathrin-dependent endocytic pathway. Although, the formation of GEECs is unaffected by inhibition of Rab5 GTPase and phosphatidylinositol-3′-kinase (PI3K) activity, their fusion with sorting endosomes is dependent on both activities. Overexpression of Rab5 reverts PI3K inhibition of fusion, providing evidence that Rab5 effectors play important roles in heterotypic fusion between the dynamin-independent GEECs and clathrin- and dynamin-dependent sorting endosomes.

Regulation of fluid-phase endocytosis in alveolar macrophages

1995 ◽  
Vol 269 (4) ◽  
pp. L520-L526 ◽  
Author(s):  
G. Pataki ◽  
L. Czopf ◽  
T. Jilling ◽  
N. Marczin ◽  
J. Catravas ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Fluid Phase ◽  
Fluorescein Isothiocyanate ◽  
The Other ◽  
Camp Content ◽  
Cyclic Monophosphate ◽  
Fluid Phase Endocytosis ◽  
Independent Mechanism ◽  
A Cell ◽  
Methyl Xanthine

We investigated whether fluid-phase endocytosis in rabbit alveolar macrophages (AM) was regulated by alterations in intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Suspensions of freshly isolated AM were incubated with anionic dextrans (mol mass = 10 kDa), coupled to fluorescein isothiocyanate (FITC), at either 37 or 4 degrees C. There was a rapid increase in AM-associated fluorescence, quantified by laser flow-cytometry and video microscopy during the first hour of incubation at 37 degrees C, which was directly proportional to the amount of tracer present in the medium. In contrast, at 4 degrees C, AM fluorescence was similar to autofluorescence. Incubation of AM with forskolin (50 microM) or 3-isobutyl-1-methyl xanthine (IBMX; 0.1 mM) increased their cAMP content by 67 +/- 2 and 52 +/- 5% (mean +/- SE; n = 4) and decreased FITC-dextran uptake by 29 +/- 4 and 31 +/- 4% (n = 3). On the other hand, incubation of AM with 0.5 mM IBMX inhibited FITC-dextran uptake by 62 +/- 4% (n = 3), without any further increase in cAMP. Incubation of AM with 0.4 mM 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP), a cell-permeable analogue of cAMP, decreased FITC-dextran uptake by 48 +/- 5% (n = 6). Pulse-chase experiments showed that the rate of FITC-dextran exocytosis was not affected by cAMP. We concluded that fluid-phase endocytosis in rabbit AM is regulated by cAMP and by an additional, cAMP-independent mechanism of IBMX.

CD28 can promote T cell survival through a phosphatidylinositol 3-kinase-independent mechanism

1997 ◽  
Vol 27 (12) ◽  
pp. 3283-3289 ◽  
Author(s):  
Yves Collette ◽  
D. Razanajaona ◽  
M. Ghiotto ◽  
Daniel Olive
Keyword(s):  
T Cell ◽  
Cell Survival ◽  
Phosphatidylinositol 3 Kinase ◽  
Independent Mechanism ◽  
T Cell Survival ◽  
Phosphatidylinositol 3

scholarly journals Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro.

1996 ◽  
Vol 7 (3) ◽  
pp. 355-367 ◽  
Author(s):  
D J Spiro ◽  
W Boll ◽  
T Kirchhausen ◽  
M Wessling-Resnick
Keyword(s):  
Transferrin Receptor ◽  
Kinase Inhibitor ◽  
Morphological Changes ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Phosphatidylinositol 3 Kinase ◽  
Vitro Assay ◽  
Phosphatidylinositol 3

Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin receptors. This effect is rapid and dose dependent, with maximal down-regulation elicited with 30 min of treatment and with an IC50 approximately 25 nM wortmannin. Wortmannin-treated cells display an increased endocytic rate constant for transferrin internalization and decreased exocytic rate constants for transferrin recycling. In addition to these effects in vivo, wortmannin is a potent inhibitor (IC50 approximately 15 nM) of a cell-free assay that detects the delivery of endocytosed probes into a common compartment. Inhibition of the in vitro assay involves the inactivation of a membrane-associated factor that can be recruited onto the surface of vesicles from the cytosol. Its effects on the cell-free assay suggest that wortmannin inhibits receptor sorting and/or vesicle budding required for delivery of endocytosed material to "mixing" endosomes. This idea is consistent with morphological changes induced by wortmannin, which include the formation of enlarged transferrin-containing structures and the disruption of the perinuclear endosomal compartment. However, the differential effects of wortmannin, specifically increased transferrin receptor internalization and inhibition of receptor recycling, implicate a role for phosphatidylinositol 3-kinase activity in multiple sorting events in the transferrin receptor's membrane traffic pathway.

FITC-dextran as a probe for endosome function and localization in kidney

1990 ◽  
Vol 258 (2) ◽  
pp. C309-C317 ◽  
Author(s):  
W. I. Lencer ◽  
P. Weyer ◽  
A. S. Verkman ◽  
D. A. Ausiello ◽  
D. Brown
Keyword(s):  
Epithelial Cells ◽  
Water Channel ◽  
Fluorescein Isothiocyanate ◽  
Endocytic Pathway ◽  
Water Permeability Coefficient ◽  
Osmotic Water ◽  
Endocytic Vesicles ◽  
Excised Tissue ◽  
Specific Membrane ◽  
Endocytic Pathways

Fluorescein isothiocyanate (FITC)-labeled endosomes were localized in kidney epithelial cells after tissue fixation and sectioning, and specific membrane transport properties of isolated endocytic vesicles were measured using the same probe. Rats were infused intravenously with 10 kDa FITC-dextran, and kidneys were fixed with paraformaldehyde lysine periodate. FITC-labeled vesicles were visualized in semithin (1 micron) frozen sections of excised tissue by epifluorescent microscopy and by electron microscopy after a photoconversion reaction. Most FITC-labeled endosomes were apically located in epithelial cells lining the urinary tubules. By immunocytochemistry the anti-lysosomal glycoprotein LGP 120 was absent from most of the FITC-labeled vesicles, although some colocalization was noted. The limiting membrane of FITC-labeled endosomes contained a vacuolar proton pump (pHmin = 6.23 +/- 0.033) and a water channel (osmotic water permeability coefficient, Pf = 0.052 +/- 0.005 cm/s) and was highly permeable to ethylene glycol and urea but relatively impermeable to glucose. Methods allowing the attribution of specific membrane functions to vesicles that can be visualized in the apical endocytic pathway of epithelial cells should be of general use for the study of endocytic pathways in a variety of systems.

Sign in / Sign up

Export Citation Format

Share Document