RESUMEN
Besides a fundamental structural role at the plasma membrane, spectrin- and actin-based skeletons have been proposed to participate in various processes including vesicular trafficking. Neuroendocrine cells release hormones and neuropeptides through calcium-regulated exocytosis, a process that is coordinated by a fine remodeling of the actin cytoskeleton. We describe here that calcium-regulated exocytosis is impaired in chromaffin and PC12 cells with reduced αII-spectrin expression levels. Using yeast two-hybrid screening, we show that neuronal Wiskott-Aldrich Syndrome protein (N-WASP) is a partner of the αII-spectrin SH3 domain and demonstrate that secretagogue-evoked N-WASP recruitment at cell periphery is blocked in the absence of αII-spectrin. Additionally, experiments performed with ectopically expressed αII-spectrin mutant unable to bind N-WASP indicated that the interaction between SH3 domain and N-WASP is pivotal for neuroendocrine secretion. Our results extend the list of spectrin interactors and strengthen the idea that αII-spectrin is an important scaffold protein that gathers crucial actin-related players of the exocytic machinery.
Asunto(s)
Proteínas Portadoras/metabolismo , Células Cromafines/metabolismo , Proteínas de Microfilamentos/metabolismo , Células Neuroendocrinas/metabolismo , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismo , Animales , Calcio/metabolismo , Proteínas Portadoras/genética , Catecolaminas/metabolismo , Bovinos , Exocitosis/fisiología , Hormona del Crecimiento/metabolismo , Proteínas de Microfilamentos/genética , Mutación , Células PC12 , Ratas , Técnicas del Sistema de Dos Híbridos , Proteína Neuronal del Síndrome de Wiskott-Aldrich/genética , Dominios Homologos srcRESUMEN
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
AlphaII-spectrin, a basic component of the spectrin-based scaffold which organizes and stabilizes membrane microdomains in most animal cells, has been recently implicated in cell adherence and actin dynamics. Here we investigated the contribution of αΙΙ-spectrin to neuritogenesis, a highly complex cellular process which requires continuous actin cytoskeleton remodeling and cross-talk between extracellular cues and their cell surface receptors, including cell adhesion molecules. Using RNA interference-mediated gene silencing to down-regulate αΙΙ-spectrin expression in human neuroblastoma SH-SY5Y cells, we observed major changes in neurite morphology and cell shape: (1) reduced mean length and a higher number of neurites per cell; occasional long neurites were thinner and displayed abnormal adhesiveness during cell migration resulting in frequent breaks; similar persisting adhesiveness and breaks were also observed in trailing edges of cell bodies; (2) irregular polygonal cell shape in parallel with loss of cortical F-actin from neuronal cell bodies; (3) reduction in protein levels of αΙ- and ßΙ-spectrins, but not ßΙΙ-spectrin (4) decreased global expression of adhesion molecule L1 and spectrin-binding adapter ankyrin-B, which links L1 to the plasma membrane. Remarkably, αΙΙ-spectrin depletion affected L1 - but not NCAM - cell surface expression, and L1 clustering at growth cones. This study demonstrates that αΙΙ-spectrin is implicated in normal morphology and adhesive properties of neuron cell bodies and neurites, and in cell surface expression and organization of adhesion molecule L1.
Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Neuritas/fisiología , Neuroblastoma/metabolismo , Neuroblastoma/patología , Animales , Ancirinas/metabolismo , Western Blotting , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/genética , ARN Interferente Pequeño/genética , Espectrina/metabolismo , Células Tumorales CultivadasRESUMEN
Lu/BCAM (Lutheran/basal cell-adhesion molecule) is a laminin 511/521 receptor expressed in erythroid and endothelial cells, and in epithelial tissues. The RK573-574 (Arg573-Lys574) motif of the Lu/BCAM cytoplasmic domain interacts with αI-spectrin, the main component of the membrane skeleton in red blood cells. In the present paper we report that Lu/BCAM binds to the non-erythroid αII-spectrin via the RK573-574 motif. Alanine substitution of this motif abolished the Lu/BCAM-spectrin interaction, enhanced the half-life of Lu/BCAM at the MDCK (Madin-Darby canine kidney) cell surface, and increased Lu/BCAM-mediated cell adhesion and spreading on laminin 511/521. We have shown that the Lu/BCAM-spectrin interaction mediated actin reorganization during cell adhesion and spreading on laminin 511/521. This interaction was involved in a laminin 511/521-to-actin signalling pathway leading to stress fibre formation. This skeletal rearrangement was associated with an activation of the small GTP-binding protein RhoA, which depended on the integrity of the Lu/BCAM laminin 511/521-binding site. It also required a Lu/BCAM-αII-spectrin interaction, since its disruption decreased stress fibre formation and RhoA activation. We conclude that the Lu/BCAM-spectrin interaction is required for stress fibre formation during cell spreading on laminin 511/521, and that spectrin acts as a signal relay between laminin 511/521 and actin that is involved in actin dynamics.
Asunto(s)
Actinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Sistema del Grupo Sanguíneo Lutheran/metabolismo , Espectrina/metabolismo , Secuencias de Aminoácidos , Animales , Adhesión Celular/fisiología , Línea Celular , Citoesqueleto/metabolismo , Perros , Eritrocitos/metabolismo , Humanos , Receptores de Laminina/metabolismoRESUMEN
Lu (Lutheran) blood group and BCAM (basal cell adhesion molecule) antigens both reside on two gp (glycoprotein) isoforms, Lu and Lu(v13), that differ by the size of their cytoplasmic tail. They are receptors of laminin-10/11 and are expressed in RBCs (red blood cells), epithelial cells of multiple tissues and vascular endothelial cells. To gain more insights into the biological function of Lu/BCAM gps, we looked for potential partners of their cytoplasmic tail. We isolated Ubc9 (ubiquitin-conjugating enzyme 9) protein by screening a human kidney library using the yeast two-hybrid system. Lu/Ubc9 interaction was validated by GST (glutathione S-transferase) pull-down and co-immunoprecipitation experiments. Endogenous Ubc9 formed a complex with endogenous or recombinant Lu gp in A498 and MDCK (Madin-Darby canine kidney) epithelial cells respectively. Replacement of Lys(585) by alanine in the Lu gp abolished in vitro and ex vivo interactions of Lu gp with Ubc9 protein. Lu K585A mutant transfected in MDCK cells exhibited a normal basolateral membrane expression but was overexpressed at the surface of polarized MDCK cells as compared with wild-type Lu. Pulse-chase experiments showed extended half-life of Lu K585A gp at the plasma membrane, suggesting an impaired endocytosis of this mutant leading to protein accumulation at the membrane. Furthermore, we showed that the ability of MDCK-Lu K585A cells to spread on immobilized laminin was dramatically decreased. Our results support a physiological role for the direct interaction between Lu gp and Ubc9 protein and reveal a role for this enzyme in regulating the stability of Lu gp at the cell membrane.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Polaridad Celular , Enzimas Ubiquitina-Conjugadoras/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Adhesión Celular , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Línea Celular , Membrana Celular/metabolismo , Forma de la Célula , Citoplasma/metabolismo , Perros , Regulación de la Expresión Génica , Humanos , Lisina/genética , Lisina/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Técnicas del Sistema de Dos Híbridos , Enzimas Ubiquitina-Conjugadoras/genéticaRESUMEN
Spectrins, components of the membrane skeleton, are implicated in various cellular functions. Understanding the diversity of these functions requires better characterization of the interacting domains of spectrins, such as the SH3 domain. Yeast two-hybrid screening of a kidney cDNA library revealed that the SH3 domain of alpha II-spectrin binds specifically isoform A of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP). The alpha II-spectrin SH3 domain does not interact with LMW-PTP B or C nor does LMW-PTP A interact with the alpha I-spectrin SH3 domain. The interaction of spectrin with LMW-PTP A led us to look for a tyrosine-phosphorylated residue in alpha II-spectrin. Western blotting showed that alpha II-spectrin is tyrosine phosphorylated in vivo. Using mutagenesis on recombinant peptides, we identified the residue Y1176 located in the calpain cleavage site of alpha II-spectrin, near the SH3 domain, as an in vitro substrate for Src kinase and LMW-PTP A. This Y1176 residue is also an in vivo target for kinases and phosphatases in COS cells. Phosphorylation of this residue decreases spectrin sensitivity to calpain in vitro. Similarly, the presence of phosphatase inhibitors in cell culture is associated with the absence of spectrin cleavage products. This suggests that the Y1176 phosphorylation state could modulate spectrin cleavage by calpain and may play an important role during membrane skeleton remodeling.
Asunto(s)
Calpaína/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Espectrina/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Animales , Fraccionamiento Celular , Línea Celular , Citoesqueleto/metabolismo , Masculino , Datos de Secuencia Molecular , Fosforilación , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , Ratas , Ratas Sprague-Dawley , Alineación de Secuencia , Técnicas del Sistema de Dos Híbridos , Levaduras/genética , Levaduras/metabolismoRESUMEN
T-lymphocyte activation after antigen presentation to the T-Cell Receptor (TCR) is a critical step in the development of proper immune responses to infection and inflammation. This dynamic process involves reorganization of the actin cytoskeleton and signaling molecules at the cell membrane, leading to the formation of the Immunological Synapse (IS). The mechanisms regulating the formation of the IS are not completely understood. Nonerythroid spectrin is a membrane skeletal protein involved in the regulation of many cellular processes, including cell adhesion, signaling and actin cytoskeleton remodeling. However, the role of spectrin in IS formation has not been explored. We used molecular, imaging and cellular approaches to show that nonerythroid αII-spectrin redistributes to the IS during T-cell activation. The redistribution of spectrin coincides with the relocation of CD45 and LFA-1, two components essential for IS formation and stability. We assessed the role of spectrin by shRNA-mediated depletion from Jurkat T cells and show that spectrin-depleted cells exhibit decreased adhesion and are defective in forming lamellipodia and filopodia. Importantly, IS formation is impaired in spectrin-depleted cells. Thus, spectrin may be engaged in regulation of distinct events necessary for the establishment and maturity of the IS: besides the involvement of spectrin in the control of CD45 and LFA-1 surface display, spectrin acts in the establishment of cell-cell contact and adhesion processes during the formation of the IS.
Asunto(s)
Sinapsis Inmunológicas/fisiología , Espectrina/fisiología , Adhesión Celular , Humanos , Células Jurkat , Transporte de Proteínas , Seudópodos/metabolismo , Seudópodos/ultraestructuraRESUMEN
Members of the protein 4.1 family of adapter proteins are expressed in a broad panel of tissues including various epithelia where they likely play an important role in maintenance of cell architecture and polarity and in control of cell proliferation. We have recently characterized the structure and distribution of three members of the protein 4.1 family, 4.1B, 4.1R and 4.1N, in mouse kidney. We describe here binding partners for renal 4.1 proteins, identified through the screening of a rat kidney yeast two-hybrid system cDNA library. The identification of putative protein 4.1-based complexes enables us to envision potential functions for 4.1 proteins in kidney: organization of signaling complexes, response to osmotic stress, protein trafficking, and control of cell proliferation. We discuss the relevance of these protein 4.1-based interactions in kidney physio-pathology in the context of their previously identified functions in other cells and tissues. Specifically, we will focus on renal 4.1 protein interactions with beta amyloid precursor protein (beta-APP), 14-3-3 proteins, and the cell swelling-activated chloride channel pICln. We also discuss the functional relevance of another member of the protein 4.1 superfamily, ezrin, in kidney physio-pathology.
Asunto(s)
Proteínas Sanguíneas/fisiología , Epitelio/metabolismo , Riñón/metabolismo , Proteínas Asociadas a Microtúbulos/fisiología , Proteínas 14-3-3/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Ancirinas/química , Proliferación Celular , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , ADN Complementario/metabolismo , Progresión de la Enfermedad , Biblioteca de Genes , Humanos , Riñón/embriología , Proteínas de la Membrana , Ratones , Familia de Multigenes , Nefronas/metabolismo , Ósmosis , Unión Proteica , Conformación Proteica , Ratas , Transducción de Señal , Técnicas del Sistema de Dos HíbridosRESUMEN
The spectrin-based membrane skeleton, a multi-protein scaffold attached to diverse cellular membranes, is presumed to be involved in the stabilization of membranes, the establishment of membrane domains as well as in vesicle trafficking and nuclear functions. Spectrin tetramers made of alpha- and beta-subunits are linked to actin microfilaments, forming a network that binds a multitude of proteins. The most prevalent alpha-spectrin subunit in non-erythroid cells, alphaII-spectrin, contains two particular spectrin repeats in its central region, alpha9 and alpha10, which host an Src homology 3 domain, a tissue-specific spliced sequence of 20 residues, a calmodulin-binding site and major cleavage sites for caspases and calpains. Using yeast two-hybrid screening of kidney libraries, we identified two partners of the alpha9-alpha10 repeats: the potential tumour suppressor Tes, an actin-binding protein mainly located at focal adhesions; and EVL (Ena/vasodilator-stimulated phosphoprotein-like protein), another actin-binding protein, equally recruited at focal adhesions. Interactions between spectrin and overexpressed Tes and EVL were confirmed by co-immunoprecipitation. In vitro studies showed that the interaction between Tes and spectrin is mediated by a LIM (Lin-11, Isl-1 and Mec3) domain of Tes and by the alpha10 repeat of alphaII-spectrin whereas EVL interacts with the Src homology 3 domain located within the alpha9 repeat. Moreover, we describe an in vitro interaction between Tes and EVL, and a co-localization of these two proteins at focal adhesions. These interactions between alphaII-spectrin, Tes and EVL indicate new functions for spectrin in actin dynamics and focal adhesions.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas de Microfilamentos/metabolismo , Espectrina/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Sitios de Unión , Adhesión Celular , Línea Celular , Membrana Celular , Proteínas del Citoesqueleto , Expresión Génica , Proteínas con Dominio LIM , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Unión al ARN , Técnicas del Sistema de Dos HíbridosRESUMEN
Molecular mechanisms underlying mineralocorticoid receptor (MR)-mediated gene expression are not fully understood but seem to largely depend upon interactions with specific coregulators. To identify novel human MR (hMR) molecular partners, yeast two-hybrid screenings performed using the N-terminal domain as bait, allowed us to isolate protein inhibitor of activated signal transducer and activator of transcription (PIAS)1 and PIASxbeta, described as SUMO (small ubiquitin-related modifier) E3-ligases. Specific interaction between PIAS1 and hMR was confirmed by glutathione-S-transferase pull-down experiments and N-terminal subdomains responsible for physical contacts were delineated. Transient transfections demonstrated that PIAS1 is a corepressor of aldosterone-activated MR transactivation but has no significant effect on human glucocorticoid receptor transactivation. The agonist or antagonist nature of the bound ligand also determines PIAS1 corepressive action. We provided evidence that PIAS1 conjugated SUMO-1 to hMR both in vitro and in vivo. Deciphering the unique sumoylation pattern of hMR, which possesses five consensus SUMO-1 binding sites, by combinatorial lysine substitutions, revealed a major impact of sumoylation on hMR properties. Using a murine mammary tumor virus promoter, PIAS1 action was independent of sumoylation whereas with glucocorticoid response element promoter, PIAS1 corepressive action depended on hMR sumoylation status. Taken together, our results identify a novel function for PIAS1 which interacts with the N-terminal domain of hMR and represses its ligand-dependent transcriptional activity, at least in part, through SUMO modifications.
Asunto(s)
Proteínas/metabolismo , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Transcripción Genética/genética , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Células HeLa , Humanos , Mutagénesis Sitio-Dirigida , Mutación Puntual , Reacción en Cadena de la Polimerasa , Proteínas Inhibidoras de STAT Activados , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Transfección , Ubiquitina/metabolismoRESUMEN
The spectrin-actin scaffold underlying the lipid bilayer is considered to participate in cell-shape stabilization and in the organization of specialized membrane subdomains. These structures are dynamic and likely to undergo frequent remodelling during changes in cell shape. Proteolysis of spectrin, which occurs during apoptosis, leads to destabilization of the scaffold. It is also one of the major processes involved in membrane remodelling. Spectrins, the main components of the membrane skeleton, are the targets for two important protease systems: m- and micro-calpains (Ca2+-activated proteases) and caspase-3 (activated during apoptosis). In this paper, we show that caspase-2 also targets spectrin in vitro, and we characterize Ca2+/calmodulin-dependent regulation of spectrin cleavage by caspases. Yeast two-hybrid screening reveals that the large isoform (1/L) of procaspase-2 specifically binds to alphaII-spectrin, while the short isoform does not. Like caspase-3, caspase-2 cleaves alphaII-spectrin in vitro at residue Asp-1185. This study emphasizes a role of executioner caspase for caspase-2. We also demonstrated that the executioner caspase-7 but not caspase-6 cleaves spectrin at residue Asp-1185 in vitro. This spectrin cleavage by caspases 2, 3 and 7 is inhibited by the Ca2+-dependent binding of calmodulin to spectrin. In contrast, calmodulin binding enhances spectrin cleavage by calpain at residue Tyr-1176. These results indicate that alphaII-spectrin cleavage is highly influenced by Ca2+ homoeostasis and calmodulin, which therefore represent potential regulators of the stability and the plasticity of the spectrin-based skeleton.
Asunto(s)
Calmodulina/metabolismo , Caspasas/metabolismo , Espectrina/metabolismo , Ácido Aspártico/análisis , Sitios de Unión , Calpaína/metabolismo , Caspasa 2 , Isoformas de Proteínas/metabolismo , Espectrina/química , Técnicas del Sistema de Dos HíbridosRESUMEN
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
A fine regulation of the amiloride-sensitive Epithelial Sodium Channel (ENaC), made of alpha, beta and gamma subunits, is crucial for maintenance of Na+ balance and blood pressure. Both beta- and gamma-ENaC participate in negative regulation by interacting with Nedd4-2, an E3 ubiquitin-ligase. Disruption of this interaction results in increased ENaC activity (Liddle syndrome). By two-hybrid screenings, we identified new potential partners of alpha-ENaC: WWP1 (E3 ubiquitin-ligase protein), UBC9 and TSG101 (E2 ubiquitin/SUMO-conjugating enzymes) and confirmed these interactions in GST pull-down assays. All these partners are implicated in protein trafficking and could be involved in the regulation of ENaC activity.
Asunto(s)
Canales de Sodio/análisis , Secuencia de Aminoácidos , Canales Epiteliales de Sodio , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Canales de Sodio/fisiologíaRESUMEN
Spectrins are ubiquitous scaffolding components of the membrane skeleton that organize and stabilize microdomains on both the plasma membrane and the intracellular organelles. By way of their numerous interactions with diverse protein families, they are implicated in various cellular functions. Using small interfering RNA strategy in the WM-266 cell line derived from human melanoma, we found that alphaII-spectrin deficiency is associated with a defect in cell proliferation, which is related to a cell cycle arrest at the G1 phase (first gap phase), as evaluated by DNA analysis and Rb phosphorylation. These observations coincided with elevated expression of the cyclin-dependent kinase inhibitor, p21Cip. Concomitantly, spectrin loss impaired cell adhesion and spreading. These cell adhesion defects were associated with modifications of the actin cytoskeleton, such as loss of stress fibers, alterations of focal adhesions, and modified expression of some integrins. Our results provide novel insights into spectrin functions by demonstrating the involvement of alphaII-spectrin in cell cycle regulation and actin organization.
Asunto(s)
Ciclo Celular , Espectrina/metabolismo , Adhesión Celular , Línea Celular Tumoral , Forma de la Célula , Regulación hacia Abajo , Humanos , ARN Interferente Pequeño/genética , Espectrina/genéticaRESUMEN
PURPOSE OF REVIEW: Malaria represents one of the most important selective factors affecting human populations. Several inherited diseases of red blood cells lead to resistance at the erythrocytic stage. Among patients who experience hereditary elliptocytosis related to mutations of erythrocyte membrane proteins, molecular studies have shown the prevalence of particular spectrin mutations in patients from black ethnic extraction, leading one to question the selection of new malaria-resistant genes. RECENT FINDINGS: Prospective epidemiological and molecular studies in West Africa have confirmed the prevalence (between 0.6 and 1.6%) of particular spectrin mutations related to hereditary elliptocytosis. These studies have also revealed the frequency of alpha-spectrin chain polymorphisms, associated in cis with elliptocytogenic spectrin mutations and defining particular spectrin allele haplotypes. Culture studies of Plasmodium falciparum in elliptocytes bearing such elliptocytogenic alleles of spectrin showed that these alleles are supplementary genetic factors of malaria resistance in vitro. SUMMARY: Certain instances of spectrin mutations or polymorphisms have not yet been shown to constitute new factors of innate resistance to malaria in vivo. Epidemiological surveys of hereditary elliptocytosis and parasite culture studies, however, have argued that the relationships between parasite and spectrin-based skeleton should be examined more closely and the molecular interactions between parasite ligands and particular spectrin chain domains should be characterized.
Asunto(s)
Malaria/inmunología , Espectrina/genética , Animales , Citoesqueleto/química , Citoesqueleto/parasitología , Eritrocitos/química , Eritrocitos/parasitología , Humanos , Inmunidad Innata/genética , Malaria/epidemiologíaRESUMEN
alpha- and beta-spectrins are components of molecular scaffolds located under the lipid bilayer and named membrane skeletons. Disruption of these scaffolds through mutations in spectrins demonstrated that they are involved in the membrane localization or the maintenance of proteins associated with them. The ubiquitous alphaII-spectrin chain bears in its central region a unique domain that is sensitive to several proteases such as calpains or caspases. The conservation of this region in vertebrates suggests that the proteolysis of alphaII-spectrin by these enzymes could be involved in important functions. To assess the role of alphaII-spectrin cleavage in vivo, we generated a murine model in which the exons encoding the region defining this cleavage sensitivity were disrupted by gene targeting. Surprisingly, homozygous mice expressing this mutant alphaII-spectrin appeared healthy, bred normally, and had no histological anomaly. Remarkably, the mutant alphaII-spectrin assembles correctly into the membrane skeleton, thus challenging the notion that this region is required for the stable biogenesis of the membrane skeleton in nonerythroid cells. Our finding also argues against a critical role of this particular alphaII-spectrin cleavage in either major cellular functions or in normal development.
Asunto(s)
Calpaína/metabolismo , Caspasas/metabolismo , Mutación/genética , Fenotipo , Espectrina/fisiología , Secuencia de Aminoácidos , Animales , Apoptosis , Secuencia de Bases , Western Blotting , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Técnicas de Transferencia de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Espectrina/genéticaRESUMEN
Proteinase 3 (PR3), a serine proteinase contained in neutrophil azurophilic granules, is considered a risk factor for vasculitides and rheumatoid arthritis when expressed on the outer leaflet of neutrophil plasma membrane and is the preferred target of antineutrophil cytoplasm autoantibodies (ANCA) in Wegener granulomatosis. ANCA binding to PR3 expressed at the surface of neutrophils activates them. Evidence is provided that neutrophil apoptosis induced significantly more membrane PR3 expression without degranulation (but no enhanced membrane CD35, CD66b, CD63, myeloperoxidase, or elastase expression). This observation was confirmed on cytoplasts, a model of granule-free neutrophils. We hypothesized that PR3 could interact with proteins involved in membrane flip-flop (eg, phospholipid scramblase 1 [PLSCR1]). PR3-PLSCR1 interaction in neutrophils was demonstrated by confocal microscopy and coimmunoprecipitation. In the RBL-2H3 rat mast-cell line stably transfected with PR3 or its inactive mutant (PR3S203A), PR3 externalization depended on PLSCR1, as shown by less PR3 externalization in the presence of rPLSCR1 siRNA, but independently of its serine-proteinase activity. Finally, apoptosis-externalized PR3 decreased the human macrophage-phagocytosis rate of apoptotic PR3 transfectants. Therefore, in addition to ANCA binding in vasculitis, the proinflammatory role of membrane PR3 expression may involve interference with macrophage clearance of apoptotic neutrophils.
Asunto(s)
Apoptosis , Macrófagos/enzimología , Mieloblastina/metabolismo , Neutrófilos/enzimología , Fagocitosis , Proteínas de Transferencia de Fosfolípidos/metabolismo , Animales , Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Anticuerpos Anticitoplasma de Neutrófilos/metabolismo , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos CD/metabolismo , Apoptosis/genética , Apoptosis/inmunología , Artritis Reumatoide/enzimología , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Línea Celular , Membrana Celular/enzimología , Membrana Celular/genética , Membrana Celular/inmunología , Regulación Enzimológica de la Expresión Génica/inmunología , Granulomatosis con Poliangitis/enzimología , Granulomatosis con Poliangitis/genética , Granulomatosis con Poliangitis/inmunología , Humanos , Macrófagos/inmunología , Mastocitos/enzimología , Mastocitos/inmunología , Mutación/inmunología , Mieloblastina/genética , Mieloblastina/inmunología , Activación Neutrófila/genética , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Elastasa Pancreática/genética , Elastasa Pancreática/inmunología , Elastasa Pancreática/metabolismo , Peroxidasa/genética , Peroxidasa/inmunología , Peroxidasa/metabolismo , Fagocitosis/genética , Fagocitosis/inmunología , Proteínas de Transferencia de Fosfolípidos/genética , Proteínas de Transferencia de Fosfolípidos/inmunología , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , Ratas , Factores de Riesgo , Vesículas Secretoras/enzimología , Vesículas Secretoras/genética , Vesículas Secretoras/inmunología , Vasculitis/enzimologíaRESUMEN
Ubiquitination of proteins such as ion transporters appears to be an important process in the regulation of their membrane expression. Recently, using two-hybrid screening, we have selected a potential partner for the alpha-subunit of the amiloride-sensitive epithelial sodium channel (ENaC): the WWP1 protein, a ubiquitin ligase belonging to the Nedd4 family. To establish whether WWP1 is co-expressed with ENaC, we employed in situ hybridisation, immunohistochemistry and Western blotting to determine the expression of WWP1 in various tissues and cell lines, including those known to express ENaC. As expected, WWP1 was expressed, like ENaC, in the bronchiolar epithelium. However it was also present in the proximal colon and the proximal part of the nephron (where ENaC is not expressed) and absent in the distal parts of the nephron (where ENaC is expressed abundantly). These results suggest that other channels or transport proteins, containing specific domains, such as PY motifs, could be the targets for regulation by WWP1.