RESUMEN
Human red blood cells contain all of the elements involved in the formation of nonmuscle actomyosin II complexes (V. M. Fowler. 1986. J. Cell. Biochem. 31:1-9; 1996. Curr. Opin. Cell Biol. 8:86-96). No clear function has yet been attributed to these complexes. Using a mathematical model for the structure of the red blood cell spectrin skeleton (M. J. Saxton. 1992. J. Theor. Biol. 155:517-536), we have explored a possible role for myosin II bipolar minifilaments in the restoration of the membrane skeleton, which may be locally damaged by major mechanical or chemical stress. We propose that the establishment of stable links between distant antiparallel actin protofilaments after a local myosin II activation may initiate the repair of the disrupted area. We show that it is possible to define conditions in which the calculated number of myosin II minifilaments bound to actin protofilaments is consistent with the estimated number of myosin II minifilaments present in the red blood cells. A clear restoration effect can be observed when more than 50% of the spectrin polymers of a defined area are disrupted. It corresponds to a significant increase in the spectrin density in the protein free region of the membrane. This may be involved in a more complex repair process of the red blood cell membrane, which includes the vesiculation of the bilayer and the compaction of the disassembled spectrin network.
Asunto(s)
Membrana Eritrocítica/metabolismo , Miosinas/sangre , Actinas/sangre , Actinas/química , Actomiosina/sangre , Actomiosina/química , Fenómenos Biofísicos , Biofisica , Simulación por Computador , Envejecimiento Eritrocítico/fisiología , Deformación Eritrocítica/fisiología , Membrana Eritrocítica/química , Humanos , Membrana Dobles de Lípidos/sangre , Membrana Dobles de Lípidos/química , Modelos Biológicos , Modelos Moleculares , Miosinas/química , Estrés Oxidativo , Conformación Proteica , Espectrina/química , Espectrina/metabolismo , Estrés MecánicoRESUMEN
Ankyrins are a multi-gene family of peripheral proteins that link ion channels and cell adhesion molecules to the spectrin-based skeleton in specialized membrane domains. In the mammalian skeletal myofiber, ankyrins were immunolocalized in several membrane domains, namely the costameres, the postsynaptic membrane and the triads. Ank1 and Ank3 transcripts were previously detected in skeletal muscle by northern blot analysis. However, the ankyrin isoforms associated with these domains were not identified, with the exception of an unconventional Ank1 gene product that was recently localized at discrete sites of the sarcoplasmic reticulum. Here we study the expression and subcellular distribution of the Ank3 gene products, the ankyrinsG, in the rat skeletal muscle fiber. Northern blot analysis of rat skeletal muscle mRNAs using domain-specific Ank3 cDNA probes revealed two transcripts of 8.0 kb and 5.6 kb containing the spectrin-binding and C-terminal, but not the serine-rich, domains. Reverse transcriptase PCR analysis of rat skeletal muscle total RNA confirmed the presence of Ank3 transcripts that lacked the serine-rich and tail domains, a major insert of 7813 bp at the junction of the spectrin-binding and C-terminal domains that was previously identified in brain Ank3 transcripts. Immunoblot analysis of total skeletal muscle homogenates using ankyrinG-specific antibodies revealed one major 100 kDa ankyrinG polypeptide. Immunofluorescence labeling of rat diaphragm cryosections showed that ankyrin(s)G are selectively associated with (1) the depths of the postsynaptic membrane folds, where the voltage-dependent sodium channel and N-CAM accumulate, and (2) the sarcoplasmic reticulum, as confirmed by codistribution with the sarcoplasmic reticulum Ca2+-ATPase (SERCA 1). At variance with ankyrin(s)G, ankyrin(s)R (ank1 gene products) accumulate at the sarcolemma and at sarcoplasmic structures, in register with A-bands. Both ankyrin isoforms codistributed over Z-lines and at the postsynaptic membrane. These data extend the notion that ankyrins are differentially localized within myofibers, and point to a role of the ankyrinG family in the organization of the sarcoplasmic reticulum and the postsynaptic membrane.
Asunto(s)
Ancirinas/análisis , Músculo Esquelético/química , Retículo Sarcoplasmático/química , Membranas Sinápticas/química , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Ancirinas/genética , Diafragma/química , Masculino , Datos de Secuencia Molecular , Fibras Musculares Esqueléticas/química , Unión Neuromuscular/química , ARN Mensajero/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
We have previously shown that in human or pig whole erythrocytes, only a single 71-kDa polypeptide cross-reacts with the affinity-purified antibody to pig platelet caldesmon (der Terrossian et al., 1989). In the present paper, we demonstrate that this polypeptide represents a genuine caldesmon which remains attached to the membrane prepared in the presence of an excess of free Mg2+ but not in its absence. Immunoreactivity of this peptide is specific towards the antibody to pig platelet caldesmon since it is not labelled with antibodies to other components of the red cell membrane. Erythrocyte caldesmon was purified to 95% homogeneity and displays well known characteristics of caldesmons from other sources. Together with tropomyosin, it has the ability to regulate platelet actin-activated rabbit skeletal muscle myosin ATPase activity. The stoichiometry of 1 caldesmon/1 tropomyosin/7-9 actin molecules indicates that the amount of caldesmon, in the red cell membrane, corresponds precisely to the amount of tropomyosin. Immunofluorescent labelling of whole erythrocytes gave similar punctate patterns with purified antibodies to myosin, to caldesmon, to tropomyosin and to actin (but not to spectrin), suggesting colocalization of these proteins. Together, and for the first time, our results give strong evidence that caldesmon, bound on the actin protofilament, might represent the inhibitory component, so far uncharacterized, of a thin-filament-like system in erythrocyte.
Asunto(s)
Actinas/sangre , Proteínas de Unión a Calmodulina/sangre , Proteínas Contráctiles/sangre , Membrana Eritrocítica/fisiología , Eritrocitos/fisiología , Adenosina Trifosfatasas/sangre , Animales , Plaquetas/metabolismo , Encéfalo/metabolismo , Calmodulina/metabolismo , Proteínas de Unión a Calmodulina/aislamiento & purificación , Proteínas Contráctiles/aislamiento & purificación , Citoesqueleto/metabolismo , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Humanos , Inmunohistoquímica , Magnesio/farmacología , Músculos/metabolismo , Miosinas/metabolismo , Conejos , PorcinosRESUMEN
The plasma membrane of nucleated erythrocytes contains a microtubular marginal band which appears to be associated with the plasma membrane skeleton. In this report, we identify two families of cytoskeletal proteins which may be involved in such an association. These proteins, of molecular mass 78 kDa and 48 kDa on SDS-PAGE, are shown to bind tubulin based on a 125I-labeled tubulin binding assay. Solubilization of isolated chicken erythrocyte plasma membranes in Triton X-100 shows that these proteins centrifuge with the pellet, indicating that they are bound to the membrane skeleton. Finally, immunofluorescence studies using antisera raised against the 78 kDa and 48 kDa proteins show that they colocalize with the marginal band in intact cells. Colocalization of cytoskeletal tubulin-binding proteins with the marginal band favors a hypothesis suggesting that the 78 kDa and 48 kDa proteins are involved in the association of the two molecular superstructures.
Asunto(s)
Membrana Eritrocítica/química , Proteínas de la Membrana/análisis , Proteínas Asociadas a Microtúbulos/análisis , Microtúbulos/química , Tubulina (Proteína)/metabolismo , Animales , Pollos , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Immunoblotting , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , SolubilidadRESUMEN
Caldesmon, a major actin- and calcium-dependent calmodulin-binding protein, is now considered as an essential inhibitory factor of the actomyosin machinery in smooth muscle cells as well as in non-muscle cells. Since its structure seems to vary with the cell in a same species and because platelet and erythrocyte have a common embryonic origin, we have used the affinity purified antibody raised against the platelet caldesmon to determine whether this protein is present in erythrocyte. Using the immunoblotting technique, we show here that, in whole erythrocytes, only a single polypeptide crossreacts with this polyclonal antibody. A 71-72 kDa Mr has been calculated from SDS-PAGE. It is therefore different from those of the gizzard (Mr 145-150 kDa) or the platelet (Mr 80 kDa) proteins. Furthermore, we also give evidence that it is not adducin since this newly discovered calcium-dependent calmodulin-binding protein of erythrocyte, does not crossreact with the polyclonal affinity purified antibody raised against platelet caldesmon.