A gene family consisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane association sites

1992 ◽  
Vol 103 (1) ◽  
pp. 131-143 ◽  
Author(s):  
N. Sato ◽  
N. Funayama ◽  
A. Nagafuchi ◽  
S. Yonemura ◽  
S. Tsukita ◽  
...  
Keyword(s):  
Gene Family ◽  
Plasma Membranes ◽  
Immunofluorescence Microscopy ◽  
Membrane Association ◽  
Protein Sequence Analysis ◽  
Teratocarcinoma Cells ◽  
Specific Localization ◽  
End Capping ◽  
Mouse Teratocarcinoma

Radixin is a barbed end-capping actin-modulating protein which was previously reported to be concentrated at cell-to-cell adherens junctions (AJ) and cleavage furrows. Recently, cDNA encoding mouse radixin was isolated, showing that radixin is highly homologous to but distinct from ezrin. From mouse teratocarcinoma cells we isolated and analyzed cDNA encoding another radixin-related protein. Sequence analysis has demonstrated that this protein is a mouse homologue of human moesin (98.3% identity) and that it shares 71.7% and 80.1% identity with ezrin and radixin, respectively. Translation experiments in vitro combined with immunoblot analyses led us to conclude that there is a gene family consisting of ezrin, radixin and moesin. These members are coexpressed in various types of cells. Then, by immunofluorescence microscopy, we closely analyzed their distribution using polyclonal and monoclonal antibodies, which could recognize all three members. In addition to cell-to-cell AJ and cleavage furrows, it was shown that they were concentrated at microvilli and ruffling membranes in various types of cells. Furthermore, the cell-to-substrate AJ (focal contacts) were clearly stained by anti-radixin pAb only after the apical/lateral membranes and cytoplasm were removed by the zinc method. We conclude that at least one of the members of the ezrin-radixin-moesin family is concentrated at specific regions where actin filaments are densely associated with plasma membranes.

scholarly journals In vitro synthesis and membrane insertion of bovine MP26, an integral protein from lens fiber plasma membrane.

1983 ◽  
Vol 96 (3) ◽  
pp. 633-638 ◽  
Author(s):  
D L Paul ◽  
D A Goodenough
Keyword(s):  
Plasma Membranes ◽  
Proteolytic Processing ◽  
Membrane Insertion ◽  
Membrane Association ◽  
Lens Fiber ◽  
In Vitro Synthesis ◽  
Reticulocyte Lysate ◽  
Immune Precipitation ◽  
Dog Pancreas

Synthesis of MP26, the principal protein of lens fiber plasma membranes, was directed in the reticulocyte lysate system by poly A mRNA enriched from whole bovine lens RNA using oligo (dt)-cellulose chromatography. Synthesized MP26 was enriched by immune precipitation. The in vitro-synthesized MP26 had an electrophoretic mobility indistinguishable from that of the native molecule. MP26 showed a cotranslational requirement for dog pancreas microsomes in order for membrane association to occur. Microsome-associated in vitro-synthesized MP26 showed a sensitivity to digestion with chymotrypsin which was similar to the sensitivity of native MP26 in isolated lens fiber plasma membranes, indicating correct insertion of the MP26 into the microsome. Synthesis and membrane insertion of MP26 using N-formyl-[35S]methionyl tRNA as label demonstrated that no proteolytic processing or significant glycosylation accompanied membrane insertion. Chymotryptic cleavage of membrane-inserted, N-formyl-[35S]methionine-labeled MP26 resulted in loss of label, suggesting that the N-terminal of the in vitro-synthesized MP26 faces the cytoplasm.

In Vitro Cytotoxicity of 24 Chemicals to Mouse Teratocarcinoma Cells

1989 ◽  
Vol 17 (1) ◽  
pp. 34-37
Author(s):  
Karen Atkinson ◽  
Lesley Hulme ◽  
Richard H. Clothier ◽  
Michael Balls
Keyword(s):  
In Vitro Cytotoxicity ◽  
Teratocarcinoma Cells ◽  
Mouse Teratocarcinoma

Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues

1999 ◽  
Vol 112 (8) ◽  
pp. 1149-1158 ◽  
Author(s):  
K. Hayashi ◽  
S. Yonemura ◽  
T. Matsui ◽  
S. Tsukita
Keyword(s):  
Plasma Membranes ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Tissue Type ◽  
Cross Linking ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Erm Proteins

Ezrin/radixin/moesin (ERM) proteins are thought to play an important role in organizing cortical actin-based cytoskeletons through cross-linkage of actin filaments with integral membrane proteins. Recent in vitro biochemical studies have revealed that ERM proteins phosphorylated on their COOH-terminal threonine residue (CPERMs) are active in their cross-linking activity, but this has not yet been evaluated in vivo. To immunofluorescently visualize CPERMs in cultured cells as well as tissues using a mAb specific for CPERMs, we developed a new fixation protocol using trichloroacetic acid (TCA) as a fixative. Immunoblotting analyses in combination with immunofluorescence microscopy showed that TCA effectively inactivated soluble phosphatases, which maintained the phosphorylation level of CPERMs during sample processing for immunofluorescence staining. Immunofluorescence microscopy with TCA-fixed samples revealed that CPERMs were exclusively associated with plasma membranes in a variety of cells and tissues, whereas total ERM proteins were distributed in both the cytoplasm and plasma membranes. Furthermore, the amounts of CPERMs were shown to be regulated in a cell and tissue type-dependent manner. These findings favored the notion that phosphorylation of the COOH-terminal threonine plays a key role in the regulation of the cross-linking activity of ERM proteins in vivo.

In vitro Differentiation of Mouse Teratocarcinoma Cells Monitored by Intermediate Filament Expression

1982 ◽  
Vol 22 (1-3) ◽  
pp. 90-99 ◽  
Author(s):  
DENISE PAULIN ◽  
HEDWIG JAKOB ◽  
FRANÇOIS JACOB ◽  
KLAUS WEBER ◽  
MARY OSBORN
Keyword(s):  
Intermediate Filament ◽  
In Vitro Differentiation ◽  
Teratocarcinoma Cells ◽  
Mouse Teratocarcinoma

scholarly journals Calpain activity in patients with thrombotic thrombocytopenic purpura is associated with platelet microparticles

Blood ◽  
1992 ◽  
Vol 80 (9) ◽  
pp. 2246-2251 ◽  
Author(s):  
JG Kelton ◽  
TE Warkentin ◽  
CP Hayward ◽  
WG Murphy ◽  
JC Moore
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Membrane Association ◽  
Membrane Glycoproteins ◽  
Calpain Activity ◽  
Thrombocytopenic Purpura ◽  
Calcium Dependent ◽  
Active Protease ◽  
Triton X ◽  
Platelet Microparticles

Abstract Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia and disseminated platelet thrombi throughout the microvasculature. Studies by our group have demonstrated calcium- dependent proteolytic activity (calpain) that is no longer detectable in the serum of patients with acute TTP after their recovery. The purpose of this study was to investigate if the protease activity of TTP was detectable in plasma and, therefore, not an in vitro phenomenon secondary to the formation of serum. Additionally, we looked for evidence of membrane association of the active protease in the patients' samples, which would explain the persistence of its activity in the presence of plasma inhibitors. Acute TTP samples, both serum and plasma, were collected from 10 patients with TTP. Calpain was measured using bioassays for enzyme activity and also by detection of the protein using immunoblotting with an anticalpain monoclonal antibody (MoAb). In all instances, calpain could be detected both functionally and antigenically in the acute TTP sera and plasma. No calpain activity could be detected in any of the controls, although antigenic calpain was detectable in one sample from a patient who had undergone cardiopulmonary bypass surgery. To investigate whether the calpain was associated with microparticles in the plasma, the TTP plasma samples were ultrafiltered and ultracentrifuged. Activity was not lost by passage across a 0.2-micron filter but was detectable only in the pellet following ultracentrifugation. Membrane association of the calpain in the microparticles also was demonstrated using solubilization with Triton X-100. Immunoprecipitation studies demonstrated that the calpain activity could be removed by MoAbs against platelet membrane glycoproteins (IX and IIb/IIa) but not by a MoAb against red blood cell membrane glycophorin. These studies indicate that active calpain is associated with platelet microparticles in plasma from patients with TTP.

scholarly journals Glycan-to-Glycan Binding: Molecular Recognition through Polyvalent Interactions Mediates Specific Cell Adhesion

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 397
Author(s):  
Gradimir Misevic ◽  
Emanuela Garbarino
Keyword(s):  
Cell Adhesion ◽  
Plasma Membranes ◽  
Specific Cell ◽  
Cellular Interactions ◽  
New Paradigm ◽  
Tumor Cell Adhesion ◽  
Self Recognition ◽  
Structural And Functional Properties ◽  
Low Valent

Glycan-to-glycan binding was shown by biochemical and biophysical measurements to mediate xenogeneic self-recognition and adhesion in sponges, stage-specific cell compaction in mice embryos, and in vitro tumor cell adhesion in mammals. This intermolecular recognition process is accepted as the new paradigm accompanying high-affinity and low valent protein-to-protein and protein-to-glycan binding in cellular interactions. Glycan structures in sponges have novel species-specific sequences. Their common features are the large size >100 kD, polyvalency >100 repeats of the specific self-binding oligosaccharide, the presence of fucose, and sulfated and/or pyruvylated hexoses. These structural and functional properties, different from glycosaminoglycans, inspired their classification under the glyconectin name. The molecular mechanism underlying homophilic glyconectin-to-glyconectin binding relies on highly polyvalent, strong, and structure-specific interactions of small oligosaccharide motifs, possessing ultra-weak self-binding strength and affinity. Glyconectin localization at the glycocalyx outermost cell surface layer suggests their role in the initial recognition and adhesion event during the complex and multistep process. In mammals, Lex-to-Lex homophilic binding is structure-specific and has ultra-weak affinity. Cell adhesion is achieved through highly polyvalent interactions, enabled by clustering of small low valent structure in plasma membranes.

scholarly journals Inter-species functional compatibility of the Theobroma cacao and Arabidopsis FT orthologs: 90 million years of functional conservation of meristem identity genes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
S. F. Prewitt ◽  
A. Shalit-Kaneh ◽  
S. N. Maximova ◽  
M. J. Guiltinan
Keyword(s):  
Gene Expression ◽  
Tissue Culture ◽  
Gene Family ◽  
Flowering Time ◽  
Molecular Mechanisms ◽  
Regulatory Pathways ◽  
Late Flowering ◽  
Precocious Flowering ◽  
Transition To Flowering

Abstract Background In angiosperms the transition to flowering is controlled by a complex set of interacting networks integrating a range of developmental, physiological, and environmental factors optimizing transition time for maximal reproductive efficiency. The molecular mechanisms comprising these networks have been partially characterized and include both transcriptional and post-transcriptional regulatory pathways. Florigen, encoded by FLOWERING LOCUS T (FT) orthologs, is a conserved central integrator of several flowering time regulatory pathways. To characterize the molecular mechanisms involved in controlling cacao flowering time, we have characterized a cacao candidate florigen gene, TcFLOWERING LOCUS T (TcFT). Understanding how this conserved flowering time regulator affects cacao plant’s transition to flowering could lead to strategies to accelerate cacao breeding. Results BLAST searches of cacao genome reference assemblies identified seven candidate members of the CENTRORADIALIS/TERMINAL FLOWER1/SELF PRUNING gene family including a single florigen candidate. cDNA encoding the predicted cacao florigen was cloned and functionally tested by transgenic genetic complementation in the Arabidopsis ft-10 mutant. Transgenic expression of the candidate TcFT cDNA in late flowering Arabidopsis ft-10 partially rescues the mutant to wild-type flowering time. Gene expression studies reveal that TcFT is spatially and temporally expressed in a manner similar to that found in Arabidopsis, specifically, TcFT mRNA is shown to be both developmentally and diurnally regulated in leaves and is most abundant in floral tissues. Finally, to test interspecies compatibility of florigens, we transformed cacao tissues with AtFT resulting in the remarkable formation of flowers in tissue culture. The morphology of these in vitro flowers is normal, and they produce pollen that germinates in vitro with high rates. Conclusion We have identified the cacao CETS gene family, central to developmental regulation in angiosperms. The role of the cacao’s single FT-like gene (TcFT) as a general regulator of determinate growth in cacao was demonstrated by functional complementation of Arabidopsis ft-10 late-flowering mutant and through gene expression analysis. In addition, overexpression of AtFT in cacao resulted in precocious flowering in cacao tissue culture demonstrating the highly conserved function of FT and the mechanisms controlling flowering in cacao.

scholarly journals Cloning of mouse ptx3, a new member of the pentraxin gene family expressed at extrahepatic sites

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 1862-1872 ◽  
Author(s):  
M Introna ◽  
VV Alles ◽  
M Castellano ◽  
G Picardi ◽  
L De Gioia ◽  
...  
Keyword(s):  
Gene Family ◽  
Tertiary Structure ◽  
Helical Structure ◽  
Distinct Pattern ◽  
Mononuclear Phagocytes ◽  
Acute Phase Reactants ◽  
Inflammatory Reactions ◽  
Reactive Protein

Abstract Pentraxins, which include C reactive protein (CRP) and serum amyloid P component (SAP), are prototypic acute phase reactants that serve as indicators of inflammatory reactions. Here we report genomic and cDNA cloning of mouse ptx3 (mptx3), a member of the pentraxin gene family and characterize its extrahepatic expression in vitro and in vivo. mptx3 is organized into three exons on chromosome 3: the first (43 aa) and second exon (175 aa) code for the signal peptide and for a protein portion with no high similarity to known sequences the third (203 aa) for a domain related to classical pentraxins, which contains the “pentraxin family signature.” Analysis of the N terminal portion predicts a predominantly alpha helical structure, while the pentraxin domain of ptx3 is accommodated comfortably in the tertiary structure fold of SAP. Normal and transformed fibroblasts, undifferentiated and differentiated myoblasts, normal endothelial cells, and mononuclear phagocytes express mptx3 mRNA and release the protein in vitro on exposure to interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF)alpha. mptx3 was induced by bacterial lipopolysaccharide in vivo in a variety of organs and, most strongly, in the vascular endothelium of skeletal muscle and heart. Thus, mptx3 shows a distinct pattern of in vivo expression indicative of a significant role in cardiovascular and inflammatory pathology.

scholarly journals Identification, Expression, and Functions of the Somatostatin Gene Family in Spotted Scat (Scatophagus argus)

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 194
Author(s):  
Peizhe Feng ◽  
Changxu Tian ◽  
Xinghua Lin ◽  
Dongneng Jiang ◽  
Hongjuan Shi ◽  
...  
Keyword(s):  
Gene Family ◽  
Growth Regulation ◽  
Expression Patterns ◽  
Open Reading Frames ◽  
Pcr Analysis ◽  
Sequence Alignments ◽  
Scatophagus Argus ◽  
Gene Structure And Expression ◽  
Liver Fragments

Somatostatins (SSTs) are a family of proteins consisting of structurally diverse polypeptides that play important roles in the growth regulation in vertebrates. In the present study, four somatostatin genes (SST1, SST3, SST5, and SST6) were identified and characterized in the spotted scat (Scatophagus argus). The open reading frames (ORFs) of SST1, SST3, SST5, and SST6 cDNA consist of 372, 384, 321, and 333 bp, respectively, and encode proteins of 123, 127, 106, and 110 amino acids, respectively. Amino acid sequence alignments indicated that all SST genes contained conserved somatostatin signature motifs. Real-time PCR analysis showed that the SST genes were expressed in a tissue specific manner. When liver fragments were cultured in vitro with synthetic peptides (SST1, SST2, or SST6 at 1 μM or 10 μM) for 3 h or 6 h, the expression of insulin-like growth factor 1 and 2 (Igf-1 and Igf-2) in the liver decreased significantly. Treatment with SST5 had no significant effect on Igf-1 and Igf-2 gene expression. This study provides an enhanced understanding of the gene structure and expression patterns of the SST gene family in S. argus. Furthermore, this study provides a foundation for future exploration into the role of SST genes in growth and development.

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