Name: purification of the membrane compartment for endoplasmic reticulum-associated degradation of exogenous antigens in cross-presentation
Uploaded: 2017-08-21T14:00:00.000+00:00
Duration: 12 min 48 s
Description: Watch this Scientific Journal Video about Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation at JoVE.com

This work is supported by the Takasaki University of Health and Welfare.

1. Growing Cells and Addition of Exogenous Antigens
<ol>
	<li>Prepare bOVA using a biotin-protein labeling kit following the manufacturer&#39;s protocol. 
	NOTE: Ordinarily, bOVA contains 2 M biotin per 1 M OVA on average.</li>
	<li>Grow DC2.4 cells in RPMI-1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/mL penicillin-streptomycin, 55 mM 2-mercaptoethanol, 10 mM HEPES (pH 7.5), and 10% fetal calf serum (hereafter RPMI) at 37 &#176;C in 5% CO2 in a humidified incubator (hereafter without mention, cells are incubated at this condition). It is also possible to use DMEM supplemented with 10% fetal calf serum, 3.7 g/L NaHCO3 (hereafter DMEM) in place of RPMI.</li>
	<li>A day before purification, split the cells into RPMI to 1 x 105 cells/mL in a tissue culture plate. Avoid keeping the cells at a confluent state. DC2.4 cells can highly incorporate exogenous antigens until a semi-confluent state, but rapidly lose the ability after reaching an over-confluent state.</li>
	<li>Before the DC2.4 cells are semi-confluent, add 250 &#181;g/mL of bOVA for 1 x 106 cells/mL and incubate for 2 - 4 h. 
	NOTE: During downstream experiments, all buffers and reagents are kept at 4 &#176;C unless otherwise indicated.</li>
</ol>
2. Preparation of Microsomes
<ol>
	<li>Harvest the DC2.4 cells by gentle pipetting from the tissue plate into a new 50 mL conical tube.</li>
	<li>Centrifuge at 1,000 x g for 5 min, 4 &#176;C and carefully remove the medium with bOVA by aspiration.</li>
	<li>Wash the DC2.4 cells twice with 40 mL of PBS buffer (1.37 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4) by centrifugation at 1,000 x g for 5 min, 4 &#176;C and discard the supernatant each time by aspiration.</li>
	<li>Resuspend the pellet in step 2.3 in 1 - 2 mL (1/20-1/10 volume of culture medium) of homogenization medium (0.25 M sucrose, 1 mM EDTA, 10 mM HEPES-NaOH [pH 7.4]) with 1/1,000 volume of protease inhibitor cocktails and transfer the resuspended DC2.4 cells into an ice-cold Dounce homogenizer.</li>
	<li>Disrupt the resuspended DC2.4 cells gently by 10 - 20 strokes with the ice-cold Dounce homogenizer. 
	NOTE: During the disruption step, the Dounce homogenizer is cooled on ice.</li>
	<li>Transfer the cell-disrupted suspension to a new 15 mL conical tube.</li>
	<li>Add 8 - 9 mL homogenization medium to 10 mL total and centrifuge the conical tube for 10 min at 2,000 x g and 4 &#176;C.</li>
	<li>Transfer the supernatant to a new 15-mL conical tube to remove unbroken cells and nuclei, and centrifuge the conical tube again for 10 min at 2,000 x g and 4 &#176;C.</li>
	<li>Transfer the supernatant into a new ultracentrifugation tube and centrifuge for 45 min at 100,000 x g and 4 &#176;C.</li>
	<li>Aspirate the supernatant and resuspend the pellet carefully in 1 - 2 mL of homogenization medium with 1/1,000 volume of protease inhibitor cocktails by pipetting the solution up and down several times to make a microsome fraction for downstream experiments.</li>
	<li>Transfer the microsome fraction into a new 5-mL round bottom tube. 
	NOTE: After this step, it is possible to enrich objective microsomes by iodixanol density gradient centrifugation according to the manufacturer&#39;s protocol, to remove non-specific microsomes. The peak fractions for exogenous antigens are collected and then subjected to the next step of purification (step 3).</li>
</ol>
3. Purification of Microsomes with bOVA Undergoing ERAD
<ol>
	<li>Add 1/100 volume of fresh SA-magnetic beads to the microsome fraction of step 2.11 in the 5 mL round bottom tube. 
	NOTE: Before this step, it is possible to pre-clear the microsome fraction by control-magnetic beads to reduce contaminations of non-specific microsomes.</li>
	<li>Gently mix well and rotate the round bottom tube slowly for 30 min at 4 &#176;C.</li>
	<li>Add 2-3 mL of homogenization medium to 4 mL total into the round bottom tube and gently mix well.</li>
	<li>Place the round bottom tube on a magnetic stand and incubate for 10 min at 4 &#176;C. Since the beads bound to the vesicles are attracted to the magnet, brown micro-beads will gradually accumulate to the tube wall closest to the magnet.</li>
	<li>With the tube remaining in the magnetic stand, carefully discard the supernatants by aspiration to remove the unbound vesicles.</li>
	<li>Wash the magnetic beads bound to the vesicles twice with 5 mL of homogenization medium by the magnetic stand for 10 min at 4 &#176;C and discard the supernatant each time by carefully aspiration. 
	NOTE: Without the magnetic stand, purification by centrifugations at 2,000 x g for 10 min, 4 &#176;C is also available.</li>
	<li>Resuspend the magnetic beads bound to the vesicles carefully in 100 &#181;L homogenization medium by pipetting the solution up and down several times.</li>
	<li>Transfer the resuspended vesicles into a new microtube as the purified microsome for downstream experiments. 
	NOTE: Typically, 50 &#181;L microsome fraction containing 5 - 20 &#181;g proteins from 1 x 107 cells can be isolated.</li>
</ol>
4. Analysis of the Purified Microsomes
<ol>
	<li>Resuspend the magnetic beads bound to the vesicles from step 3.8 by 100 - 200 &#181;L of TNE buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5 M EDTA, 1% Nonidet P-40) with 1/1,000 volume of protease inhibitor cocktails instead of the homogenization medium.</li>
	<li>Transfer the lysate from step 4.1 to a new 1.5-mL microtube.</li>
	<li>Determine the protein concentration of step 4.2 by using a BCA assay kit per the manufacturer&#39;s protocol. 
	NOTE: Typically, 5 - 10 &#181;L lysate from step 4.2 is enough to determine the protein concentration.</li>
	<li>Transfer the lysate from step 4.2 (2 &#181;g of protein for silver staining and 10 &#181;g of protein for Western blotting) into new microtubes.</li>
	<li>Put the microtubes on a heat block at 95 &#176;C and boil proteins in 1x SDS gel-loading buffer (100 mM Tris-HCl pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) for 5 min.</li>
	<li>Centrifuge the microtubes for 10 min at 21,500 x g and 4 &#176;C. Collect the supernatants into new microtubes to remove the insoluble fractions.</li>
	<li>Analyze the resolved proteins in step 4.6 (2 &#181;g) by standard SD-PAGE.</li>
	<li>Visualize the protein bands by the silver staining using silver staining kit following the manufacturer&#39;s protocol.</li>
	<li>Analyze the resolved proteins in step 4.6 (10 &#181;g) by standard SD-PAGE and Western-blotting. Use the reagent (SA-HRP) per the manufacturer&#39;s protocol and visualize by fluorography.</li>
</ol>
5. In Vitro Reconstitution of ERAD Ubiquitination of bOVA Using Purified Microsomes
<ol>
	<li>Transfer the purified microsomes (5 - 10 &#181;g of protein) from step 3.8 with a 50% volume of reticulocyte lysate (RL), in 1x reaction buffer (50 mM Tris pH 7.4, 3 mM ATP, 0.5 mM MgCl2), and 0.2 pM Flag-tagged ubiquitin in a new microtube. The final volume of this experiment is 20 - 40 &#181;L.</li>
	<li>Incubate the microtube for 2 h at 37 &#176;C. 
	NOTE: If the amount of ubiquitinated bOVA in step 5.12 is too small to detect by Western blotting, add 10 &#181;M of MG132 or 2 &#181;M of lactacystin to inhibit the processing of the ubiquitinated bOVA by proteasomes.</li>
	<li>Stop the reaction by placing the microtube on ice.</li>
	<li>Resolve the microsomes by adding 500 &#181;L TNE buffer with 1/1,000 volume of protease inhibitor cocktails.</li>
	<li>Centrifuge the microtube for 10 min at 21,500 x g and 4 &#176;C. Collect the supernatants into a new microtube to remove the insoluble fractions, which contain ubiquitinated bOVA in DC2.4 before the in vitro ubiquitination assay.</li>
	<li>Transfer the supernatant to a new microtube and add 1/100 volume of new SA-magnetic beads (usually 5 &#181;L for 500 &#181;L of supernatants).</li>
	<li>Gently mix well and rotate the microtube slowly for 30 min at 4 &#176;C.</li>
	<li>Centrifuge the microtube for 10 min at 21,500 x g and 4 &#176;C. Discard the supernatant by aspiration to recover the bOVA and ubiquitinated bOVA bound with the SA-magnetic beads.</li>
	<li>Wash twice the collected SA-magnetic beads with 1 mL of TNE buffer by centrifugation for 10 min at 21,500 x g and 4 &#176;C. Discard the supernatant each time by aspiration.</li>
	<li>Boil the SA-magnetic beads in 1x SDS gel loading buffer for 5 min at 95 &#176;C on a heat-block to resolve the purified proteins by the SA-magnetic beads.</li>
	<li>Centrifuge the microtube for 10 min at 21,500 x g and 4 &#176;C. Collect the supernatants into a new microtube to remove the insoluble fractions.</li>
	<li>Analyze the SA-magnetic beads bound to the proteins by standard SD-PAGE and Western-blotting. The use of antibodies (anti-Flag, anti-multi-Ub, and anti-mouse IgG-HRP) and the reagent (SA-HRP) is per the manufacturer&#39;s protocol and visualized by fluorography.</li>
</ol>

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The authors have nothing to disclose.

In previous studies of CP, the incorporated exogenous antigens accumulated in the restricted area of the late endosome or ER by immunofluorescent microscopy16,30,31,32. It is estimated that ERAD-like transport and processing of exogenous antigens are carried out in these specialized areas of the ER or late endosome, as the cellular compartment was identified by sucrose or iodixanol density grad...

The MHC I molecules are expressed on the surface of all nucleated cells, with short antigenic peptides derived from endogenous antigens, which are processed by the ubiquitin-proteasome system in the cytosol1. After processing, antigenic peptides are transported into the endoplasmic reticulum (ER) lumen by the peptide transporter TAP. In the ER lumen, a series of specific chaperones assist the peptide loading and the correct folding of the MHC I complex. This series of molecules is called the peptide-loading complex (PLC), indicating that the ER is a central compartment for peptide loading upon MHC I2. After peptide loading, the MHC I molecules are transported to the cell surface and play a key role in the adaptive immune system as self-markers, and enables the CD8+ cytotoxic T lymphocytes (CTLs) to detect cancer cells or infectious agents by antigenic peptides from non-self proteins3.
In antigen presenting cells (APC), antigenic peptides from exogenous antigens are also presented upon MHC I4,5,6,7,8 via CP, which is mainly carried out by DCs9,10,11. CP is essential both for the activation of na&#239;ve CD8+ T cells and memory CD8+ T cells into anti-infectious and anti-tumoral CTLs12,13, and in the maintenance of immune tolerance by the inactivating of self-acting na&#239;ve T cells14,15. The CP plays many important roles in the adaptive immune system, however the molecular mechanisms of CP have yet to be described in detail. Previous studies of CP revealed that exogenous antigens were localized both in the ER and the endosome and were processed by ERAD, suggesting that exogenous antigens are transported from the endosome to the ER for ERAD-like processing and peptide loading16. However, accumulating evidence indicates that the peptide loading of CP is carried out not in the ER but rather in non-classical endocytic compartments, which also have distinctive features of the ER (Figure 1)17,18,19,20,21. To avoid degradation of the antigenic peptide precursors by the high activity of aminopeptidase22 in the cytosol, processing and peptide loading in CP occurs in the proximal area of these non-classical endocytic compartments (Figure 1). Though the characterizations of these endocytic compartments are controversial, there are no existing specific molecules other than exogenous antigens in this compartment.
ERAD is a cellular pathway, which specifically removes misfolded proteins from the ER. In the ERAD pathway, misfolded proteins are retrogradely transported through the ER membrane to the cytoplasm and processed by the ubiquitin-proteasome system23,24,25. When large molecules, such as proteins, are transported through the lipid bilayer, these molecules pass through a molecular apparatus called a translocon, such as the Sec61 complex and Derlin complex in the ER26, and the Tom complex and Tim complex in the mitochondria27. When exogenously-added antigens are transported through the ER membrane, they must penetrate the lipid bilayer in complex with translocons, such as the Sec61 complex. The method described here purified the targeted vesicle by utilizing these membrane-penetrating molecules as markers for the endocytic compartments.
The method described here is a new vesicle purification protocol using the DC-like cell line DC2.428 and biotinylated ovalbumin (bOVA) as an exogenous antigen. The endocytic compartments were purified by streptavidin (SA)-magnetic beads using the membrane-penetrating bOVA as a maker. In this purified microsome, some exogenously added bOVA was still preserved in membrane fractions but were transported to the outside of microsome, and then ubiquitinated and processed in vitro29. This purified microsome contained not only endocytic compartment-specific proteins but also ER-resident proteins for ERAD and the peptide loading complex; suggesting that the cellular compartment is the prospective endocytic compartment for CP29. This protocol is not dependent on the kind of exogenous antigens, and is also applicable for other DC subsets and other cell types, such as macrophages, B cells, and endothelial cells, to clarify the precise molecular mechanism of DCs for proficient CP.

<table><tbody><tr><td>RPMI 1640</td><td>gibco by life technologies</td><td>11875-093</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Equitech bio</td><td>SFB30</td><td></td></tr><tr><td>Sodium pyruvate</td><td>gibco by life technologies</td><td>11360-070</td><td></td></tr><tr><td>MEM non-essential amino acids</td><td>gibco by life technologies</td><td>11140-050</td><td></td></tr><tr><td>HEPES</td><td>gibco by life technologies</td><td>15630-080</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>gibco by life technologies</td><td>21985-023</td><td></td></tr><tr><td>L-glutamine</td><td>gibco by life technologies</td><td>25030-164</td><td></td></tr><tr><td>Penisicillin-Sreptomycin</td><td>gibco by life technologies</td><td>15140-122</td><td></td></tr><tr><td>DMEM</td><td>gibco by life technologies</td><td>12100-46</td><td></td></tr><tr><td>OVA</td><td>SIGMA</td><td>A5503</td><td></td></tr><tr><td>Biotin-protein labelling kit</td><td>Thermo Fisher Scientific</td><td>F6347</td><td></td></tr><tr><td>MG-132</td><td>Santa Cruz Biotechnology</td><td>201270</td><td></td></tr><tr><td>lactacystin</td><td>SIGMA</td><td>L6785</td><td></td></tr><tr><td>Dounce homogenizer</td><td>IUCHI</td><td>131703</td><td></td></tr><tr><td>protease inhibitor cocktails</td><td>SIGMA</td><td>P8340</td><td></td></tr><tr><td>iodixanol</td><td>Cosmo bio</td><td>1114542</td><td></td></tr><tr><td>SA-magnetic beads</td><td>New England Biolabs</td><td>201270</td><td></td></tr><tr><td>control magnetic beads</td><td>Chemagen</td><td>M-PVA012</td><td></td></tr><tr><td>magnetic stand</td><td>BD Biosciences</td><td>552311</td><td></td></tr><tr><td>BCA protein assay kit</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>silver staining kits</td><td>Cosmo bio</td><td>423413</td><td></td></tr><tr><td>Reticulocyte Lysate</td><td>Promega</td><td>1730714</td><td></td></tr><tr><td>Flag-tagged ubiquitin</td><td>SIGMA</td><td>U5382</td><td></td></tr><tr><td>anti-ovalbumin (OVA,mouse)</td><td>Antibody Shop</td><td>HYB 094-06</td><td></td></tr><tr><td>ant-multi-ubiquitin (mouse)</td><td>MBL</td><td>D058&amp;minus;3</td><td></td></tr><tr><td>anti-Flag (mouse)</td><td>SIGMA</td><td>F3165</td><td></td></tr><tr><td>trypsin</td><td>SIGMA</td><td>85450C</td><td></td></tr></tbody></table>

purification of the membrane compartment for endoplasmic reticulum-associated degradation of exogenous antigens in cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

To elucidate the molecular mechanism of CP, it is necessary to identify the cellular compartments, where exogenous antigens undergo ERAD-like transport and processing. While observations by immunofluorescent microscopy or by electron microscopy identified the cellular compartment where exogenous antigens accumulated16,17,18,19,...

Watch this Scientific Journal Video about Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation at JoVE.com

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

purification-membrane-compartment-for-endoplasmic-reticulum

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

8.2K Views.  Takasaki University of Health and Welfare. The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

Video: Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain protein homeostasis (proteostasis). Declines in mitochondrial proteostasis have been linked to cancer, aging, neurodegeneration, and many other diseases. Msp1 is a AAA+ ATPase anchored in the outer mitochondrial membrane that maintains proteostasis by removing mislocalized tail-anchored proteins. Using purified components reconstituted into proteoliposomes, we have shown that Msp1 is necessary and sufficient to extract a model tail-anchored protein from a lipid bilayer. Our simplified reconstituted system overcomes several of the technical barriers that have hindered detailed study of membrane protein extraction. Here, we provide detailed methods for the generation of liposomes, membrane protein reconstitution, and the Msp1 extraction assay.

Here, we present a detailed protocol for reconstitution of Msp1 extraction activity with fully purified components in defined proteoliposomes.

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

Biochemistry

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both ...

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

Intralumenal Vesicles and Multivesicular Bodies

Intraluminal vesicles (ILVs) are small vesicles 50-80 nm in diameter formed during the maturation of early endosomes. A specialized endosome containing numerous ILVs is called a multivesicular body (MVB). ILVs contain internalized molecules such as antigens, nucleic acids, proteins, and metabolites. Some of these molecules are released from the MVBs inside exosomes and are transported to other cells. Other MVBs contain molecules that are retained in the ILVs and are later degraded within the cell.
MVBs were first observed in neuronal cells in 1955 using electron microscopy. MVBs have a diameter of 400-500 nm, are spherical, and were initially thought to be a form of the late endosome; however, recent studies have shown that MVBs are an intermediate form of these organelles between the early and the late endosomes.
ILV formation takes place when a ubiquitin-tagged protein enters the cell in an endocytic vesicle and fuses with the early endosome. As the early endosome matures, the endosomal membrane bends inwards to form ILVs. An ESCRT protein complex mediates ILV formation. Phosphatidylinositol 3-phosphate (PtdIns3P) is also required for ILV formation. PtdIns3P acts as a docking site for ESCRTs for the bending of the endosomal membrane.
The ESCRT complex and the ATPase, Vps4, required for ILV formation can be hijacked by enveloped retroviruses that infect host cells. Normally, ILVs are formed inside maturing endosomes with the help of ESCRTs within the cell, and the ubiquitin-tagged proteins are recognized and sorted into ILVs. In contrast, retroviruses use the ESCRTs to be released outside the cell. For example, the human immunodeficiency virus (HIV) codes for proteins, such as Gag, NC-p2, and Tat, that require ubiquitination for export. This ubiquitination is essential because ESCRTs and Vps4 recognize and sort only ubiquitin-tagged proteins into viral buds. Once the viral particles bud off from an infected cell, they are released into the extracellular space to infect other host cells.

Intraluminal vesicles (ILVs) are small vesicles 50-80 nm in diameter formed during the maturation of early endosomes. A specialized endosome containing ...

Education

JoVE Core

Cell Biology

Unit 1

Endocytosis and Exocytosis

KEY TERMS AND CONCEPTS

Receptor Downregulation in MVBs

Multivesicular bodies (MVBs) are mature endosomes that sort ubiquitinated proteins and then fuse with lysosomes to degrade the sorted proteins. Epidermal growth factor (EGF) and its receptor (EGFR) form a complex that can be internalized through endocytosis, sorted into an MVB, and later degraded.
The EGFR can initiate signaling pathways that&#160; lead to cell proliferation, migration, and differentiation. Overexpression of EGFR&#160; stimulates cells to proliferate. Excessive&#160; EGFR activation may result in cancer; therefore, EGFR expression must be tightly regulated. The degradation of the EGF-EGFR complex through the endocytic pathway is a means of downregulating EGFR and preventing its overexpression.
EGF binding to EGFR induces EGFR dimerization. EGFR is ubiquitinated by a ubiquitin ligase. Ubiquitination triggers the endocytosis of the dimerized EGF-EGFR complex. Once inside the cell, the complex is first sorted into the early endosome. As the early endosome matures, ESCRT proteins, or endosomal sorting complexes required for transport, translocate&#160; the EGFR-EGF complex into intraluminal vesicles (ILVs). Several ILVs are formed inside the maturing endosome, eventually becoming multivesicular bodies (MVBs). MVBs containing the EGF-EGFR complex then fuses with a lysosome for degradation of the EGFR-EGF complex.
Similar to EGFR, cytokine receptors are also internalized and downregulated by MVBs. Ubiquitinated receptor-cytokine complexes are endocytosed and later delivered to MVBs for degradation. The degradation weakens the cytokine signaling. In the case of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF&#593;) or interleukin-1 (IL-1), any defect in ubiquitination, endocytosis, or degradation of the receptor-cytokine complex through MVB can cause enhanced or sustained signaling. This may lead to inflammatory diseases like Crohn&#39;s disease or rheumatoid arthritis.

Multivesicular bodies (MVBs) are mature endosomes that sort ubiquitinated proteins and then fuse with lysosomes to degrade the sorted proteins. Epidermal ...

Export of Misfolded Proteins out of the ER

After folding, the ER assesses the quality of secretory and membrane proteins. The correctly folded proteins are cleared by the calnexin cycle for transport to their final destination, while misfolded proteins are held back in the ER lumen. The ER chaperones attempt to unfold and refold the misfolded proteins but sometimes fail to achieve the correct native conformation. Such terminally misfolded proteins are then exported to the cytosol by ER-associated degradation or ERAD pathway for degradation.
Different modes of ERAD pathways
Hrd1 is a ubiquitin ligase and a retro-translocation channel for misfolded polypeptides. It complexes with another membrane protein, Hrd3, to form the core ERAD machinery. The other players associated with these core proteins depend upon the topology of the misfolded protein. The retrotranslocation complex depends on the luminal lectin chaperones to identify misfolded proteins. Studies in the yeast system suggest the presence of ERAD-C, ERAD-L, and ERAD-M pathways to export misfolded proteins from various parts of the ER. The target proteins of the ERAD-L and ERAD-M pathways use the Hrd1 channel to exit the ER lumen and membrane, respectively, while the ERAD-C pathway utilizes the Doa10p ubiquitin ligase complex to degrade the cytoplasmic domains of the ER membrane proteins. Sometimes, these pathways can overlap as some misfolded proteins need translocation machinery from two different ERAD pathways to exit the ER. The mammalian ERAD machinery is more complex than yeast and has multiple proteins that export and mark proteins for proteasomal degradation. Eventually, all ERAD pathways converge due to their dependence on a cytoplasmic AAA-ATPase to provide the mechanical force to extract proteins through the membrane.
Degradation of non-glycosylated proteins
The ER has another lectin-independent system that monitors and clears the non-glycosylated proteins from ER. It consists of the resident ER chaperone, BiP protein, and the ER-localized DnaJ family members (ERdjs). ER stress induces the production of HERP, a ubiquitin-like membrane protein, which assembles with other ERAD proteins like the Hrd1 and AAA-ATPase, forming a complex. BiP uses some ERdj proteins as cofactors to recruit non-glycosylated misfolded proteins to the HERP complex for retrotranslocation and ubiquitination of the misfolded protein. HERP is associated with the proteasome on the cytosolic side, where it delivers the misfolded proteins for degradation.

After folding, the ER assesses the quality of secretory and membrane proteins. The correctly folded proteins are cleared by the calnexin cycle for ...

Transmembrane Transport in Endoplasmic Reticulum and Peroxisomes

Antigen Presenting Cells

The immune system is a complex network of cells and molecules that protects the body from foreign invaders. T cells, a type of white blood cell, play a crucial role in this process. They recognize and attack foreign substances, such as pathogens, that enter the body.
T cells require the help of antigen-presenting cells (APCs), which process foreign antigens into smaller fragments that can be recognized by T cells. These APCs are highly specialized cells that efficiently internalize antigens through phagocytosis or receptor-mediated endocytosis.
Dendritic cells, a type of professional APC, are particularly efficient at antigen processing and presentation. They are highly mobile and can be found in tissues first exposed to foreign antigens, such as the skin, mucosal surfaces, and lymphoid organs.
Macrophages may also function as APCs, and are found in all tissues. They can engulf and process foreign antigens and release cytokines, which activate T cells and other immune cells, thereby coordinating the immune response.
B cells are another type of APC that can present antigens to T cells. When an antigen is presented to a B cell, it activates the B cell to produce antibodies against the antigen. This activation process involves the binding of the antigen to the B cell receptor, followed by a series of molecular signaling events that produce antibodies.
When an APC engulfs an antigen, it breaks into smaller fragments and combines them with major histocompatibility complex (MHC) molecules. The antigen-MHC complex is then transported to the APC&#39;s plasma membrane, where it is presented to a T cell.
The appropriate T cell receptor recognizes the processed antigen complex, triggering an immune response against the foreign invader. This process is crucial for protecting the body from infection and disease.

Antigen-presenting cells, also known as APCs, process foreign antigens, such as fragments from pathogens, for recognition by T cells.
When an APC engulfs ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Summary

Explore More Videos

Chapters in this video

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

Intralumenal Vesicles and Multivesicular Bodies

Receptor Downregulation in MVBs

Export of Misfolded Proteins out of the ER

Antigen Presenting Cells