Isolation of Lipid Rafts From B Lymphocytes

Cherukuri, Anu; Tzeng, Shiang-Jong; Gidwani, Arun; Sohn, Hyung Sun; Tolar, Pavel; Snyder, Michelle

doi:10.1385/1-59259-796-3:213

Cited by 7 publications

(6 citation statements)

References 25 publications

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“…DRM preparation was performed as described (13), except that OptiPrep (Axis-Shield) was used instead of sucrose. Fractions 4 to 11 were separated on 12% SDS NuPAGE gels (Invitrogen) and transferred to polyvinylidene difluoride (PVDF) membrane (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Cutting Edge: NKG2D-Dependent Cytotoxicity Is Controlled by Ligand Distribution in the Target Cell Membrane

Martinez

Brzostowski

Long

et al. 2011

The Journal of Immunology

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While the importance of membrane microdomains in receptor-mediated activation of lymphocytes has been established, much less is known about the role of receptor ligand distribution on APC and target cells. Detergent-resistant membrane (DRM) domains, into which glycophosphatidylinositol (GPI)-linked proteins partition, are enriched in cholesterol and glycosphingolipids. ULBP1 is a GPI-linked ligand for natural cytotoxicity receptor NKG2D. To investigate how ULBP1 distribution on target cells affects NKG2D-dependent NK cell activation, we fused the extracellular domain of ULBP1 to the transmembrane domain of CD45. Introduction of this transmembrane domain eliminated the association of ULBP1 with the DRM fraction and caused a significant reduction of cytotoxicity and degranulation by NK cells. Clustering and lateral diffusion of ULBP1 was not affected by changes in the membrane anchor. These results show that the partitioning of receptor ligands in discrete membrane domains of target cells is an important determinant of NK cell activation.

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Section: Methodsmentioning

confidence: 99%

Cutting Edge: NKG2D-Dependent Cytotoxicity Is Controlled by Ligand Distribution in the Target Cell Membrane

Martinez

Brzostowski

Long

et al. 2011

The Journal of Immunology

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“…The TX-100 extraction procedure was performed as described [43]. Briefly, after coclustering, 1 ϫ 10 8 NHHT-39 cells were lysed directly for 30 min on ice in a final volume of 1 ml TNEV ϩ [10 mM Tris, 150 mM NaCl, 5 mM EDTA, 1 mM Na 3 VO 4 , and protease inhibitors (see above), pHϭ7.5], supplemented with 1% TX-100.…”

Section: Extraction Of Drmsmentioning

confidence: 99%

Coalescence of B cell receptor and invariant chain MHC II in a raft-like membrane domain

Hauser

Lindner

2014

Journal of Leukocyte Biology

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The BCR binds antigen for processing and subsequent presentation on MHC II molecules. Polyvalent antigen induces BCR clustering and targeting to endocytic processing compartments, which are also accessed by Ii-MHC II. Here, we report that clustered BCR is able to team up with Ii-MHC II already at the plasma membrane of mouse B-lymphocytes. Colocalization of BCR and Ii-MHC II on the cell surface required clustering of both types of molecules. The clustering of only one type did not trigger the recruitment of the other. Ii-bound MIF (a ligand of Ii) also colocalized with clustered BCR upon oligomerization of MIF on the surface of the B cell. Abundant surface molecules, such as B220 or TfnR, did not cocluster with the BCR. Some membrane raft-associated molecules, such as peptide-loaded MHC II, coclustered with the BCR, whereas others, such as GM1, did not. The formation of a BCR- and Ii-MHC II-containing membrane domain by antibody-mediated clustering was independent of F-actin and led to the coendocytosis of its constituents. With a rapid Brij 98 extraction method, it was possible to capture this membrane domain biochemically as a DRM. Ii and clustered BCR were present on the same DRM, as shown by immunoisolation. The coalescence of BCR and Ii-MHC II increased tyrosine phosphorylation, indicative of enhanced BCR signaling. Our work suggests a novel role for MIF and Ii-MHC II in BCR-mediated antigen processing.

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“…Pervanadate treatment inhibits tyrosine phosphatases and thereby enables proteins to remain in phosphorylated state. The cells were washed in PBS and processed for lipid rafts isolation as described previously (18). For SDS-PAGE analyses, lipid raft fractions and soluble fractions were pooled and separated on a 4 -12% gradient NuPAGE polyacrylamide gel (Invitrogen Life Technologies).…”

Section: Pervanadate Treatment Lipid Rafts Isolation Sds-page and mentioning

confidence: 99%

CD94/NKG2A Inhibits NK Cell Activation by Disrupting the Actin Network at the Immunological Synapse

Masilamani

Nguyen

Kabát

et al. 2006

The Journal of Immunology

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An adequate immune response is the result of the fine balance between activation and inhibitory signals. The exact means by which inhibitory signals obviate activation signals in immune cells are not totally elucidated. Human CD94/NKG2A is an ITIM-containing inhibitory receptor expressed by NK cells and some CD8+ T cells that recognize HLA-E. We show that the engagement of this receptor prevents NK cell activation by disruption of the actin network and exclusion of lipid rafts at the point of contact with its ligand (inhibitory NK cell immunological synapse, iNKIS). CD94/NKG2A engagement leads to recruitment and activation of src homology 2 domain-bearing tyrosine phosphatase 1. This likely explains the observed dephosphorylation of guanine nucleotide exchange factor and regulator of actin, Vav1, as well as ezrin-radixin-moesin proteins that connect actin filaments to membrane structures. In contrast, NK cell activation by NKG2D induced Vav1 and ezrin-radixin-moesin phosphorylation. Thus, CD94/NKG2A prevents actin-dependent recruitment of raft-associated activation receptors complexes to the activating synapse. This was further substantiated by showing that inhibition of actin polymerization abolished lipid rafts exclusion at the iNKIS, whereas cholesterol depletion had no effect on actin disruption at the iNKIS. These data indicate that the lipid rafts exclusion at the iNKIS is an active process which requires an intact cytoskeleton to maintain lipid rafts outside the inhibitory synapse. The net effect is to maintain an inhibitory state in the proximity of the iNKIS, while allowing the formation of activation synapse at distal points within the same NK cell.

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Isolation of Lipid Rafts From B Lymphocytes

Cited by 7 publications

References 25 publications

Cutting Edge: NKG2D-Dependent Cytotoxicity Is Controlled by Ligand Distribution in the Target Cell Membrane

Cutting Edge: NKG2D-Dependent Cytotoxicity Is Controlled by Ligand Distribution in the Target Cell Membrane

Coalescence of B cell receptor and invariant chain MHC II in a raft-like membrane domain

CD94/NKG2A Inhibits NK Cell Activation by Disrupting the Actin Network at the Immunological Synapse

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