Different endocytic compartments are involved in the tight association of class II molecules with processed hen egg lysozyme and ribonuclease A in B cells

1995 ◽  
Vol 108 (6) ◽  
pp. 2337-2345 ◽  
Author(s):  
J.M. Escola ◽  
J.C. Grivel ◽  
P. Chavrier ◽  
J.P. Gorvel
Keyword(s):  
Fluid Phase ◽  
Subcellular Fractionation ◽  
Class Ii ◽  
Ribonuclease A ◽  
Endocytic Pathway ◽  
Late Endosomes ◽  
Stable Class ◽  
Endocytic Compartments ◽  
Hen Egg ◽  
Hen Egg Lysozyme

The processing of exogenous antigens and the association of peptides with class II molecules both occur within the endocytic pathway. 2A4 B lymphoma cells of the H-2k haplotype were grown in the presence or the absence of two different exogenous antigens (hen egg lysozyme and ribonuclease A) internalized by fluid-phase endocytosis. Using subcellular fractionation techniques, we demonstrate that, in the presence of hen egg lysozyme, newly synthesized SDS-stable class II molecules are detected in a dense endocytic compartment which does not have the characteristics of neither early and late endosomes nor lysosomes. In contrast, no SDS-stable class II molecules are observed between ribonuclease A and newly synthesized class II molecules. Interestingly, when class II molecules are analyzed at steady state, SDS-stable class II molecules induced by ribonuclease A are found in a compartment cosedimenting with late endosomes. These results suggest that the tight associations between ribonuclease A or hen egg lysozyme with class II molecules occur in distinct endocytic compartments and that these associations may depend on the sensitivity of antigens to proteolysis.

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.

Trafficking of interleukin 2 and transferrin in endosomal fractions of T lymphocytes

1994 ◽  
Vol 107 (5) ◽  
pp. 1289-1295 ◽  
Author(s):  
V. Duprez ◽  
M. Smoljanovic ◽  
M. Lieb ◽  
A. Dautry-Varsat
Keyword(s):  
Horseradish Peroxidase ◽  
Interleukin 2 ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Recycling Endosomes ◽  
High Affinity ◽  
Human T Cell ◽  
Late Endosomes ◽  
Discontinuous Sucrose Gradient ◽  
Acidic Organelles

The T lymphocyte growth factor interleukin 2 binds to surface high-affinity receptors and is rapidly internalized and degraded in acidic organelles. The alpha and beta chains of high-affinity interleukin 2 receptors are internalized together with interleukin 2. To identify the intracellular pathway followed by interleukin 2, we have compared the subcellular distribution of interleukin 2, transferrin and a fluid-phase marker, horseradish peroxidase, in the human T cell line IARC 301.5. Transferrin was used as a marker of early and recycling endosomes, and horseradish peroxidase to probe for the whole endocytic pathway. Fractionation of intracellular organelles on a discontinuous sucrose gradient showed that internalized interleukin 2 is initially mostly found in compartments with similar densities to transferrin, e.g. early and recycling endosomes. The kinetics of entry and exit of interleukin 2 from such organelles was much slower than that of transferrin. Later on, interleukin 2 is predominantly found in dense lysosome-containing fractions. Very little, if any, interleukin 2 was found in fractions corresponding to late endosomes containing horseradish peroxidase. These results suggest that, after endocytosis, interleukin 2 enters early or recycling endosomes before it reaches dense lysosomes.

Rab22a affects the morphology and function of the endocytic pathway

2001 ◽  
Vol 114 (22) ◽  
pp. 4041-4049 ◽  
Author(s):  
Rosana Mesa ◽  
Cristina Salomón ◽  
Marcelo Roggero ◽  
Philip D. Stahl ◽  
Luis S. Mayorga
Keyword(s):  
Fluorescent Protein ◽  
Fluid Phase ◽  
Small Gtpases ◽  
Endocytic Pathway ◽  
Site Directed Mutagenesis ◽  
Rab Proteins ◽  
Rab Protein ◽  
Late Endosomes ◽  
And Function ◽  
Negative Mutant

Soon after endocytosis, internalized material is sorted along different pathways in a process that requires the coordinated activity of several Rab proteins. Although abundant information is available about the subcellular distribution and function of some of the endocytosis-specific Rabs (e.g. Rab5 and Rab4), very little is known about some other members of this family of proteins. To unveil some of the properties of Rab22a, one of the less studied endosome-associated small GTPases, we have expressed the protein tagged with the green fluorescent protein in CHO cells. The results indicate that Rab22a associates with early and late endosomes (labeled by a 5 minute rhodamine-transferrin uptake and the cation-independent mannose 6-phosphate receptor, respectively) but not with lysosomes (labeled by 1 hour rhodamine horseradish peroxidase uptake followed by 1 hour chase). Overexpression of the protein causes a prominent morphological enlargement of the early and late endosomes. Two mutants were generated by site-directed mutagenesis, a negative mutant (Rab22aS19N, with reduced affinity for GTP) and a constitutively active mutant (Rab22aQ64L, with reduced endogenous GTPase activity). The distribution of the negative mutant was mostly cytosolic, whereas the positive mutant associated with early and late endosomes and, interestingly also with lysosomes and autophagosomes (labeled with monodansylcadaverine). Cells expressing Rab22a wild type and Rab22aS19N displayed decreased endocytosis of a fluid phase marker. Conversely, overexpression of Rab22aQ64L, which strongly affects the morphology of endosomes, did not inhibit bulk endocytosis. Our results show that Rab22a has a unique distribution along the endocytic pathway that is not shared by any other Rab protein, and that it strongly affects the morphology and function of endosomes.

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.

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.

scholarly journals Molecular modeling of hen egg lysozyme HEL[52-61] peptide binding to I-Ak MHC class II molecule.

1998 ◽  
Vol 10 (12) ◽  
pp. 1753-1764 ◽  
Author(s):  
P. Weber ◽  
I. Raynaud ◽  
L. Ettouati ◽  
M. C. Trescol-Biemont ◽  
P. A. Carrupt ◽  
...  
Keyword(s):  
Molecular Modeling ◽  
Mhc Class Ii ◽  
Peptide Binding ◽  
Class Ii ◽  
Hen Egg ◽  
Hen Egg Lysozyme

scholarly journals Class II-restricted presentation of an endogenously derived immunodominant T-cell determinant of hen egg lysozyme.

1991 ◽  
Vol 88 (8) ◽  
pp. 3290-3294 ◽  
Author(s):  
A. Brooks ◽  
S. Hartley ◽  
L. Kjer-Nielsen ◽  
J. Perera ◽  
C. C. Goodnow ◽  
...  
Keyword(s):  
T Cell ◽  
Class Ii ◽  
Hen Egg ◽  
Hen Egg Lysozyme

Cytosolic targeting of hen egg lysozyme gives rise to a short-lived protein presented by class I but not class II major histocompatibility complex molecules

1991 ◽  
Vol 21 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Veronique Calin-Laurens ◽  
Frédérique Forquet ◽  
Denis Gerlier ◽  
Chantal Rabourdin-Combe ◽  
Estelle Mottez ◽  
...  
Keyword(s):  
Major Histocompatibility Complex ◽  
Class Ii ◽  
Class I ◽  
Major Histocompatibility ◽  
Complex Molecules ◽  
Histocompatibility Complex ◽  
Hen Egg ◽  
Hen Egg Lysozyme

scholarly journals Association of Rap1a and Rap1b proteins with late endocytic/phagocytic compartments and Rap2a with the Golgi complex

1994 ◽  
Vol 107 (6) ◽  
pp. 1661-1670 ◽  
Author(s):  
V. Pizon ◽  
M. Desjardins ◽  
C. Bucci ◽  
R.G. Parton ◽  
M. Zerial
Keyword(s):  
Golgi Complex ◽  
Mammalian Cells ◽  
Subcellular Fractionation ◽  
Small Gtpases ◽  
Intracellular Location ◽  
Confocal Immunofluorescence Microscopy ◽  
Late Endosomes ◽  
Confocal Immunofluorescence ◽  
Ras Family ◽  
Endocytic Compartments

Among the small GTPases of the Ras family, Rap proteins exhibit the highest homology with p21Ras. The four Rap proteins so far identified constitute two subgroups, comprising the Rap1(A,B) and the Rap2(A,B) proteins. The intracellular location of Rap1A, Rap1B and Rap2A proteins was investigated in mammalian cells by confocal immunofluorescence microscopy. Using a specific anti-Rap1 affinity-purified antibody, both Rap1A and Rap1B proteins were localized to late endocytic compartments (late endosomes/lysosomes) in fibroblasts. The localization of the Rap1A and B proteins transiently overexpressed with the vaccinia T7 system was identical to that observed for endogenous Rap1 proteins. In contrast, epitope-tagged Rap2A protein colocalized with several markers of the Golgi complex, thus indicating that its site of function was distinct from that of Rap1A. In addition, morphological and subcellular fractionation studies provided evidence for the association of Rap1 proteins with phagosomes displaying biochemical features of late endocytic structures in J774 macrophages. Thus, the localization of Rap1A and Rap1B implicates their involvement in late endocytic/phagocytic processes.

Characterization of endocytosis in intact cells by quantitative fluorescence microscopy

1991 ◽  
Vol 49 ◽  
pp. 234-235
Author(s):  
Frederick R. Maxfield
Keyword(s):  
Lipid Membrane ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Intact Cells ◽  
Full Field ◽  
Intensity Measurements ◽  
Endocytic Compartments ◽  
Microscopy Techniques ◽  
Fluorescent Molecules

Endocytosis, the process by which extracellular macromolecules are taken into the cell is particularly amenable to quantitative analysis using fluorescence techniques. Fluorescently-labeled proteins and other macromolecules are taken up by cells via receptor-mediated endocytosis or fluid phase pinocytosis. Recently, fluorescent lipid probes have also been synthesized which label the endocytic pathway, allowing observation of lipid membrane traffic.Digital imaging allows the manipulation of images, background and autofluorescence subtractions, and fluorescence intensity measurements of individual endocytic compartments. Image intensification microscopy and digital image processing have been used extensively for studies of endocytosis, including the measurements of endosome pH, and the identification and quantitation of endocytic vesicle fusion. Recently, pathways taken by endocytosed fluorescent molecules have been followed using the confocal microscope, adding a powerful tool to investigate questions about endocytosis which were very difficult to study with conventional, full-field microscopy techniques.

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