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.

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 Early Endosomes Are Required for Major Histocompatiblity Complex Class II Transport to Peptide-loading Compartments

1999 ◽  
Vol 10 (9) ◽  
pp. 2891-2904 ◽  
Author(s):  
Valérie Brachet ◽  
Gérard Péhau-Arnaudet ◽  
Catherine Desaymard ◽  
Graça Raposo ◽  
Sebastian Amigorena
Keyword(s):  
Mhc Class Ii ◽  
Class Ii ◽  
Endocytic Pathway ◽  
Cross Linking ◽  
Histocompatibility Complex ◽  
Early Endosomes ◽  
Peptide Loading ◽  
Golgi Network ◽  
Mhc Class Ii Molecules

Antigen presentation to CD4+ T lymphocytes requires transport of newly synthesized major histocompatibility complex (MHC) class II molecules to the endocytic pathway, where peptide loading occurs. This step is mediated by a signal located in the cytoplasmic tail of the MHC class II-associated Ii chain, which directs the MHC class II-Ii complexes from the trans-Golgi network (TGN) to endosomes. The subcellular machinery responsible for the specific targeting of MHC class II molecules to the endocytic pathway, as well as the first compartments these molecules enter after exit from the TGN, remain unclear. We have designed an original experimental approach to selectively analyze this step of MHC class II transport. Newly synthesized MHC class II molecules were caused to accumulate in the Golgi apparatus and TGN by incubating the cells at 19°C, and early endosomes were functionally inactivated by in vivo cross-linking of transferrin (Tf) receptor–containing endosomes using Tf-HRP complexes and the HRP-insoluble substrate diaminobenzidine. Inactivation of Tf-containing endosomes caused a marked delay in Ii chain degradation, peptide loading, and MHC class II transport to the cell surface. Thus, early endosomes appear to be required for delivery of MHC class II molecules to the endocytic pathway. Under cross-linking conditions, most αβIi complexes accumulated in tubules and vesicles devoid of γ-adaptin and/or mannose-6-phosphate receptor, suggesting an AP1-independent pathway for the delivery of newly synthesized MHC class II molecules from the TGN to 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.

Mucolipidosis Type III α/β: The First Characterization of This Rare Disease by Autopsy

2011 ◽  
Vol 135 (4) ◽  
pp. 503-510
Author(s):  
Darcy A Kerr ◽  
Vincent A Memoli ◽  
Sara S Cathey ◽  
Brent T Harris
Keyword(s):  
Electron Microscopy ◽  
Heart Valves ◽  
Light And Electron Microscopy ◽  
Lysosomal Storage ◽  
Cytoplasmic Granules ◽  
Type Iii ◽  
Mucolipidosis Type ◽  
Electron Lucent ◽  
And Cartilage

Abstract We report findings from an autopsy of a 45-year-old woman with the rare lysosomal storage disease mucolipidosis type III α/β. Her disease manifested most notably as multiple bone and cartilage problems with tracheal and bronchial malacia. Principal autopsy findings included gross abnormalities in bone and cartilage with corresponding microscopic cytoplasmic lysosomal granules. These cytoplasmic granules were also seen in histologic preparations of the brain, myocardium, heart valves, and fibroblasts of the liver and skin by light and electron microscopy. By electron microscopy there were scattered, diffuse vesicular cytoplasmic granules in neurons and glia and an increase in lysosomal structures with fine electron lucent granularity in the above tissue types. Our findings help elaborate current understanding of this disease and differentiate it from the mucopolysaccharidoses and related disorders. To our knowledge, this is the first report to document pathologic findings in a patient with mucolipidosis type III α/β by autopsy.

scholarly journals The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3289-3298 ◽  
Author(s):  
Wolfram Antonin ◽  
Claudia Holroyd ◽  
Ritva Tikkanen ◽  
Stefan Höning ◽  
Reinhard Jahn
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Subcellular Fractionation ◽  
Immunogold Electron Microscopy ◽  
Fusion Reactions ◽  
Coated Pits ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Golgi Network

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

scholarly journals A versatile nanobody-based toolkit to analyze retrograde transport from the cell surface

2018 ◽  
Vol 115 (27) ◽  
pp. E6227-E6236 ◽  
Author(s):  
Dominik P. Buser ◽  
Kai D. Schleicher ◽  
Cristina Prescianotto-Baschong ◽  
Martin Spiess
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
Adaptor Protein ◽  
Retrograde Transport ◽  
Transport Pathways ◽  
Consensus Sequences ◽  
Retrograde Traffic ◽  
Biochemical Detection ◽  
Golgi Network ◽  
Kinetics Of

Retrograde transport of membranes and proteins from the cell surface to the Golgi and beyond is essential to maintain homeostasis, compartment identity, and physiological functions. To study retrograde traffic biochemically, by live-cell imaging or by electron microscopy, we engineered functionalized anti-GFP nanobodies (camelid VHH antibody domains) to be bacterially expressed and purified. Tyrosine sulfation consensus sequences were fused to the nanobody for biochemical detection of trans-Golgi arrival, fluorophores for fluorescence microscopy and live imaging, and APEX2 (ascorbate peroxidase 2) for electron microscopy and compartment ablation. These functionalized nanobodies are specifically captured by GFP-modified reporter proteins at the cell surface and transported piggyback to the reporters’ homing compartments. As an application of this tool, we have used it to determine the contribution of adaptor protein-1/clathrin in retrograde transport kinetics of the mannose-6-phosphate receptors from endosomes back to the trans-Golgi network. Our experiments establish functionalized nanobodies as a powerful tool to demonstrate and quantify retrograde transport pathways.

Characterization of Photochemically Induced Spinal Cord Injury in the Rat by Light and Electron Microscopy

1994 ◽  
Vol 127 (1) ◽  
pp. 76-93 ◽  
Author(s):  
Mary Bartlett Bunge ◽  
Vicky R. Holets ◽  
Margaret L. Bates ◽  
Theresa S. Clarke ◽  
Brant D. Watson
Keyword(s):  
Electron Microscopy ◽  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Light And Electron Microscopy ◽  
Cord Injury

Characterization of the Uptake and Intracellular Trafficking of G4 Polyamidoamine Dendrimers

2011 ◽  
Vol 64 (3) ◽  
pp. 302
Author(s):  
Chin-Ling Pai ◽  
Ming-Jium Shieh ◽  
Pei-Jen Lou ◽  
Fei-Hong Huang ◽  
Tzu-Wen Wang ◽  
...  
Keyword(s):  
Intracellular Trafficking ◽  
Fluorescein Isothiocyanate ◽  
Pamam Dendrimers ◽  
Endocytic Pathway ◽  
Lysosomal Ph ◽  
Cellular Environment ◽  
Polyamidoamine Dendrimers ◽  
Energy Dependent ◽  
Endocytic Pathways

Polyamidoamine (PAMAM) dendrimers are highly branched spherical polymers that have emerged as potent synthetic drug and gene carriers; however, much remains to be learned about the mechanism of dendrimer-mediated cellular uptake. In this study, the endocytic pathway and intracellular trafficking of generation 4 (G4) PAMAM dendrimers were evaluated via fluorescein isothiocyanate (FITC) conjugation. We found that the G4-FITC dendrimers were internalized by energy-dependent and non-specific endocytic pathways. Interesting, G4-FITC dendrimers can not only buffer the endosomal/lysosomal pH but also co-localize with lysosomal markers over a period of 3 to 12 h, after which the signal decreased in the lysosomes and began to co-localize with the mitochondrial marker. This study contributes to the understanding of the molecular behaviour of G4 PAMAM dendrimers in a cellular environment and will facilitate the development of more effective PAMAM-mediated drug and gene delivery systems.

Production and characterization of monoclonal antibodies to the sapstaining fungus Ophiostoma piceae

1994 ◽  
Vol 40 (1) ◽  
pp. 35-44 ◽  
Author(s):  
Srabani Banerjee ◽  
Judy Little ◽  
Maria Chan ◽  
Brian T. Luck ◽  
Colette Breuil ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Monoclonal Antibodies ◽  
Light And Electron Microscopy ◽  
Cross Reactivity ◽  
Cell Wall Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Enzymatic Modification ◽  
Ophiostoma Piceae

A sensitive immunological tool has been developed to detect the sapstaining fungus Ophiostoma piceae 3871, which plagues the wood industry. Monoclonal antibodies (1F3(1), 4G3(14), 4G2(4), and 2B6(24)) produced against cell wall protein extracts of this fungus were specific. Specificity was estimated by enzyme linked immunosorbent assay, western blotting, and light and electron microscopy using the immunogold technique. Electron microscopy revealed gold particles localized on the outer surface of the cell wall. When screened against 24 biological control fungi the antibodies showed pratically no cross-reactivity (< 4%). When tested against 19 other staining fungi, the antibodies recognized three strains of Ophiostoma piceae, 1F3(1) recognized Phialophora botulispora, and the antibodies showed less than 5% reactivity with the other fungi. Chemical and enzymatic modification of the antigen revealed that the epitopes recognized by the monoclonal antibodies were glycospecific. Although the antibodies were produced against the cell wall protein extracts of the fungus grown in liquid culture, they also recognized the fungus growing in wood and therefore can be employed to investigate wood colonization by this fungus.Key words: Ophiostoma piceae, monoclonal antibodies, glycoprotein.

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