RESUMEN
Erythrocyte tropomodulin (E-Tmod), a globular protein of 359 residues, is highly expressed in the erythrocyte, heart and skeletal muscle. By binding to the N-terminus of tropomyosin (TM) and actin, E-Tmod blocks the elongation and depolymerization of the actin filaments at the pointed end. In erythrocytes, the E-Tmod/TM complex contributes to the formation of the short actin protofilament, which in turn defines the geometry of the membrane skeleton. In juvenile mice, over-expression of E-Tmod is associated with dilated cardiomyopathy. We have previously cloned the human E-Tmod cDNA, identified its TM-binding region, and mapped its gene to chromosome 9q22. Through genomic library screening and PCR-based genomic walking we have now cloned the mouse E-Tmod gene, whose coding region spans approximately 60kb containing nine exons and eight introns. The human E-Tmod gene obtained by PCR has an identical exon-intron organization. In sanpodo, a Tmod homologue in Drosophila, the exon boundaries are also conserved except that exons 2-5 and 6-7 are 'fused' and alternative splicing of two additional 5' exons and the 3' exons may give rise to several sanpodo isoforms. In a Tmod-like gene of C. elegans, exons 2-3 are 'fused', boundaries of exons 1, 7, 8, and 9 are conserved and exon/intron junctions of exons 4, 5 and 6 are shifted by a few residues. Analyses of 15 Tmod members from six species show no insertions or deletions of residues in the region of exons 6 and 7. A 5' rapid amplification of cDNA ends reveals that mouse E-Tmod transcripts obtained from embryonic stem cells, skeletal muscle and heart, but not smooth muscle, contain an additional 86bp untranslated cDNA sequence further upstream from exon 1. Thus, alternative promoters may provide a possible mechanism for tissue-specific expression and regulation of E-Tmod. This study is the first to report the exon organization of E-Tmod genes, which allows their regulation, manipulation, and disease relevance to be further investigated.
Asunto(s)
Actinas/metabolismo , Proteínas Portadoras/genética , Genes/genética , Proteínas de Microfilamentos , Regiones no Traducidas 5' , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/genética , Proteínas Portadoras/metabolismo , ADN/química , ADN/genética , ADN/aislamiento & purificación , Drosophila/genética , Eritrocitos/química , Exones , Biblioteca Genómica , Humanos , Intrones , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Mutagénesis Insercional , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , TropomodulinaRESUMEN
Tropomodulin is a globular protein that caps the pointed end of actin filaments by complexing with the N-terminus of a tropomyosin (TM) molecule. TM consists of coiled coils except for the N-terminus, which may be globular. Here we report that human TM isoform 5 (hTM5) lacking the N-terminal 18 residues lost its binding activity toward tropomodulin. We further characterized the tropomodulin-binding site by creating a series of deletion and missense mutations within this region, followed by a solid-phase binding assay. I7, V10, and I14, hydrophobic residues located at the a and d positions of N-terminal heptad repeats involving intertwine, are essential for tropomodulin binding. R12, a positively charged residue at the f position, is also involved in recognition. In contrast, A2R and G3Y mutations, each creating a bulky N-terminus, did not alter the binding. In addition, rat TM5b, which differs from hTM5 in residues 4-6, exhibits a similar binding affinity. The tropomodulin-binding site, therefore, is mapped to residues 7-14 at the beginning of the long heptad repeats. Column chromatography revealed that hTM5 mutants remained capable of dimerization. Results also suggest tropomodulin has a groove-type, rather than a cavity-type, binding site for hTM5. We also mapped the epitope of monoclonal antibody LC1 to residues 4-10 of hTM5 and showed the competition between mAb LC1 and tropomodulin in hTM5 binding. Since the N-terminal residues need to overlap with the C-terminus of TM in their head-to-tail association, this investigation elucidates the mechanisms by which the tropomodulin-hTM5 complex is formed and functions in regulating the actin filaments.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Microfilamentos , Tropomiosina/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Sitios de Unión/genética , Unión Competitiva , Cartilla de ADN/genética , Mapeo Epitopo , Escherichia coli/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Aminoácido , Tropomodulina , Tropomiosina/genética , Tropomiosina/metabolismoRESUMEN
The human erythrocyte membrane skeleton consists of hexagonal lattices with junctional complexes containing F-actin protofilaments of approximately 33-37 nm in length. We hypothesize that complexes formed by tropomodulin, a globular capping protein at the pointed end of actin filaments, and tropomyosin (TM), a rod-like molecule of approximately 33-35 nm, may contribute to the formation of protofilaments. We have previously cloned the human tropomodulin complementary DNA and identified human TM isoform 5 (hTM5), a product of the gamma-TM gene, as one of the major TM isoforms in erythrocytes. We now identify TM5b, a product of the alpha-TM gene, to be the second major TM isoform. TM5a, the alternatively spliced isoform of the alpha-TM gene, which differs by 1 exon and has a weaker actin-binding affinity, however, is not present. TM4, encoded by the delta-TM gene, is not present either. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, hTM5 comigrated with the slower TM major species in erythrocyte membranes, and hTM5b comigrated with the faster TM major species. TM5b, like TM5, binds strongly to tropomodulin, more so than other TM isoforms. The 2 major TM isoforms, therefore, share several common features: They have 248 residues, are approximately 33-35 nm long, and have high affinities toward F-actin and tropomodulin. These common features may be the key to the mechanism by which protofilaments are formed. Tropomodulin-TM5 or tropomodulin-TM5b complexes may stabilize F-actin in segments of approximately 33-37 nm during erythroid terminal differentiation and may, therefore, function as a molecular ruler. TM5 and TM5b further define the hexagonal geometry of the skeletal network and allow actin-regulatory functions of TMs to be modulated by tropomodulin. (Blood. 2000;95:1473-1480)
Asunto(s)
Proteínas Portadoras/sangre , Membrana Eritrocítica/ultraestructura , Eritrocitos/metabolismo , Tropomiosina/sangre , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Clonación Molecular , Citoesqueleto/ultraestructura , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/ultraestructura , Humanos , Proteínas de Microfilamentos/sangre , Proteínas de Microfilamentos/química , Datos de Secuencia Molecular , Isoformas de Proteínas/sangre , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Tropomodulina , Tropomiosina/química , Tropomiosina/genéticaRESUMEN
PURPOSE: Protein 4.2 is a major erythrocyte membrane skeletal protein, playing an important role in maintaining the integrity and stability of the membrane. It is a transglutaminase-like molecule with no enzymatic cross-linking activity. Several protein 4.2-associated proteins (i.e. band 3, ankyrin, and protein 4.1) and transglutaminase activities have been detected in the lens. The purpose of this study is to find out if protein 4.2 is also expressed in lens fiber membranes. METHODS: Western blot analysis of cell membranes isolated from bovine and chicken lens fibers and erythrocytes, and immunocytochemistry of frozen sections of bovine and chicken lens fibers were carried out using two protein 4.2-specific antibodies. These two peptide antibodies have been used to identify two alternatively spliced protein 4.2 isoforms in human erythrocyte membranes: the short (P4.2S, or hP4.2(691)) and the long (P4.2L, or hP4.2(721)) isoforms. RESULTS: Western blot analysis using anti-P4.2(L) antibody demonstrated specific immunoreactive polypeptides in bovine and chicken lens fiber membranes and erythrocyte membranes, co-migrating with hP4.2(721). Immunofluorescence staining of bovine and chicken lenses, using anti-P4.2(L) antibody, revealed specific signals along the cell membranes of cortical fibers. The signals exhibited a unique, patchy pattern along the cortical fiber cell membranes in both cross-sectional and longitudinal views. In cross sections, the labeling of anti-P4.2(L) along the entire cell membranes gave an appearance of a hexagonal shape of fiber cells. CONCLUSIONS: Protein 4.2, or its analogs, is present in the lens fiber membranes. Its specific staining pattern in the lens fibers suggests that it participates in the architecture of the lens fiber cell membranes, and may play a role in the lens mechanics and pathology.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Membrana Eritrocítica/metabolismo , Proteínas del Ojo/metabolismo , Cristalino/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Bovinos , Pollos , Proteínas del Citoesqueleto , Técnica del Anticuerpo Fluorescente Indirecta , Cristalino/citología , Datos de Secuencia Molecular , Fragmentos de PéptidosRESUMEN
We have determined the effects of tropomodulin (Tmod), talin, vinculin, and alpha-actinin on ligament fibroblast adhesion. The anterior cruciate ligament (ACL), which lacks a functional healing response, and the medial collateral ligament (MCL), a functionally healing ligament, were selected for this study. The micropipette aspiration technique was used to determine the forces needed to separate ACL and MCL cells from a fibronectin-coated surface. Delivery of exogenous tropomodulin, an actin-filament capping protein, into MCL fibroblasts significantly increased adhesion, whereas its monoclonal antibody (mAb) significantly decreased cell adhesiveness. However, for ACL fibroblasts, Tmod significantly reduced adhesion, whereas its mAb had no effect. mAbs to talin, vinculin, and alpha-actinin significantly decreased the adhesion of both ACL and MCL cells with increasing concentrations of antibody, and also reduced stress fiber formation and cell spreading rate as revealed by immunofluorescence microscopy. Disruption of actin filament and microtubule assembly with cytochalasin D and colchicine, respectively, also significantly reduced adhesion in ACL and MCL cells. In conclusion, both ACL and MCL fibroblast adhesion depends on cytoskeletal assembly; however, this dependence differs between ACL and MCL fibroblasts in many ways, especially in the role of Tmod. These results add yet another possible factor in explaining the clinical differences in healing between the ACL and the MCL.
Asunto(s)
Ligamento Cruzado Anterior/fisiología , Adhesión Celular/fisiología , Proteínas del Citoesqueleto/fisiología , Fibroblastos/fisiología , Ligamento Colateral Medial de la Rodilla/fisiología , Proteínas de Microfilamentos , Actinina/inmunología , Actinina/fisiología , Adulto , Ligamento Cruzado Anterior/citología , Anticuerpos Monoclonales/farmacología , Proteínas Portadoras/inmunología , Proteínas Portadoras/fisiología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Proteínas del Citoesqueleto/inmunología , Citoesqueleto/fisiología , Femenino , Fibronectinas/fisiología , Humanos , Recién Nacido , Masculino , Ligamento Colateral Medial de la Rodilla/citología , Persona de Mediana Edad , Talina/inmunología , Talina/fisiología , Tropomodulina , Vinculina/inmunología , Vinculina/fisiología , Cicatrización de Heridas/fisiologíaRESUMEN
A major adaptation to exercise is new capillary formation in skeletal muscle. On the basis of angiogenesis in tumors and during development, several angiogenic growth factors may be involved, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta 1 (TGF-beta 1). In 9-wk-old female Wistar rats, mRNA expression for these three growth factors in gastrocnemius muscle was examined by quantitative Northern analysis after a single 1-h run at 15 or 20 m/min at 10 degrees incline in room air. A third group ran at 15 m/min in 12% O2, and resting control groups were included at inspired O2 fractions of 0.21 and 0.12. Exercise significantly increased mRNA levels two- to fourfold, which was evident over the first 4 h postexercise; by 8 and 24 h, mRNA levels returned to baseline. For all three factors, mRNA levels were significantly higher after exercise at 20 than at 15 m/min. Hypoxia at rest doubled VEGF and TGF-beta 1 message but had no effect on bFGF. Hypoxic exercise further raised VEGF mRNA levels but had no effect on the other factors. We suggest that VEGF, bFGF, and TGF-beta 1 may be involved in the angiogenic response to exercise and that reduced intracellular PO2 (as occurs during normoxic exercise) may be part of the stimulus to such growth factor production.
Asunto(s)
Factores de Crecimiento Endotelial/biosíntesis , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Músculo Esquelético/metabolismo , Esfuerzo Físico/fisiología , ARN Mensajero/biosíntesis , Factor de Crecimiento Transformador beta/biosíntesis , Animales , Northern Blotting , Femenino , Semivida , Hipoxia/metabolismo , Hibridación in Situ , Músculo Esquelético/fisiología , Ratas , Ratas WistarRESUMEN
Tropomodulin is a newly characterized pointed end capping protein for actin filaments. It binds specifically to the N terminus of tropomyosin and blocks the elongation and depolymerization of tropomyosin-coated actin filaments. A 1.9-kb human tropomodulin cDNA clone was used to map its gene by fluorescence in situ hybridization. The tropomodulin gene was assigned to human chromosome 9q22.2-q22.3, a region that is also known to contain several other genes and disease loci and is proximal to the loci for gelsolin and alpha-fodrin. The gene for tropomodulin is expressed in major human tissues at different levels in the following order: heart and skeletal muscle much greater than that in brain, lung, and pancreas, which is greater than that in placenta, liver, and kidney. Human tropomodulin and a 64-kDa autoantigen in Graves disease (1D) are related: tropomodulin has 42 and 41% identity with the Graves protein in the N-terminal (69 residue) and C-terminal (194 residue) regions, respectively. The insertion of several homologous repeats in the midsection of the Graves protein, together with the extension of a proline-rich C terminus, accounts for the differences in length between the Graves protein (572 residues) and tropomodulin (359 residues). The significant sequence identity indicates that these two genes are evolved from a common ancestral gene.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Cromosomas Humanos Par 9 , Proteínas de Microfilamentos , Secuencia de Aminoácidos , Autoantígenos/biosíntesis , Autoantígenos/química , Autoantígenos/genética , Northern Blotting , Mapeo Cromosómico , Enfermedad de Graves/inmunología , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Distribución Tisular , TropomodulinaRESUMEN
The adhesive energy in lectin-induced agglutination can be assessed by the deformation of erythrocytes in aggregates. Helix pomatia (HPA) and Dolichos biflorus (DBA) specifically agglutinated blood group A erythrocytes and induced a change in curvatures of cells at the end of the aggregates. The curvatures changed from concavity to convexity with increasing lectin concentrations. HPA-induced aggregates achieved the theoretical maximal end cell curvature of 0.27 microns-1 at 2-3 micrograms/ml; DBA-induced aggregates approached 0.23 microns-1, requiring 400 micrograms/ml. At any given lectin concentration, HPA showed greater surface binding, caused higher cell curvatures, induced larger aggregate size, and had greater adhesive energies, as compared to DBA. HPA and DBA are globular proteins with a diameter of approximately 5.5 nm and approximately 6.1 nm, respectively, as revealed by the negative staining electron microscopy. The former induced uniform intercellular spacing (approximately 15 nm), whereas the latter induced both smooth (approximately 20 nm) and ruffled spacings. The uniform intercellular spacing was not a function of lectin concentrations. Microscopic studies of erythrocyte deformation provided morphological correlates of biophysical findings of differential adhesive energies induced by these two blood group A-specific lectins.
Asunto(s)
Sistema del Grupo Sanguíneo ABO , Agregación Eritrocitaria , Deformación Eritrocítica , Eritrocitos/ultraestructura , Hemaglutininas/farmacología , Lectinas de Plantas , Eritrocitos/efectos de los fármacos , Humanos , Lectinas/farmacología , Microscopía Electrónica , Microscopía Electrónica de RastreoRESUMEN
Tropomodulin is a 40.6-kDa protein that binds to one end of the rod-like tropomyosin and inhibits its cooperativity and binding to actin. In myofibrils, tropomodulin has been localized at or near the free end of thin filaments. Using recombinant and chimeric molecules in a solid-phase binding assay, we demonstrate that it is the N-terminus of tropomyosin that interacts with tropomodulin. Among several tropomyosin isoforms tested, hTM5 encoded by the human gamma-tropomyosin gene has the highest affinity toward human erythrocyte tropomodulin. Tropomodulin may, therefore, regulate the length and/or organization of actin filaments by differential binding to tropomyosin isoforms. hTM5 exists in the human erythrocyte membrane skeleton. In non-muscle cells, tropomodulin may block the head-to-tail association of tropomyosins and their interaction with actin at the pointed end of actin filaments by preferentially binding to TM5 at its N-terminus.
Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Eritrocítica/metabolismo , Proteínas de Microfilamentos , Tropomiosina/metabolismo , Actinas/química , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Autorradiografía , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Pollos , Electroforesis en Gel de Poliacrilamida , Humanos , Modelos Estructurales , Datos de Secuencia Molecular , Músculo Liso , Músculos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Radioisótopos de Azufre , Tropomodulina , Tropomiosina/química , Tropomiosina/genéticaRESUMEN
The elasticity of red cell membrane was determined in a rectangular flow channel under controlled shear flow. The relation between shear stress and cell extension ratio (lambda) has been analyzed with the use of Evans' two-dimensional model. The deformed cell shapes observed experimentally agreed well with the model with lambda up to 1.4. The best correlation was found at lambda = 1.2. The analysis suggests a nonlinear extensional membrane modulus in the low stress range encountered in the flow channel. In terms of an appropriate strain parameter, the elastic modulus is shown to rise toward the level encountered in micropipette aspiration experiments. The implications of the present findings in modeling of cell mechanics and in cell hemolysis are discussed.
Asunto(s)
Deformación Eritrocítica/fisiología , Membrana Eritrocítica/fisiología , Modelos Cardiovasculares , Elasticidad , Eritrocitos/ultraestructura , Hemólisis/fisiología , Humanos , Estrés MecánicoRESUMEN
Human protein 4.2 (P4.2) is a major membrane skeletal protein in erythrocytes. Individuals with P4.2 deficiency exhibit spherocytosis and experience various degrees of hemolytic anemia, suggesting a role for this protein in maintaining stability and integrity of the membrane. Molecular cloning of P4.2 cDNAs showed that P4.2 is a transglutaminaselike molecule in erythrocytes but lacks the essential cysteine for cross-linking activity. Two cDNA isoforms have been identified from a human reticulocyte cDNA library, with the long isoform containing a 90-base pair (bp) in-frame insertion encoding an extra 30 amino acids near the N-terminus. Characterization of the P4.2 gene suggests differential splicing as the mechanism for generating these two cDNA isoforms. The donor site for the short isoform (P4.2S) agrees better with the consensus than the donor site for the long isoform (P4.2L) does. Expression of P4.2L was detected by a long-isoform-specific antibody raised against a peptide within the 30-amino acid insert. Western blot analyses showed P4.2L to be a minor membrane skeletal protein in human erythrocytes with an apparent molecular weight (mol wt) of approximately 3 Kd larger than the major protein 4.2, P4.2S. By in situ hybridization of a full-length 2.4-kilobase (kb) cDNA to human metaphase chromosomes, the gene for P4.2 was mapped to bands q15-q21 of chromosome 15, and it is not linked to the gene for coagulation factor XIIIa (plasma transglutaminase, TGase).
Asunto(s)
Proteínas Sanguíneas/genética , Cromosomas Humanos Par 15 , Membrana Eritrocítica/fisiología , Empalme del ARN , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Sanguíneas/análisis , Western Blotting , Bandeo Cromosómico , Proteínas del Citoesqueleto , ADN/sangre , ADN/genética , ADN/aislamiento & purificación , Humanos , Cariotipificación , Proteínas de la Membrana/sangre , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Oligonucleótidos Antisentido , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Transglutaminasas/genéticaRESUMEN
Human erythrocyte tropomodulin is a novel tropomyosin regulatory protein that binds to the end of erythrocyte tropomyosin and blocks heat-to-tail association of tropomyosin along actin filaments. It has been proposed to play a role in modulating the association of tropomyosin with the spectrin-actin complex in the erythrocyte membrane skeleton. Immunoscreening of a human fetal liver cDNA expression library in lambda gt11, followed by 5'-end extension by polymerase chain reaction from the same library, yielded a composite cDNA sequence of 2665 base pairs (bp). It contains a 34-bp 5'-untranslated region, a 1.6-kilobase (kb) 3'-untranslated region, and a complete open reading frame of 1077 bp that encodes a protein of 359 amino acids with a calculated molecular mass of 40.6 kDa and a pI of 4.8. Authenticity of the tropomodulin cDNA was confirmed by a complete sequence match of 49 predicted amino acids with the sequences of three tryptic peptides of the erythrocyte tropomodulin. The sequence has no internal repeats and no significant homology with any known proteins. Secondary structure predictions indicate that tropomodulin may consist of a series of seven or eight short alpha-helical segments and fold into a somewhat compact shape. The tropomyosin binding activity has been mapped to an N-terminal region containing residues 39-138. Nine independent PCR clones, five from a human reticulocyte cDNA library and four from the fetal liver cDNA library, revealed identical N-terminal 103 amino acids, suggesting that the sequence reported here may also be of erythrocyte tropomodulin. Northern analysis of human reticulocyte RNA showed two hybridizing bands of 2.7 and 1.6 kb, indicating that the 2665-bp cDNA sequence reported here was that of the longer transcript.
Asunto(s)
Proteínas Portadoras/genética , Hígado/metabolismo , Proteínas de Microfilamentos , Tropomiosina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Northern Blotting , Southern Blotting , Western Blotting , Proteínas Portadoras/metabolismo , Clonación Molecular , ADN/genética , Electroforesis en Gel de Poliacrilamida , Humanos , Hígado/embriología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN/genética , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , TropomodulinaRESUMEN
Cell adhesion plays a fundamental role in the organization of cells in differentiated organs, cell motility, and immune response. A novel micromanipulation method is employed to quantify the direct contribution of surface adhesion receptors to the physical strength of cell adhesion. In this technique, a cell is brought into contact with a glass-supported planar membrane reconstituted with a known concentration of a given type of adhesion molecules. After a period of incubation (5-10 min), the cell is detached from the planar bilayer by pulling away the pipette holding the cell in the direction perpendicular to the glass-supported planar bilayer. In particular, we investigated the adhesion between a Jurkat cell expressing CD2 and a glass-supported planar bilayer containing either the glycosyl-phosphatidylinositol (GPI) or the transmembrane (TM) isoform of the counter-receptor lymphocyte function-associated antigen 3 (LFA-3) at a concentration of 1,000 molecules/microns 2. In response to the pipette force the Jurkat cells that adhered to the planar bilayer containing the GPI isoform of LFA-3 underwent extensive elongation. When the contact radius was reduced by approximately 50%, the cell then detached quickly from its substrate. The aspiration pressure required to detach a Jurkat cell from its substrate was comparable to that required to detach a cytotoxic T cell from its target cell. Jurkat cells that had been separated from the substrate again adhered strongly to the planar bilayer when brought to proximity by micromanipulation. In experiments using the planar bilayer containing the TM isoform of LFA-3, Jurkat cells detached with little resistance to micromanipulation and without changing their round shape.
Asunto(s)
Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos T/fisiología , Antígenos de Superficie/fisiología , Adhesión Celular , Membrana Dobles de Lípidos , Glicoproteínas de Membrana/fisiología , Receptores Inmunológicos/fisiología , Linfocitos T/fisiología , Antígenos CD2 , Antígenos CD58 , Glucolípidos , Glicosilfosfatidilinositoles , Humanos , Linfoma de Células T , Fosfatidilinositoles , Linfocitos T/inmunología , Células Tumorales CultivadasRESUMEN
Protein 4.2 (P4.2) comprises approximately 5% of the protein mass of human erythrocyte (RBC) membranes. Anemia occurs in patients with RBCs deficient in P4.2, suggesting a role for this protein in maintaining RBC stability and integrity. We now report the molecular cloning and characterization of human RBC P4.2 cDNAs. By immunoscreening a human reticulocyte cDNA library and by using the polymerase chain reaction, two cDNA sequences of 2.4 and 2.5 kilobases (kb) were obtained. These cDNAs differ only by a 90-base-pair insert in the longer isoform located three codons downstream from the putative initiation site. The 2.4- and 2.5-kb cDNAs predict proteins of approximately 77 and approximately 80 kDa, respectively, and the authenticity was confirmed by sequence identity with 46 amino acids of three cyanogen bromide-cleaved peptides of P4.2. Northern blot analysis detected a major 2.4-kb RNA species in reticulocytes. Isolation of two P4.2 cDNAs implies existence of specific regulation of P4.2 expression in human RBCs. Human RBC P4.2 has significant homology with human factor XIII subunit a and guinea pig liver transglutaminase. Sequence alignment of P4.2 with these two transglutaminases, however, revealed that P4.2 lacks the critical cysteine residue required for the enzymatic crosslinking of substrates.
Asunto(s)
Proteínas Sanguíneas/genética , Clonación Molecular , Proteínas de la Membrana/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas del Citoesqueleto , ADN/genética , Membrana Eritrocítica/metabolismo , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , ARN/sangre , ARN/genética , Mapeo Restrictivo , Reticulocitos/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
Red blood cell (RBC) rouleaux were formed in a flow channel in the presence of 2 g/dl dextran (molecular weight 76,000). The partial separation of RBC rouleau doublets adhering to the floor of the flow channel in response to small oscillatory shear stresses was observed experimentally. Theoretical analyses on displacement and drag force were performed to determine whether the motion of the cell involves membrane rotation (i.e., rolling) or sliding. From the experimental data and the results of theoretical analyses, it is concluded that, under the conditions of the experiments, the RBCs in a doublet separate from each other by rolling, rather than sliding of the sheared cell.
Asunto(s)
Agregación Eritrocitaria , Reología , Fenómenos Biomecánicos , HumanosRESUMEN
The biophysical properties of cytotoxic T lymphocytes during the killing of their target cells was investigated by using a human cytotoxic T lymphocyte clone, F1, and the target cell, JY, for which it is specific. In single cytotoxic cell/target cell pairs after their conjugation there are changes in the viscoelastic properties of the target cell in association with the lethal hit delivery and post-binding cytolytic steps. On the basis of these changes in the target cell, the complex cytolytic event can be divided into stages: the viscoelastic coefficients exhibited an initial increase followed by a return to resting values; thereafter these coefficients decreased below control and then rose again prior to lysis. The eventual killing of the target cell involves bubbling and swelling of the nucleus, clustering of granules, damage to the cytoplasmic membrane, cell swelling, and lysis. The viscoelastic changes involved in target cell death suggest the loss of integrity of its cytoskeletal apparatus.
Asunto(s)
Citotoxicidad Inmunológica , Linfocitos T Citotóxicos/fisiología , Línea Celular Transformada , Elasticidad , Humanos , Micromanipulación , Microscopía Electrónica , Linfocitos T/ultraestructura , ViscosidadRESUMEN
The alterations in the viscoelastic properties of the hemoglobin S solution following deoxygenation can be correlated, on the one hand, with the gelation of the HbS molecules and, on the other hand, with changes in the rheological behavior of the deoxygenated SS erythrocytes. The abnormal rheology of SS erythrocytes forms the basis of their altered flow behavior through filter pores in vitro and microcirculation in vivo. The results support the notion that the rheological behavior of sickle cells is an important link between the molecular events in hemoglobin S following deoxygenation and the behavior of the cell in the microcirculation. Although recent advances have shed considerable lights on the rheology of sickle cells in a variety of conditions, further studies are needed in order to elucidate the pathophysiological role of cell rheology in this disease, especially in terms of the influence of individual SS cells in the heterogeneous populations and the correlation between in vitro and in vivo behavior of SS cells.
Asunto(s)
Anemia de Células Falciformes/fisiopatología , Eritrocitos/fisiología , Anemia de Células Falciformes/sangre , Viscosidad Sanguínea , Elasticidad , Filtración , Humanos , Microcirculación/fisiopatología , ReologíaRESUMEN
A direct measurement of the avidity of the junction between a cytotoxic T lymphocyte and its target cell was achieved by using a biophysical approach. A micromanipulation technique was used to determine the force required to separate a cytotoxic T cell (human clone F1, with specificity for HLA-DRw6) from its specific target cell (JY: HLA-A2, -B7, -DR4, w6) prior to delivery of the lethal hit. The force required to separate the F1-JY pair is 1.5 X 10(4) dynes per square centimeter. This junction avidity for F1-JY pairs is 6 to 13 times greater than that for F1-F1 and JY-JY pairs; the F1-JY conjugate requires a stronger separating force and is more easily rejoined than the homologous cell pairs. This study provides an estimate of the avidity of cytotoxic T cells for their target cells and insights into the biophysical correlates of the molecular complexes formed in the interaction of cytotoxic T cells and their targets during the cytotoxic process.
Asunto(s)
Comunicación Celular , Linfocitos T Citotóxicos/citología , Citotoxicidad Inmunológica , Antígenos HLA-DR/inmunología , Antígeno HLA-DR6 , Humanos , Matemática , Linfocitos T Citotóxicos/inmunología , Factores de TiempoRESUMEN
The normal, discoid shape of red blood cells represents an equilibrium between two opposing factors, i.e., stomatocytic and echinocytic transformations. Most stomatocytic agents were found to be inhibitors of calmodulin, a regulator of the phosphorylation of membrane proteins. We determined whether red cell shape transformations could be caused by changes in phosphorylation of membrane proteins, specifically the cAMP-dependent phosphorylation of ankyrin and band 4.1. Red blood cells were incubated with 32P and 100 microM chlorpromazine (stomatocytic transformation) or 30 mM sodium salicylate (echinocytic transformation) for various time intervals. Ghost membrane proteins were examined by polyacrylamide gel electrophoresis and autoradiography. Spectrin (beta-chain), ankyrin, band 3, band 4.1 and 4.9 were phosphorylated. No change was found in the degree and pattern of phosphorylation after stomatocytic transformation. Salicylate caused a reversible inhibition of transmembranous phosphate transport in both directions. The results indicate that the stomatocytic transformation induced by chlorpromazine and the echinocytic transformation induced by salicylate do not involve a change in phosphorylation, but that the echinocytic transformation induced by salicylate is associated with an inhibition of transmembranous transport of phosphate. Studies with salicylate suggest that the phosphorylation sites of band 3 are found mainly on the endofacial side of the membrane.
Asunto(s)
Eritrocitos/metabolismo , Proteínas de la Membrana/metabolismo , Aspirina/farmacología , Clorpromazina/farmacología , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Humanos , Técnicas In Vitro , FosforilaciónRESUMEN
The interactions of human genotype AO erythrocytes (red blood cells) (RBCs) with N-acetylgalactosamine-reactive lectins isolated from Helix pomatia (HPA) and from Dolichos biflorus (DBA) were studied. Binding curves obtained with the use of tritium-labeled lectins showed that the maximal numbers of lectin molecules capable of binding to human genotype AO RBCs were 3.8 X 10(5) and 2.7 X 10(5) molecules/RBC for HPA and DBA, respectively. The binding of one type of lectin may influence the binding of another type. HPA was found to inhibit the binding of DBA, but not vice versa. The binding of HPA was weakly inhibited by a beta-D-galactose-reactive lectin isolated from Ricinus communis (designated RCA1). Limulus polyphemus lectin (LPA), with specificity for N-acetylneuraminic acid, did not influence the binding of HPA but enhanced the binding of DBA. About 80% of LPA receptors (N-acetylneuraminic acid) were removed from RBC surfaces by neuraminidase treatment. Neuraminidase treatment of RBCs resulted in increases of binding of both HPA and DBA, but through different mechanisms. An equal number (7.6 X 10(5) of new HPA sites were generated on genotypes AO and OO RBCs by neuraminidase treatment, and these new sites accounted for the enhancement (AO cells) and appearance (OO cells) of hemagglutinability by HPA. Neuraminidase treatment did not generate new DBA sites, but increased the DBA affinity for the existing receptors; as a result, genotype AO cells increased their hemagglutinability by DBA, while OO cells remained unagglutinable. The use of RBCs of different genotypes in binding assays with 3H-labeled lectins of known specificities provides an experimental system for studying cell-cell recognition and association.