Membrane rafts are microdomains of the plasma membrane that have multiple biological functions. The involvement of these structures in the biology of T cells, namely in signal transduction by the TCR, has been widely studied. However, the role of membrane rafts in immunoreceptor signaling in NK cells is less well known. We studied the distribution of the activating NKG2D receptor in lipid rafts by isolating DRMs in a sucrose density gradient or by raft fractionation by β-OG-selective solubility in the NKL cell line. We found that the NKG2D-DAP10 complex and pVav are recruited into rafts upon receptor stimulation. Qualitative proteomic analysis of these fractions showed that the actin cytoskeleton is involved in this process. In particular, we found that the actin-bundling protein L-plastin plays an important role in the clustering of NKG2D into lipid rafts. Moreover, coengagement of the inhibitory receptor NKG2A partially disrupted NKG2D recruitment into rafts. Furthermore, we demonstrated that L-plastin participates in NKG2D-mediated inhibition of NK cell chemotaxis.

• Keywords
• chemotaxis, membrane rafts,
• MeSH
• Cell Membrane drug effects   metabolism   MeSH
• Killer Cells, Natural cytology   metabolism   MeSH
• Centrifugation, Density Gradient MeSH
• Chemotaxis, Leukocyte physiology   MeSH
• Detergents pharmacology   MeSH
• Cells, Cultured MeSH
• NK Cell Lectin-Like Receptor Subfamily C metabolism   MeSH
• NK Cell Lectin-Like Receptor Subfamily K physiology   MeSH
• Humans MeSH
• RNA, Small Interfering pharmacology   MeSH
• Membrane Microdomains drug effects   physiology   MeSH
• Actin Cytoskeleton physiology   MeSH
• Microfilament Proteins antagonists & inhibitors   genetics   physiology   MeSH
• Multiprotein Complexes MeSH
• Proteome MeSH
• Receptors, Immunologic metabolism   MeSH
• RNA Interference MeSH
• Signal Transduction immunology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Detergents MeSH
• HCST protein, human MeSH Browser
• KLRK1 protein, human MeSH Browser
• LCP1 protein, human MeSH Browser
• NK Cell Lectin-Like Receptor Subfamily C MeSH
• NK Cell Lectin-Like Receptor Subfamily K MeSH
• RNA, Small Interfering MeSH
• Microfilament Proteins MeSH
• Multiprotein Complexes MeSH
• Proteome MeSH
• Receptors, Immunologic MeSH

More than one 80S monosome can translate an mRNA molecule at a time producing polysomes. The most widely used method to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes is a high-velocity centrifugation of whole cell extracts in linear sucrose gradients. This polysome profile analysis technique has been routinely used to monitor translational fitness of cells under a variety of physiological conditions, to investigate functions of initiation factors involved in translation, to reveal defects in ribosome biogenesis, to determine roles of 5' UTR structures on mRNA translatability, and more recently for examination of miRNA-mediated translational repression (see an application of this protocol on Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae).

• Keywords
• Grow yeast cultures, Polysome profile analysis, Sucrose density gradient centrifugation, Western and Northern blotting, Yeast whole cell extracts (WCEs),
• MeSH
• Centrifugation, Density Gradient methods   MeSH
• Blotting, Northern methods   MeSH
• Polyribosomes chemistry   genetics   MeSH
• Saccharomyces cerevisiae chemistry   cytology   genetics   MeSH
• Sucrose chemistry   MeSH
• Blotting, Western methods   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Sucrose MeSH

Lck is the principal signal-generating tyrosine kinase of the T cell activation mechanism. We have previously demonstrated that induced Lck activation outside of lipid rafts (LR) results in the rapid translocation of a fraction of Lck to LR. While this translocation predicates the subsequent production of IL-2, the mechanism underpinning this process is unknown. Here, we describe the main attributes of this translocating pool of Lck. Using fractionation of Brij58 lysates, derived from primary naive non-activated CD4(+) T cells, we show that a significant portion of Lck is associated with high molecular weight complexes representing a special type of detergent-resistant membranes (DRMs) of relatively high density and sensitivity to laurylmaltoside, thus called heavy DRMs. TcR/CD4 coaggregation-mediated activation resulted in the redistribution of more than 50% of heavy DRM-associated Lck to LR in a microtubular network-dependent fashion. Remarkably, in non-activated CD4(+) T-cells, only heavy DRM-associated Lck is phosphorylated on its activatory tyrosine 394 and this pool of Lck is found to be membrane confined with CD45 phosphatase. These data are the first to illustrate a lipid microdomain-based mechanism concentrating the preactivated pool of cellular Lck and supporting its high stoichiometry of colocalization with CD45 in CD4(+) T cells. They also provide a new structural framework to assess the mechanism underpinning the compartmentalization of critical signaling elements and regulation of spatio-temporal delivery of Lck function during the T cell proximal signaling.

• MeSH
• Enzyme Activation MeSH
• Lymphocyte Activation MeSH
• Leukocyte Common Antigens metabolism   MeSH
• Cell Membrane metabolism   MeSH
• CD4-Positive T-Lymphocytes immunology   MeSH
• Centrifugation, Density Gradient MeSH
• Detergents pharmacology   MeSH
• Membrane Microdomains metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Signal Transduction * MeSH
• Protein Transport MeSH
• Lymphocyte Specific Protein Tyrosine Kinase p56(lck) immunology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Leukocyte Common Antigens MeSH
• Detergents MeSH
• PTPRC protein, human MeSH Browser
• Lymphocyte Specific Protein Tyrosine Kinase p56(lck) MeSH

Prochlorothrix hollandica is one of the three known species of an unusual clade of cyanobacteria (formerly called "prochlorophytes") that contain chlorophyll a and b molecules bound to intrinsic light-harvesting antenna proteins. Here, we report the structural characterization of supramolecular complex consisting of Photosystem I (PSI) associated with the chlorophyll a/b-binding Pcb proteins. Electron microscopy and single particle image analysis of negatively stained preparations revealed that the Pcb-PSI supercomplex consists of a central trimeric PSI surrounded by a ring of 18 Pcb subunits. We conclude that the formation of the Pcb ring around trimeric PSI represents a mechanism for increasing the light-harvesting efficiency in chlorophyll b-containing cyanobacteria.

• MeSH
• Centrifugation, Density Gradient MeSH
• Microscopy, Electron MeSH
• Photosystem I Protein Complex ultrastructure   MeSH
• Protein Structure, Quaternary MeSH
• Prochlorothrix ultrastructure   MeSH
• Light-Harvesting Protein Complexes chemistry   ultrastructure   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Photosystem I Protein Complex MeSH
• Light-Harvesting Protein Complexes MeSH

Membrane domains are highly specialized parts of the cell plasma membrane, carrying on and augmenting the incoming signals. To study their structural and functional properties, it is crucial to find the least damaging mode of their isolation. Using two different cell lines, epithelial HEK cells (clone E2M11) and S49 lymphoma cells, three methods of membrane domain isolation (i.e., detergent extraction, alkaline treatment, and "drastic" homogenization) were tested for similarity and reproducibility by 2-D electrophoresis. Our data show that the protein composition of membrane domains obtained by different isolation methods is similar and that approximately 60% of the spots are present in all membrane domain preparations. Furthermore, the same degree of similarity of 2-D profiles of the most intensively silver stained spots found in membrane domains of the two cell lines derived from different tissues suggests that the composition of a large part of membrane domains proteins is conservative. We suggest that these proteins may either be involved in the organization of membrane domain structure or represent the conservative component of signal transduction machinery.

• MeSH
• Electrophoresis, Gel, Two-Dimensional MeSH
• Hyaluronan Receptors analysis   immunology   MeSH
• CD59 Antigens analysis   immunology   MeSH
• Cell Membrane metabolism   MeSH
• Centrifugation, Density Gradient MeSH
• Detergents chemistry   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Membrane Proteins isolation & purification   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hyaluronan Receptors MeSH
• CD59 Antigens MeSH
• Detergents MeSH
• Membrane Proteins MeSH

• MeSH
• Centrifugation, Density Gradient MeSH
• Electrophoresis, Agar Gel methods   MeSH
• Kinetoplastida genetics   MeSH
• DNA, Kinetoplast chemistry   genetics   isolation & purification   MeSH
• Molecular Sequence Data MeSH
• Blotting, Southern MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Kinetoplast MeSH

The role of detergent-insensitive membrane domains (DIMs) in desensitisation of the G protein-coupled receptor-mediated hormone response was studied in clone E2M11 of HEK293 cells which stably express high levels of both thyrotropin-releasing hormone (TRH) receptors and G(11)alpha G protein. DIMs were prepared by flotation in equilibrium sucrose density gradients and characterised by a panel of membrane markers representing peripheral, glycosylphosphatidylinositol-bound as well as integral membrane proteins (caveolin, CD29, CD55, CD59, CD147, the alpha subunit of Na, K-ATPase) and enzyme activities (alkaline phosphatase, adenylyl cyclase). Caveolin-containing DIMs represented only a small fraction of the overall pool of G(q)alpha/G(11)alpha-rich domains. Prolonged stimulation of E2M11 cells with TRH resulted in dramatic depletion of G(q)alpha/G(11)alpha from all DIMs, which was paralleled by a concomitant G(q)alpha/G(11)alpha increase in the high-density gradient fractions containing the bulk-phase membrane constituents soluble in 1% Triton X-100. Distribution of membrane markers was unchanged under these conditions. Membrane domains thus represent a substantial structural determinant of the G protein pool relevant to desensitisation of hormone action.

• MeSH
• Adenylyl Cyclases metabolism   MeSH
• Alkaline Phosphatase metabolism   MeSH
• Integrin beta1 metabolism   MeSH
• CD55 Antigens metabolism   MeSH
• CD59 Antigens metabolism   MeSH
• Cell Membrane drug effects   metabolism   MeSH
• Cell Line MeSH
• Centrifugation, Density Gradient MeSH
• Detergents pharmacology   MeSH
• Thyrotropin-Releasing Hormone pharmacology   MeSH
• Immunoblotting MeSH
• Caveolin 1 MeSH
• Caveolins * MeSH
• Humans MeSH
• Membrane Proteins metabolism   MeSH
• Octoxynol pharmacology   MeSH
• GTP-Binding Protein alpha Subunits, Gq-G11 MeSH
• GTP-Binding Proteins chemistry   metabolism   MeSH
• Sodium-Potassium-Exchanging ATPase metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenylyl Cyclases MeSH
• Alkaline Phosphatase MeSH
• Integrin beta1 MeSH
• CD55 Antigens MeSH
• CD59 Antigens MeSH
• CAV1 protein, human MeSH Browser
• Detergents MeSH
• Thyrotropin-Releasing Hormone MeSH
• Caveolin 1 MeSH
• Caveolins * MeSH
• Membrane Proteins MeSH
• Octoxynol MeSH
• GTP-Binding Protein alpha Subunits, Gq-G11 MeSH
• GTP-Binding Proteins MeSH
• Sodium-Potassium-Exchanging ATPase MeSH

Nucleoprotein (NP) complexes constituting the three basic components (A, B, C) of the postmicrosomal sediment (POMS) of chicken leukemic myeloblasts (CHLMs) which contain extrachromosomal DNA closely related to avian myeloblastosis virus DNA were analyzed electron microscopically. It was shown that these NP complexes resemble micromorphologically, depending on the origin of their POMS components, NP structures involved in three successive stages of early DNA synthesis. Nucleic acids harbored in these NP complexes exhibited micromorphological features typical for replicative structures. It was confirmed electron microscopically that the extrachromosomal DNA of CHLMs replicative in nature and of three length classes is organized into special NP complexes, each of which, as demonstrated, represents a unique reaction machinery of early DNA synthesis.

• MeSH
• Centrifugation, Density Gradient MeSH
• Cytoplasm MeSH
• DNA, Viral chemistry   ultrastructure   MeSH
• Cell Fractionation MeSH
• Bone Marrow pathology   MeSH
• Chickens MeSH
• Neoplasm Proteins chemistry   ultrastructure   MeSH
• Nucleoproteins isolation & purification   ultrastructure   MeSH
• Avian Leukosis pathology   MeSH
• Avian Myeloblastosis Virus ultrastructure   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• DNA, Viral MeSH
• Neoplasm Proteins MeSH
• Nucleoproteins MeSH

We have shown that the unusual CsCl-buoyant density and velocity sedimentation properties of the isolated host 7 S DNA species associated with the core fraction of avian myeloblastosis virus (AMV) are made mainly by tight association of RNA pieces prevalently joined to the single-stranded portion of this material. It was shown indirectly on sedimentation patterns of [methyl-3H]thymidine and [14C]uridine double-labelled and glyoxylated total AMV DNA, and directly in phosphorylation experiments with T4 polynucleotide kinase performed on the single-stranded portion of AMV DNA that the RNA-DNA link in AMV DNA is of a covalent nature and that the 5'-terminal end of DNA at the RNA-DNA junction is occupied by all four common deoxyribonucleotides. This first evidence of the presence of Okazaki fragments in 7 S AMV DNA clearly indicates that this DNA does not represent a randomly fragmented host DNA included by chance into virions but special fragments of host DNA having the properties of DNA replicative structures with possible consequences for some viral function(s) including those involved in virus-cell interactions.

• MeSH
• Centrifugation, Isopycnic MeSH
• DNA, Viral biosynthesis   isolation & purification   MeSH
• DNA isolation & purification   MeSH
• DNA, Single-Stranded isolation & purification   MeSH
• Cells, Cultured MeSH
• Chickens MeSH
• DNA Replication MeSH
• Virus Replication MeSH
• Ribonucleases MeSH
• RNA, Viral isolation & purification   MeSH
• Avian Myeloblastosis Virus chemistry   physiology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Viral MeSH
• DNA MeSH
• DNA, Single-Stranded MeSH
• Okazaki fragments MeSH Browser
• Ribonucleases MeSH
• RNA, Viral MeSH

Strain Sumava Af of murine alpha-herpesvirus (MHV) banded in density gradient at the boundary between 10-20% and 20-30% Ficoll solution. In electron microscope, the partially purified virus visualized by negative staining exhibited intact or disrupted envelope structures and capsid particles of about 110 nm in outer diameter. The DNA extracted from partially purified virus and further purified in CsCl density gradient had the Tm-value of 79.0 degrees C. The molecular weight of about 90 million daltons was calculated for viral DNA according to its migration in agarose gel.

• MeSH
• Centrifugation, Density Gradient MeSH
• DNA, Viral isolation & purification   MeSH
• Microscopy, Electron MeSH
• Herpesviridae isolation & purification   MeSH
• Molecular Weight MeSH
• Base Composition MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Viral MeSH