RESUMEN
Previously, we showed that a hierarchy of spectrin cytoskeletal proteins maintains nodal Na+ channels (Liu et al., 2020). Here, using mice lacking ß1, ß4, or ß1/ß4 spectrins, we show this hierarchy does not function at axon initial segments (AIS). Although ß1 spectrin, together with AnkyrinR (AnkR), compensates for loss of nodal ß4 spectrin, it cannot compensate at AIS. We show AnkR lacks the domain necessary for AIS localization. Whereas loss of ß4 spectrin causes motor impairment and disrupts AIS, loss of ß1 spectrin has no discernable effect on central nervous system structure or function. However, mice lacking both neuronal ß1 and ß4 spectrin show exacerbated nervous system dysfunction compared to mice lacking ß1 or ß4 spectrin alone, including profound disruption of AIS Na+ channel clustering, progressive loss of nodal Na+ channels, and seizures. These results further define the important role of AIS and nodal spectrins for nervous system function.
Asunto(s)
Segmento Inicial del Axón/metabolismo , Proteínas Portadoras/metabolismo , Hipocampo/metabolismo , Proteínas de Microfilamentos/metabolismo , Espectrina/metabolismo , Canales de Sodio Activados por Voltaje/metabolismo , Animales , Ancirinas/metabolismo , Conducta Animal , Proteínas Portadoras/genética , Células Cultivadas , Femenino , Hipocampo/fisiopatología , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Actividad Motora , Dominios Proteicos , Prueba de Desempeño de Rotación con Aceleración Constante , Convulsiones/genética , Convulsiones/metabolismo , Convulsiones/fisiopatología , Espectrina/deficiencia , Espectrina/genéticaRESUMEN
BACKGROUND: Precise coordination of cytoskeletal components and dynamic control of cell adhesion and migration are required for crucial cell processes such as differentiation and morphogenesis. We investigated the potential involvement of αII-spectrin, a ubiquitous scaffolding element of the membrane skeleton, in the adhesion and angiogenesis mechanism. METHODS: The cell models were primary human umbilical vein endothelial cells (HUVECs) and a human dermal microvascular endothelial cell line (HMEC-1). After siRNA- and shRNA-mediated knockdown of αII-spectrin, we assessed its expression and that of its partners and adhesion proteins using western blotting. The phenotypes of the control and spectrin-depleted cells were examined using immunofluorescence and video microscopy. Capillary tube formation was assessed using the thick gel Matrigel matrix-based method and a microscope equipped with a thermostatic chamber and a Nikon Biostation System camera. RESULTS: Knockdown of αII-spectrin leads to: modified cell shape; actin cytoskeleton organization with the presence of peripheral actin patches; and decreased formation of stress fibers. Spectrin deficiency affects cell adhesion on laminin and fibronectin and cell motility. This included modification of the localization of adhesion molecules, such as αVß3- and α5-integrins, and organization of adhesion structures, such as focal points. Deficiency of αII-spectrin can also affect the complex mechanism of in vitro capillary tube formation, as demonstrated in a model of angiogenesis. Live imaging revealed that impairment of capillary tube assembly was mainly associated with a significant decrease in cell projection length and stability. αII-spectrin depletion is also associated with significantly decreased expression of three proteins involved in capillary tube formation and assembly: VE-cadherin, MCAM and ß3-integrin. CONCLUSION: Our data confirm the role of αII-spectrin in the control of cell adhesion and spreading. Moreover, our findings further support the participation of αII-spectrin in capillary tube formation in vitro through control of adhesion molecules, such as integrins. This indicates a new function of αII-spectrin in angiogenesis.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Capilares/metabolismo , Adhesión Celular/fisiología , Células Endoteliales/metabolismo , Neovascularización Fisiológica , Espectrina/metabolismo , Antígenos CD/metabolismo , Antígeno CD146/metabolismo , Cadherinas/metabolismo , Capilares/crecimiento & desarrollo , Adhesión Celular/genética , Diferenciación Celular/genética , Movimiento Celular/genética , Forma de la Célula , Células Endoteliales/citología , Fibronectinas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Cadenas alfa de Integrinas/metabolismo , Integrina beta3/metabolismo , Laminina/metabolismo , Morfogénesis/genética , Morfogénesis/fisiología , Neovascularización Fisiológica/genética , ARN Interferente Pequeño , Espectrina/deficiencia , Espectrina/genética , Fibras de Estrés/metabolismoRESUMEN
STAT3 gain-of-function (GOF) mutations can be responsible for an incomplete phenotype mainly characterized by hematological autoimmunity, even in the absence of other organ autoimmunity, growth impairment, or severe infections. We hereby report a case with an incomplete form of STAT3 GOF intensified by a concomitant hereditary hematological disease, which misleads the diagnosis. The patient presented with lymphadenopathy, splenomegaly, hypogammaglobulinemia, and severe autoimmune hemolytic anemia (AIHA) with critical complications, including stroke. A Primary Immune Regulatory Disorders (PIRD) was suspected, and molecular analysis revealed a de novo STAT3 gain-of-function mutation. The response to multiple immune suppressive treatments was ineffective, and further investigations revealed a spectrin deficiency. Ultimately, hematopoietic stem cell transplantation from a matched unrelated donor was able to cure the patient. Our case shows an atypical presentation of STAT3 GOF associated with hereditary spherocytosis, and how achievement of a good long-term outcome depends on a strict clinical and laboratory monitoring, as well as on prompt therapeutic intervention.
Asunto(s)
Agammaglobulinemia/genética , Anemia Hemolítica Autoinmune/genética , Mutación con Ganancia de Función , Trastornos Linfoproliferativos/genética , Factor de Transcripción STAT3/genética , Espectrina/deficiencia , Agammaglobulinemia/inmunología , Edad de Inicio , Anemia Hemolítica Autoinmune/inmunología , Niño , Ciclosporina/efectos adversos , Ciclosporina/farmacocinética , Ciclosporina/uso terapéutico , Citocromo P-450 CYP3A/genética , Femenino , Mutación de Línea Germinal , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/etiología , Trastornos del Crecimiento/genética , Trasplante de Células Madre Hematopoyéticas , Humanos , Inmunoglobulinas Intravenosas/uso terapéutico , Inmunosupresores/uso terapéutico , Trastornos Linfoproliferativos/tratamiento farmacológico , Trastornos Linfoproliferativos/inmunología , Mutación Missense , Polimorfismo de Nucleótido Simple , Prednisolona/uso terapéutico , Hemorragia Retiniana/inducido químicamente , Factor de Transcripción STAT3/fisiología , Espectrina/genética , Donante no EmparentadoRESUMEN
Increased fibrosis is a characteristic remodeling response to biomechanical and neurohumoral stress and a determinant of cardiac mechanical and electrical dysfunction in disease. Stress-induced activation of cardiac fibroblasts (CFs) is a critical step in the fibrotic response, although the precise sequence of events underlying activation of these critical cells in vivo remain unclear. Here, we tested the hypothesis that a ßIV-spectrin/STAT3 complex is essential for maintenance of a quiescent phenotype (basal nonactivated state) in CFs. We reported increased fibrosis, decreased cardiac function, and electrical impulse conduction defects in genetic and acquired mouse models of ßIV-spectrin deficiency. Loss of ßIV-spectrin function promoted STAT3 nuclear accumulation and transcriptional activity, and it altered gene expression and CF activation. Furthermore, we demonstrate that a quiescent phenotype may be restored in ßIV-spectrin-deficient fibroblasts by expressing a ßIV-spectrin fragment including the STAT3-binding domain or through pharmacological STAT3 inhibition. We found that in vivo STAT3 inhibition abrogates fibrosis and cardiac dysfunction in the setting of global ßIV-spectrin deficiency. Finally, we demonstrate that fibroblast-specific deletion of ßIV-spectrin is sufficient to induce fibrosis and decreased cardiac function. We propose that the ßIV-spectrin/STAT3 complex is a determinant of fibroblast phenotype and fibrosis, with implications for remodeling response in cardiovascular disease (CVD).
Asunto(s)
Enfermedades Cardiovasculares/fisiopatología , Fibroblastos/patología , Ventrículos Cardíacos/patología , Factor de Transcripción STAT3/metabolismo , Espectrina/deficiencia , Animales , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/patología , Modelos Animales de Enfermedad , Femenino , Fibrosis , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/fisiopatología , Humanos , Masculino , Ratones , Ratones Noqueados , Factor de Transcripción STAT3/antagonistas & inhibidores , Espectrina/genética , Remodelación VentricularRESUMEN
Sensory neurons embedded in skin are responsible for the sense of touch. In humans and other mammals, touch sensation depends on thousands of diverse somatosensory neurons. By contrast, Caenorhabditis elegans nematodes have six gentle touch receptor neurons linked to simple behaviors. The classical touch assay uses an eyebrow hair to stimulate freely moving C. elegans, evoking evasive behavioral responses. This assay has led to the discovery of genes required for touch sensation, but does not provide control over stimulus strength or position. Here, we present an integrated system for performing automated, quantitative touch assays that circumvents these limitations and incorporates automated measurements of behavioral responses. The Highly Automated Worm Kicker (HAWK) unites a microfabricated silicon force sensor holding a glass bead forming the contact surface and video analysis with real-time force and position control. Using this system, we stimulated animals along the anterior-posterior axis and compared responses in wild-type and spc-1(dn) transgenic animals, which have a touch defect due to expression of a dominant-negative α-spectrin protein fragment. As expected from prior studies, delivering large stimuli anterior and posterior to the mid-point of the body evoked a reversal and a speed-up, respectively. The probability of evoking a response of either kind depended on stimulus strength and location; once initiated, the magnitude and quality of both reversal and speed-up behavioral responses were uncorrelated with stimulus location, strength, or the absence or presence of the spc-1(dn) transgene. Wild-type animals failed to respond when the stimulus was applied near the mid-point. These results show that stimulus strength and location govern the activation of a characteristic motor program and that the C. elegans body surface consists of two receptive fields separated by a gap.
Asunto(s)
Caenorhabditis elegans/fisiología , Animales , Animales Modificados Genéticamente , Conducta Animal/fisiología , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Sistemas de Computación , Mecanorreceptores/fisiología , Mecanotransducción Celular/fisiología , Estimulación Física/instrumentación , Células Receptoras Sensoriales/fisiología , Espectrina/deficiencia , Espectrina/genética , Espectrina/fisiología , Tacto/fisiologíaRESUMEN
Congenital myopathies are a heterogeneous group of muscle disorders that are often genetically determined. Here, we investigated a boy with congenital myopathy, deafness, and neuropathy from a consanguineous Kurdish family by autozygosity mapping and whole exome sequencing. We found a homozygous nonsense mutation in SPTBN4 [c.1597C>T, NM_020971.2; p.(Q533*), NP_066022.2; ClinVar SUB2292235] encoding ßIV-spectrin, a non-erythrocytic member of the ß-spectrin family. Western blot confirmed the absence of the full-length 288 kDa isoform in muscle and of a specific 72 kDa isoform in fibroblasts. Clinical symptoms of the patient largely corresponded to those described for the quivering mouse, a loss-of-function animal model. Since the human phenotype of ßIV-spectrin deficiency included a myopathy with incomplete congenital fiber-type disproportion, we investigated muscle of the quivering (qv4J) mouse and found complete absence of type 1 fibers (fiber-type 2 uniformity). Immunohistology confirmed expression of ßIV-spectrin in normal human and mouse muscle at the sarcolemma and its absence in patient and quivering (qv4J) mouse. SPTBN4 mRNA-expression levels in healthy skeletal muscle were found in the range of other regulatory proteins. More patients have to be described to confirm the triad of congenital myopathy, neuropathy and deafness as the defining symptom complex for ßIV-spectrin deficiency.
Asunto(s)
Anomalías Congénitas/genética , Sordera/genética , Genes Recesivos , Enfermedades Musculares/genética , Proteínas del Tejido Nervioso/genética , Espectrina/genética , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Codón sin Sentido , Perfilación de la Expresión Génica , Genoma Humano , Humanos , Masculino , Ratones , Músculo Esquelético/metabolismo , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/metabolismo , Linaje , Análisis de Secuencia de ADN , Espectrina/deficiencia , Espectrina/metabolismoRESUMEN
Our bodies are in constant motion and so are the neurons that invade each tissue. Motion-induced neuron deformation and damage are associated with several neurodegenerative conditions. Here, we investigated the question of how the neuronal cytoskeleton protects axons and dendrites from mechanical stress, exploiting mutations in UNC-70 ß-spectrin, PTL-1 tau/MAP2-like and MEC-7 ß-tubulin proteins in Caenorhabditis elegans. We found that mechanical stress induces supercoils and plectonemes in the sensory axons of spectrin and tau double mutants. Biophysical measurements, super-resolution, and electron microscopy, as well as numerical simulations of neurons as discrete, elastic rods provide evidence that a balance of torque, tension, and elasticity stabilizes neurons against mechanical deformation. We conclude that the spectrin and microtubule cytoskeletons work in combination to protect axons and dendrites from mechanical stress and propose that defects in ß-spectrin and tau may sensitize neurons to damage.
Asunto(s)
Axones/fisiología , Caenorhabditis elegans/fisiología , Proteínas Asociadas a Microtúbulos/deficiencia , Movimiento , Espectrina/deficiencia , Torque , Tubulina (Proteína)/deficiencia , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans , Estrés MecánicoRESUMEN
Invadosomes are actin-rich adhesion structures involved in tissue invasion and extracellular matrix (ECM) remodelling. αII-Spectrin, an ubiquitous scaffolding component of the membrane skeleton and a partner of actin regulators (ABI1, VASP and WASL), accumulates highly and specifically in the invadosomes of multiple cell types, such as mouse embryonic fibroblasts (MEFs) expressing SrcY527F, the constitutively active form of Src or activated HMEC-1 endothelial cells. FRAP and live-imaging analysis revealed that αII-spectrin is a highly dynamic component of invadosomes as actin present in the structures core. Knockdown of αII-spectrin expression destabilizes invadosomes and reduces the ability of the remaining invadosomes to digest the ECM and to promote invasion. The ECM degradation defect observed in spectrin-depleted-cells is associated with highly dynamic and unstable invadosome rings. Moreover, FRAP measurement showed the specific involvement of αII-spectrin in the regulation of the mobile/immobile ß3-integrin ratio in invadosomes. Our findings suggest that spectrin could regulate invadosome function and maturation by modulating integrin mobility in the membrane, allowing the normal processes of adhesion, invasion and matrix degradation. Altogether, these data highlight a new function for spectrins in the stability of invadosomes and the coupling between actin regulation and ECM degradation.
Asunto(s)
Matriz Extracelular/metabolismo , Podosomas/metabolismo , Espectrina/metabolismo , Animales , Línea Celular , Técnicas de Silenciamiento del Gen , Humanos , Integrina beta3/química , Integrina beta3/metabolismo , Ratones , Imagen Molecular , Mutación , Estabilidad Proteica , Espectrina/deficiencia , Espectrina/genéticaRESUMEN
The diagnosis of hereditary spherocytosis (HS) in a newborn infant is generally made on the basis of a positive family history, spherocytes on blood film and Coombs-negative hemolytic jaundice of variable severity with an elevated mean corpuscular hemoglobin concentration (MCHC) and a low mean corpuscular volume (MCV). In general, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) quantification of erythrocyte membrane proteins is not needed to make the clinical diagnosis of HS. However, we observed that a neonate with no family history of HS, but with abundant spherocytosis on repeated blood films, Coombs-negative hemolytic jaundice and normal MCHC and MCV measurements, where SDS-PAGE revealed alpha-spectrin deficiency, a rare autosomal-recessive variety of HS that generally has a severe clinical phenotype.
Asunto(s)
Ictericia/sangre , Espectrina/deficiencia , Esferocitosis Hereditaria/etiología , Prueba de Coombs , Índices de Eritrocitos , Humanos , Recién Nacido , Masculino , Esferocitos , Esferocitosis Hereditaria/sangreRESUMEN
Hereditary spherocytosis (HS) is a congenital hemolytic anemia which is characterized by spherocytes in peripheral blood and increased osmotic fragility test. The disease is caused by defects in red cell membrane cytoskeleton. In this study, we investigated erythrocyte membrane protein defects in 50 Turkish HS patients and 42 controls. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to identify the protein defects causing HS. The patients were from 27 families (39 kindred and 11 unrelated patients). They were aged between 6 months and 53 years and the mean age was 18.75 (±14.70) years. Protein deficiencies related to HS were demonstrated in 42% of study group. There was not any statistically significant relation between the protein deficiency and hemoglobin levels. Isolated or combined spectrin deficiency was the most common protein abnormality among our patients. Spectrin deficiency was detected in 22% of cases (11/50), ankyrin deficiency in 8% (4/50), protein 4.2 deficiency in 8% (4/50), combined spectrin and protein 4.2 deficiency in 2% (1/50), combined spectrin and ankyrin deficiency in 2% (1/50). Fifty-eight percent of cases (29/50) showed normal protein contents.
Asunto(s)
Membrana Eritrocítica/metabolismo , Proteínas de la Membrana/metabolismo , Esferocitosis Hereditaria/metabolismo , Adolescente , Adulto , Ancirinas/deficiencia , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Proteínas de la Membrana/deficiencia , Persona de Mediana Edad , Espectrina/deficiencia , Turquía , Adulto JovenRESUMEN
Although columnar epithelial cells are known to acquire an elongated shape, the mechanisms involved in this morphological feature have not yet been completely elucidated. Using columnar human intestinal Caco2 cells, it was established here that the levels of drebrin E, an actin-binding protein, increase in the terminal web both in vitro and in vivo during the formation of the apical domain. Drebrin E depletion was found to impair cell compaction and elongation processes in the monolayer without affecting cell polarity or the formation of tight junctions. Decreasing the drebrin E levels disrupted the normal subapical F-actin-myosin-IIB-ßII-spectrin network and the apical accumulation of EB3, a microtubule-plus-end-binding protein. Decreasing the EB3 levels resulted in a similar elongation phenotype to that resulting from depletion of drebrin E, without affecting cell compaction processes or the pattern of distribution of F-actin-myosin-IIB. In addition, EB3, myosin IIB and ßII spectrin were found to form a drebrin-E-dependent complex. Taken together, these data suggest that this complex connects the F-actin and microtubule networks apically during epithelial cell morphogenesis, while drebrin E also contributes to stabilizing the actin-based terminal web.
Asunto(s)
Forma de la Célula/fisiología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Neuropéptidos/metabolismo , Espectrina/metabolismo , Actinas/metabolismo , Células CACO-2 , Polaridad Celular/fisiología , Humanos , Proteínas Asociadas a Microtúbulos , Neuropéptidos/deficiencia , Miosina Tipo IIB no Muscular/metabolismo , Espectrina/deficiencia , Uniones EstrechasRESUMEN
Mutations in the gene encoding ß-III spectrin give rise to spinocerebellar ataxia type 5, a neurodegenerative disease characterized by progressive thinning of the molecular layer, loss of Purkinje cells and increasing motor deficits. A mouse lacking full-length ß-III spectrin (ß-IIIâ»/â») displays a similar phenotype. In vitro and in vivo analyses of Purkinje cells lacking ß-III spectrin, reveal a critical role for ß-III spectrin in Purkinje cell morphological development. Disruption of the normally well ordered dendritic arborization occurs in Purkinje cells from ß-IIIâ»/â» mice, specifically showing a loss of monoplanar organization, smaller average dendritic diameter and reduced densities of Purkinje cell spines and synapses. Early morphological defects appear to affect distribution of dendritic, but not axonal, proteins. This study confirms that thinning of the molecular layer associated with disease pathogenesis is a consequence of Purkinje cell dendritic degeneration, as Purkinje cells from 8-month-old ß-IIIâ»/â» mice have drastically reduced dendritic volumes, surface areas and total dendritic lengths compared with 5- to 6-week-old ß-IIIâ»/â» mice. These findings highlight a critical role of ß-III spectrin in dendritic biology and are consistent with an early developmental defect in ß-IIIâ»/â» mice, with abnormal Purkinje cell dendritic morphology potentially underlying disease pathogenesis.
Asunto(s)
Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Dendritas/ultraestructura , Espinas Dendríticas/metabolismo , Células de Purkinje/citología , Espectrina/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Calbindinas , Transportador 4 de Aminoácidos Excitadores/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Transportador de Glucosa de Tipo 2/metabolismo , Técnicas In Vitro , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Depresión Sináptica a Largo Plazo/genética , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Canal de Sodio Activado por Voltaje NAV1.6 , Proteínas del Tejido Nervioso/metabolismo , Técnicas de Placa-Clamp , Proteínas de Transporte de Fosfato/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Tinción con Nitrato de Plata/métodos , Canales de Sodio/metabolismo , Espectrina/deficiencia , Proteína 1 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
During peripheral nerve development, Schwann cells ensheathe axons and form myelin to enable rapid and efficient action potential propagation. Although myelination requires profound changes in Schwann cell shape, how neuron-glia interactions converge on the Schwann cell cytoskeleton to induce these changes is unknown. Here, we demonstrate that the submembranous cytoskeletal proteins αII and ßII spectrin are polarized in Schwann cells and colocalize with signaling molecules known to modulate myelination in vitro. Silencing expression of these spectrins inhibited myelination in vitro, and remyelination in vivo. Furthermore, myelination was disrupted in motor nerves of zebrafish lacking αII spectrin. Finally, we demonstrate that loss of spectrin significantly reduces both F-actin in the Schwann cell cytoskeleton and the Nectin-like protein, Necl4, at the contact site between Schwann cells and axons. Therefore, we propose αII and ßII spectrin in Schwann cells integrate the neuron-glia interactions mediated by membrane proteins into the actin-dependent cytoskeletal rearrangements necessary for myelination.
Asunto(s)
Vaina de Mielina/fisiología , Nervios Periféricos/fisiología , Células de Schwann/fisiología , Espectrina/fisiología , Actinas/antagonistas & inhibidores , Actinas/fisiología , Animales , Secuencia de Bases , Polaridad Celular , Citoesqueleto/fisiología , Técnicas de Silenciamiento del Gen , Mutación , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Células de Schwann/citología , Nervio Ciático/citología , Nervio Ciático/lesiones , Nervio Ciático/fisiología , Espectrina/antagonistas & inhibidores , Espectrina/deficiencia , Espectrina/genética , Pez Cebra/genética , Pez Cebra/fisiología , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiologíaRESUMEN
Lu/BCAM glycoproteins (gps) are the unique erythroid receptors of laminin alpha5 chain, a major component of the extracellular matrix. They interact with the membrane skeleton by binding directly to spectrin via the Lu/BCAM RK573-574 motif. Lu/BCAM gps are involved in abnormal sickle red blood cell (RBC) adhesion to components of the vascular wall. This adhesion is activated by the phosphorylation of the Lu/BCAM long isoform Lu in a protein kinase A-dependent manner. A similar high adhesion to laminin was also observed with RBCs from Hereditary Spherocytosis (HS) patients suffering from haemolytic anaemia subsequent to spectrin deficiencies. We investigated the molecular mechanisms responsible for the Lu/BCAM-mediated abnormal RBC adhesion to laminin in sickle cell disease (SCD) and HS. We showed that SCD patients treated with hydroxycarbamide (HC) had a diminished RBC adhesion to laminin that was associated with reduced levels of the PKA upstream effector cAMP and a severe decrease in Lu isoform phosphorylation. On the other hand, we showed that increased Lu/BCAM-mediated HS RBC adhesion to laminin was independent of Lu/BCAM phosphorylation. A cellular model expressing the RK573-574AA Lu/BCAM mutant, which is unable to bind to spectrin, showed increased Lu/BCAM detergent extractability and enhanced cell adhesion to laminin. Similar results were obtained with HS RBCs, strongly suggesting that their increased adhesion could result from alteration of the Lu/BCAM-spectrin interaction following the severe spectrin deficiency.
Asunto(s)
Moléculas de Adhesión Celular/genética , Sistema del Grupo Sanguíneo Lutheran/genética , Anemia Hemolítica/genética , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/metabolismo , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/metabolismo , Eritrocitos Anormales/metabolismo , Humanos , Laminina/metabolismo , Sistema del Grupo Sanguíneo Lutheran/metabolismo , Glicoproteínas de Membrana/sangre , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Espectrina/deficiencia , Espectrina/metabolismo , Esferocitosis Hereditaria/genéticaRESUMEN
Mutations in SPTBN2, the gene encoding beta-III spectrin, cause spinocerebellar ataxia type 5 in humans (SCA5), a neurodegenerative disorder resulting in loss of motor coordination. How these mutations give rise to progressive ataxia and what the precise role beta-III spectrin plays in normal cerebellar physiology are unknown. We developed a mouse lacking full-length beta-III spectrin and found that homozygous mice reproduced features of SCA5 including gait abnormalities, tremor, deteriorating motor coordination, Purkinje cell loss, and cerebellar atrophy (molecular layer thinning). In vivo analysis reveals an age-related reduction in simple spike firing rate in surviving beta-III(-/-) Purkinje cells, whereas in vitro studies show these neurons to have reduced spontaneous firing, smaller sodium currents, and dysregulation of glutamatergic neurotransmission. Our data suggest an early loss of EAAT4- (protein interactor of beta-III spectrin) and a subsequent loss of GLAST-mediated uptake may play a role in neuronal pathology. These findings implicate a loss of beta-III spectrin function in SCA5 pathogenesis and indicate that there are at least two physiological effects of beta-III spectrin loss that underpin a progressive loss of inhibitory cerebellar output, namely an intrinsic Purkinje cell membrane defect due to reduced sodium currents and alterations in glutamate signaling.
Asunto(s)
Actividad Motora/genética , Células de Purkinje/metabolismo , Células de Purkinje/patología , Espectrina/deficiencia , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/patología , Potenciales de Acción/genética , Animales , Atrofia/genética , Cerebelo/patología , Marcha/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Espectrina/genética , Ataxias Espinocerebelosas/metabolismo , Ataxias Espinocerebelosas/fisiopatología , Temblor/genéticaRESUMEN
The spectrin membrane skeleton controls the disposition of selected membrane channels, receptors, and transporters. In the brain betaIII spectrin binds directly to the excitatory amino acid transporter (EAAT4), the glutamate receptor delta, and other proteins. Mutations in betaIII spectrin link strongly to human spinocerebellar ataxia type 5 (SCA5), correlating with alterations in EAAT4. We have explored the mechanistic basis of this phenotype by targeted gene disruption of Spnb3. Mice lacking intact betaIII spectrin develop normally. By 6 months they display a mild nonprogressive ataxia. By 1 year most Spnb3(-/-) animals develop a myoclonic seizure disorder with significant reductions of EAAT4, EAAT1, GluRdelta, IP3R, and NCAM140. Other synaptic proteins are normal. The cerebellum displays increased dark Purkinje cells (PC), a thin molecular layer, fewer synapses, a loss of dendritic spines, and a 2-fold expansion of the PC dendrite diameter. Membrane and expanded Golgi profiles fill the PC dendrite and soma, and both regions accumulate EAAT4. Correlating with the seizure disorder are enhanced hippocampal levels of neuropeptide Y and EAAT3 and increased calpain proteolysis of alphaII spectrin. It appears that betaIII spectrin disruption impairs synaptogenesis by disturbing the intracellular pathways selectively regulating protein trafficking to the synapse. The mislocalization of these proteins secondarily disrupts glutamate transport dynamics, leading to seizures, neuronal damage, and compensatory changes in EAAT3 and neuropeptide Y.
Asunto(s)
Ataxia/etiología , Convulsiones/etiología , Espectrina/deficiencia , Animales , Ataxia/genética , Ataxia/fisiopatología , Secuencia de Bases , Encéfalo/metabolismo , Encéfalo/fisiopatología , Encéfalo/ultraestructura , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Transportador 4 de Aminoácidos Excitadores/metabolismo , Femenino , Marcación de Gen , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Degeneración Nerviosa/genética , Degeneración Nerviosa/fisiopatología , Fenotipo , Convulsiones/genética , Convulsiones/fisiopatología , Espectrina/genética , Espectrina/fisiología , Ataxias Espinocerebelosas/etiología , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/fisiopatología , Sinapsis/fisiología , Sinapsis/ultraestructuraRESUMEN
Hereditary Spherocytosis (HS) is a haemolytic anaemia caused by erythrocyte protein membrane defects - spectrin, ankyrin, band 3 or protein 4.2 - that lead to membrane destabilization. This study aimed to evaluate the prevalence of protein deficiencies and the role of membrane proteins or membrane-linked proteins in membrane disturbance and in HS clinical outcome. A total of 215 Portuguese individuals were studied - 203 from 71 families plus 12 individual unrelated subjects; 160 of them were diagnosed with HS. They were classified as presenting mild, moderate or severe forms of HS according to the degree of haemolytic anaemia. Standardized electrophoretic erythrocyte membrane protein analysis was used to identify and quantify protein deficiencies. Band 3 and ankyrin were found to account for the majority of the erythrocyte protein defects underlying HS. Increasing isolated protein deficiency or increasing imbalance between combined protein deficiencies seemed to underlie HS severity, by increasing membrane destabilization. There was an increased membrane linkage of the cytosolic proteins, glyceraldehyde-3-phosphate dehydrogenase and peroxiredoxin 2, and of denatured haemoglobin, suggesting that this linkage could interfere with membrane structure. Our data suggest that the quantification and the analysis of RBC membrane proteins may be helpful in predicting the clinical outcome of HS.
Asunto(s)
Membrana Eritrocítica/metabolismo , Proteínas de la Membrana/deficiencia , Esferocitosis Hereditaria/sangre , Adulto , Proteína 1 de Intercambio de Anión de Eritrocito/deficiencia , Ancirinas/deficiencia , Niño , Electroforesis en Gel de Poliacrilamida , Hemoglobinas/metabolismo , Proteínas de Homeodominio/sangre , Humanos , Pronóstico , Índice de Severidad de la Enfermedad , Espectrina/deficienciaRESUMEN
Five spontaneous, allelic mutations in the alpha-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph(1J), sph(2J), sph(2BC), sph(Dem)). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph(3J), a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sph(Ihj), a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent beta-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph(4J), a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, beta-adducin. The severity of anemia in sph(4J) indicates that the highly conserved cysteine residue at the C-terminus of alpha-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3.
Asunto(s)
Proteína 1 de Intercambio de Anión de Eritrocito/deficiencia , Proteína 1 de Intercambio de Anión de Eritrocito/genética , Proteínas de Unión a Calmodulina/deficiencia , Proteínas de Unión a Calmodulina/genética , Mutación , Espectrina/deficiencia , Espectrina/genética , Esferocitosis Hereditaria/sangre , Esferocitosis Hereditaria/genética , Alelos , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Unión a Calmodulina/sangre , Codón sin Sentido , Cartilla de ADN/genética , Membrana Eritrocítica/metabolismo , Eritrocitos Anormales/metabolismo , Eritrocitos Anormales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Mutantes , Datos de Secuencia Molecular , Mutación Missense , Estabilidad del ARN/genética , Homología de Secuencia de Aminoácido , Esferocitosis Hereditaria/patologíaRESUMEN
Lu/BCAM, the unique erythroid receptor for laminin 511/521, interacts with the erythrocyte membrane skeleton through spectrin binding. It has been reported that Hereditary Spherocytosis red blood cells (HS RBC) exhibit increased adhesion to laminin. We investigated the role of Lu/BCAM-spectrin interaction in the RBC adhesion properties of 2 splenectomised HS patients characterized by 40% spectrin deficiency. Under physiological flow conditions, HS RBC exhibited an exaggerated adhesion to laminin that was completely abolished by soluble Lu/BCAM. Triton extraction experiments revealed that a greater fraction of Lu/BCAM was unlinked to the membrane skeleton of HS RBC, as compared to normal RBC. Disruption of the spectrin interaction site in Lu/BCAM expressed in the transfected K562 cell line resulted in a weakened interaction to the skeleton and an enhanced interaction to laminin. These results demonstrated that the adhesion of HS RBC to laminin was mediated by Lu/BCAM and that its interaction with the spectrin-based skeleton negatively regulated cell adhesion to laminin. Finally, the results of this study strongly suggest that the reinforced adhesiveness of spectrin-deficient HS RBC to laminin is partly brought about by an impaired interaction between Lu/BCAM and the membrane skeleton.
Asunto(s)
Moléculas de Adhesión Celular/sangre , Membrana Eritrocítica/metabolismo , Laminina/sangre , Sistema del Grupo Sanguíneo Lutheran/sangre , Espectrina/metabolismo , Esferocitosis Hereditaria/sangre , Adhesión Celular/fisiología , Eritrocitos/metabolismo , Femenino , Hemorreología , Humanos , Recién Nacido , Células K562 , Masculino , Fosforilación , Proteínas Recombinantes/metabolismo , Espectrina/deficienciaRESUMEN
Transforming growth factor-beta (TGF-beta) signaling members, TGF-beta receptor type II (TBRII), Smad2, Smad4 and Smad adaptor, embryonic liver fodrin (ELF), are prominent tumor suppressors in gastrointestinal cancers. Here, we show that 40% of elf(+/-) mice spontaneously develop hepatocellular cancer (HCC) with markedly increased cyclin D1, cyclin-dependent kinase 4 (Cdk4), c-Myc and MDM2 expression. Reduced ELF but not TBRII, or Smad4 was observed in 8 of 9 human HCCs (P<0.017). ELF and TBRII are also markedly decreased in human HCC cell lines SNU-398 and SNU-475. Restoration of ELF and TBRII in SNU-398 cells markedly decreases cyclin D1 as well as hyperphosphorylated-retinoblastoma (hyperphosphorylated-pRb). Thus, we show that TGF-beta signaling and Smad adaptor ELF suppress human hepatocarcinogenesis, potentially through cyclin D1 deregulation. Loss of ELF could serve as a primary event in progression toward a fully transformed phenotype and could hold promise for new therapeutic approaches in human HCCs.