RESUMEN
Within the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a highly Ca2+ -selective channel important for active reabsorption of Ca2+ in the kidney. Its channel activity is controlled by a negative feedback mechanism involving calmodulin (CaM) binding. Combining advanced microscopy techniques and biochemical assays, this study characterized the dynamic lobe-specific CaM regulation. We demonstrate for the first time that functional (full-length) TRPV5 interacts with CaM in the absence of Ca2+ , and this interaction is intensified at increasing Ca2+ concentrations sensed by the CaM C-lobe that achieves channel pore blocking. Channel inactivation occurs without requiring CaM N-lobe calcification. Moreover, we show a Ca2+ -dependent binding stoichiometry at the single channel level. In conclusion, our study proposes a new model for CaM-dependent regulation - calmodulation - of this uniquely Ca2+ -selective TRP channel TRPV5 that involves apoCaM interaction and lobe-specific actions, which may be of significant physiological relevance given its role as gatekeeper of Ca2+ transport in the kidney. KEY POINTS: The renal Ca2+ channel TRPV5 is an important player in maintenance of the body's Ca2+ homeostasis. Activity of TRPV5 is controlled by a negative feedback loop that involves calmodulin (CaM), a protein with two Ca2+ -binding lobes. We investigated the dynamics of the interaction between TRPV5 and CaM with advanced fluorescence microscopy techniques. Our data support a new model for CaM-dependent regulation of TRPV5 channel activity with CaM lobe-specific actions and demonstrates Ca2+ -dependent binding stoichiometries. This study improves our understanding of the mechanism underlying fast channel inactivation, which is physiologically relevant given the gatekeeper function of TRPV5 in Ca2+ reabsorption in the kidney.
Asunto(s)
Calmodulina , Canales Catiónicos TRPV , Calcio/metabolismo , Canales de Calcio/metabolismo , Calmodulina/metabolismo , Unión Proteica , Canales Catiónicos TRPV/metabolismoRESUMEN
Thrombosis is a frequent, life-threatening complication of systemic infection associated with multiple organ damage. We have previously described a novel mechanism of inflammation-driven thrombosis induced by Salmonella Typhimurium infection of mice. Thrombosis in the liver develops 7 days after infection, persisting after the infection resolves, and is monocytic cell dependent. Unexpectedly, thrombosis was not prominent in the spleen at this time, despite carrying a similar bacterial burden as the liver. In this study, we show that thrombosis does occur in the spleen but with strikingly accelerated kinetics compared with the liver, being evident by 24 hours and resolving rapidly thereafter. The distinct kinetics of thrombosis and bacterial burden provides a test of the hypothesis that thrombi form in healthy vessels to trap or remove bacteria from the circulation, often termed immunothrombosis. Remarkably, despite bacteria being detected throughout infected spleens and livers in the early days of infection, immunohistological analysis of tissue sections show that thrombi contain very low numbers of bacteria. In contrast, bacteria are present throughout platelet aggregates induced by Salmonella in vitro. Therefore, we show that thrombosis develops with organ-specific kinetics and challenge the universality of immunothrombosis as a mechanism to capture bacteria in vivo.
Asunto(s)
Hígado/microbiología , Infecciones por Salmonella/complicaciones , Salmonella typhimurium/patogenicidad , Bazo/microbiología , Trombosis/microbiología , Animales , Hígado/inmunología , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Infecciones por Salmonella/microbiología , Bazo/inmunología , Bazo/patología , Trombosis/inmunología , Trombosis/patologíaRESUMEN
The platelet C-type lectin-like receptor CLEC-2 drives inflammation-driven venous thrombosis in mouse models of thrombo-inflammatory disease with a minimal effect on hemostasis identifying it as a target for a new class of antiplatelet agent. Here, we discuss how the protein structure and dynamic arrangement of CLEC-2 on the platelet membrane helps the receptor, which has a single YxxL motif (known as a hemITAM), to trigger intracellular signaling. CLEC-2 exists as a monomer and homo-dimer within resting platelets and forms higher-order oligomers following ligand activation, a process that is mediated by the multivalent nature of its ligands and the binding of the tandem SH2 domains of Syk to the phosphorylated hemITAM and concomitantly to PIP2 or PIP3 to localize it to the membrane. We propose that a low level of active Syk is present at the membrane in resting platelets due to phosphorylation by Src family kinases and that clustering of receptors disturbs the equilibrium between kinases and phosphatases, triggering phosphorylation of the CLEC-2 hemITAM and recruitment of Syk. Knowledge of the structure of CLEC-2 and the mechanism of platelet activation has important implications for development of therapeutics.
Asunto(s)
Lectinas Tipo C/metabolismo , Animales , Dimerización , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
An organized and dynamic cytoskeleton is required for platelet formation and function. Formins are a large family of actin regulatory proteins which are also able to regulate microtubule dynamics. There are four formin family members expressed in human and mouse megakaryocytes and platelets. We have previously shown that the actin polymerization activity of formin proteins is required for cytoskeletal dynamics and platelet spreading using a small molecule inhibitor. In the current study, we analyze transgenic mouse models deficient in two of these proteins, mDia1 and Fhod1, along with a model lacking both proteins. We demonstrate that double knockout mice display macrothrombocytopenia which is due to aberrant megakaryocyte function and a small decrease in platelet lifespan. Platelet function is unaffected by the loss of these proteins. This data indicates a critical role for formins in platelet and megakaryocyte function.
Asunto(s)
Plaquetas/metabolismo , Proteínas Fetales/metabolismo , Forminas/metabolismo , Microtúbulos/metabolismo , Pruebas de Función Plaquetaria/métodos , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones NoqueadosRESUMEN
The platelet and megakaryocyte cytoskeletons are essential for formation and function of these cells. A dynamic, properly organised tubulin and actin cytoskeleton is critical for the development of the megakaryocyte and the extension of proplatelets. Tubulin in particular plays a pivotal role in the extension of these proplatelets and the release of platelets from them. Tubulin is further required for the maintenance of platelet size, and actin is the driving force for shape change, spreading and platelet contraction during platelet activation. Whilst several key proteins which regulate these cytoskeletons have been described in detail, the formin family of proteins has received less attention. Formins are intriguing as, although they were initially believed to simply be a nucleator of actin polymerisation, increasing evidence shows they are important regulators of the crosstalk between the actin and microtubule cytoskeletons. In this review, we will introduce the formin proteins and consider the recent evidence that they play an important role in platelets and megakaryocytes in mediating both the actin and tubulin cytoskeletons.
Asunto(s)
Plaquetas/metabolismo , Megacariocitos/metabolismo , Proteínas de Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Forminas , Expresión Génica , Humanos , Proteínas de Microfilamentos/genética , Microtúbulos/metabolismoRESUMEN
Thrombosis is a hallmark of the fatal fungal infection mucormycosis. Yet, the platelet activation pathway in response to mucormycetes is unknown. In this study we determined the platelet aggregation potential of Mucor circinelloides (M. circinelloides) NRRL3631, characterized the signaling pathway facilitating aggregation in response to fungal spores, and identified the influence of the spore developmental stage upon platelet aggregation potential. Using impedance and light-transmission aggregometry, we showed that M. circinelloides induced platelet aggregation in whole blood and in platelet-rich plasma, respectively. The formation of large spore-platelet aggregates was confirmed by light-sheet microscopy, which showed spores dispersed throughout the aggregate. Aggregation potential was dependent on the spore's developmental stage, with the strongest platelet aggregation by spores in mid-germination. Inhibitor studies revealed platelet aggregation was mediated by the low affinity IgG receptor FcγRIIA and integrin αIIbß3; Src and Syk tyrosine kinase signaling; and the secondary mediators TxA2 and ADP. Flow cytometry of antibody stained platelets showed that interaction with spores increased expression of platelet surface integrin αIIbß3 and the platelet activation marker CD62P. Together, this is the first elucidation of the signaling pathways underlying thrombosis formation during a fungal infection, highlighting targets for therapeutic intervention.
Asunto(s)
Mucor/patogenicidad , Agregación Plaquetaria/inmunología , Receptores de IgG/genética , Trombosis/inmunología , HumanosRESUMEN
Breast cancer is one of the most common causes of cancer-related deaths in women. The estrogen receptor (ERα) is well known for having growth promoting effects in breast cancer. Recently, we have identified DC-SCRIPT (ZNF366) as a co-suppressor of ERα and as a strong and independent prognostic marker in ESR1 (ERα gene)-positive breast cancer patients. In this study, we further investigated the molecular mechanism on how DC-SCRIPT inhibits breast cancer cell growth. DC-SCRIPT mRNA levels from 190 primary ESR1-positive breast tumors were related to global gene expression, followed by gene ontology and pathway analysis. The effect of DC-SCRIPT on breast cancer cell growth and cell cycle arrest was investigated using novel DC-SCRIPT-inducible MCF7 breast cancer cell lines. Genome-wide expression profiling of DC-SCRIPT-expressing MCF7 cells was performed to investigate the effect of DC-SCRIPT on cell cycle-related gene expression. Findings were validated by real-time PCR in a cohort of 1,132 ESR1-positive breast cancer patients. In the primary ESR1-positive breast tumors, DC-SCRIPT expression negatively correlated with several cell cycle gene ontologies and pathways. DC-SCRIPT expression strongly reduced breast cancer cell growth in vitro, breast tumor growth in vivo, and induced cell cycle arrest. In addition, in the presence of DC-SCRIPT, multiple cell cycles related genes were differentially expressed including the tumor suppressor gene CDKN2B. Moreover, in 1,132 primary ESR1-positive breast tumors, DC-SCRIPT expression also correlated with CDKN2B expression. Collectively, these data show that DC-SCRIPT acts as a novel regulator of CDKN2B and induces cell cycle arrest in ESR1-positive breast cancer cells.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas Portadoras/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Receptor alfa de Estrógeno/genética , Biomarcadores de Tumor/genética , Neoplasias de la Mama/patología , Proteínas Portadoras/metabolismo , Puntos de Control del Ciclo Celular/genética , Proliferación Celular/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Células MCF-7 , Proteínas de Neoplasias/biosíntesis , ARN Mensajero/biosíntesisRESUMEN
Multispectral imaging is a novel microscopy technique that combines imaging with spectroscopy to obtain both quantitative expression data and tissue distribution of different cellular markers. Tetraspanins CD37 and CD53 are four-transmembrane proteins involved in cellular and humoral immune responses. However, comprehensive immunohistochemical analyses of CD37 and CD53 in human lymphoid organs have not been performed so far. We investigated CD37 and CD53 protein expression on primary human immune cell subsets in blood and in primary and secondary lymphoid organs. Both tetraspanins were prominently expressed on antigen-presenting cells, with highest expression of CD37 on B lymphocytes. Analysis of subcellular distribution showed presence of both tetraspanins on the plasma membrane and on endosomes. In addition, CD53 was also present on lysosomes. Quantitative analysis of expression and localization of CD37 and CD53 on lymphocytes within lymphoid tissues by multispectral imaging revealed high expression of both tetraspanins on CD20(+) cells in B cell follicles in human spleen and appendix. CD3(+) T cells within splenic T cell zones expressed lower levels of CD37 and CD53 compared to T cells in the red pulp of human spleen. B cells in human bone marrow highly expressed CD37, whereas the expression of CD53 was low. In conclusion, we demonstrate differential expression of CD37 and CD53 on primary human immune cells, their subcellular localization and their quantitative distribution in human lymphoid organs. This study provides a solid basis for better insight into the function of tetraspanins in the human immune response.
Asunto(s)
Antígenos de Neoplasias/análisis , Tejido Linfoide/química , Tejido Linfoide/metabolismo , Tetraspanina 25/análisis , Tetraspaninas/análisis , Antígenos de Neoplasias/biosíntesis , Humanos , Inmunohistoquímica , Tejido Linfoide/citología , Microscopía Confocal , Bazo/química , Bazo/citología , Bazo/metabolismo , Tetraspanina 25/biosíntesis , Tetraspaninas/biosíntesisRESUMEN
Dendritic cells (DCs) are the professional APCs of the immune system that dictate the type and course of an immune response. Molecular understanding of DC biology is important for the design of DC-based immunotherapies and optimal clinical applications in vaccination settings. Previously, we isolated and characterized the cDNA-encoding dendritic cell-specific transcript (DC-SCRIPT; also known as ZNF366). DC-SCRIPT mRNA expression in the immune system was confined to DCs and was reported to be an early hallmark of DC differentiation. In this study, we demonstrate IL-4 to be the dominant factor for DC-SCRIPT expression in human monocyte-derived DCs. In addition, to our knowledge, we show for the first time endogenous DC-SCRIPT protein expression in human DCs both in vitro and in situ. DC-SCRIPT protein is detected early upon differentiation of monocytes into DCs and is also present in multiple freshly isolated DC subsets. Maturation of DCs with TLR ligands further increased DC-SCRIPT mRNA expression, suggesting a role in DC maturation. Indeed, small interfering RNA-mediated knockdown of DC-SCRIPT affected the cytokine response upon TLR stimulation. These DCs displayed enhanced IL-10 and decreased IL-12 production, compared with wild-type DCs. Silencing of IL-10 in DC-SCRIPT knockdown DCs rescued IL-12 expression, suggesting a primary role for DC-SCRIPT in the regulation of IL-10 production.
Asunto(s)
Proteínas Portadoras/fisiología , Citocinas/biosíntesis , Células Dendríticas/inmunología , Receptores Toll-Like/fisiología , Biomarcadores/metabolismo , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Células Cultivadas , Citocinas/genética , Células Dendríticas/metabolismo , Regulación de la Expresión Génica/inmunología , Humanos , Interleucina-10/biosíntesis , Interleucina-4/fisiología , Simulación de Dinámica Molecular , Monocitos/inmunología , Monocitos/metabolismo , ARN Mensajero/biosíntesisRESUMEN
AIM: Klotho, a key component of the endocrine fibroblast growth factor receptor-fibroblast growth factor axis, is a multi-functional protein that impacts renal electrolyte handling. The physiological significance of Klotho will be highlighted in the regulation of calcium, phosphate, and potassium metabolism. METHODS: In this review, we compare several murine models with different renal targeted deletions of Klotho and the insights into the molecular and physiological function that these models offer. RESULTS: In vivo, Klotho deficiency is associated with severely impaired mineral metabolism, with consequences on growth, longevity and disease development. Additionally, we explore the perspectives of Klotho in renal pathology and vascular events, as well as potential Klotho treatment options. CONCLUSION: This comprehensive review emphasizes the use of Klotho to shed light on deciphering the renal molecular in vivo mechanisms in electrolyte handling, as well as novel therapeutic interventions.
RESUMEN
CLEC-2 is a target for a new class of antiplatelet agent. Clustering of CLEC-2 leads to phosphorylation of a cytosolic YxxL and binding of the tandem SH2 domains in Syk, crosslinking two receptors. We have raised 48 nanobodies to CLEC-2 and crosslinked the most potent of these to generate divalent and tetravalent nanobody ligands. Fluorescence correlation spectroscopy (FCS) was used to show that the multivalent nanobodies cluster CLEC-2 in the membrane and that clustering is reduced by inhibition of Syk. Strikingly, the tetravalent nanobody stimulated aggregation of human platelets, whereas the divalent nanobody was an antagonist. In contrast, in human CLEC-2 knock-in mouse platelets, the divalent nanobody stimulated aggregation. Mouse platelets express a higher level of CLEC-2 than human platelets. In line with this, the divalent nanobody was an agonist in high-expressing transfected DT40 cells and an antagonist in low-expressing cells. FCS, stepwise photobleaching and non-detergent membrane extraction show that CLEC-2 is a mixture of monomers and dimers, with the degree of dimerisation increasing with expression thereby favouring crosslinking of CLEC-2 dimers. These results identify ligand valency, receptor expression/dimerisation and Syk as variables that govern activation of CLEC-2 and suggest that divalent ligands should be considered as partial agonists.
Asunto(s)
Lectinas Tipo C , Anticuerpos de Dominio Único , Animales , Humanos , Ratones , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Transducción de Señal/fisiología , Anticuerpos de Dominio Único/farmacología , Quinasa Syk/metabolismoRESUMEN
Background: Disturbances in magnesium homeostasis are common in patients with chronic kidney disease (CKD) and are associated with increased mortality. The kidney is a key organ in maintaining normal serum magnesium concentrations. To this end, fractional excretion of magnesium (FEMg) increases as renal function declines. Despite recent progress, the hormonal regulation of renal magnesium handling is incompletely understood. Fibroblast Growth Factor 23 (FGF23) is a phosphaturic hormone that has been linked to renal magnesium handling. However, it has not yet been reported whether FGF23 is associated with renal magnesium handling in CKD patients. Methods: The associations between plasma FGF23 levels, plasma and urine magnesium concentrations and FEMg was investigated in a cross-sectional cohort of 198 non-dialysis CKD patients undergoing renal biopsy. Results: FGF23 was significantly correlated with FEMg (Pearson's correlation coefficient = 0.37, p<0.001) and urinary magnesium (-0.14, p=0.04), but not with plasma magnesium. The association between FGF23 and FEMg remained significant after adjusting for potential confounders, including estimated glomerular filtration rate (eGFR), parathyroid hormone and 25-hydroxyvitamin D. Conclusions: We report that plasma FGF23 is independently associated with measures of renal magnesium handling in a cohort of non-dialysis CKD patients. A potential causal relationship should be investigated in future studies.
Asunto(s)
Magnesio , Insuficiencia Renal Crónica , Humanos , Factor-23 de Crecimiento de Fibroblastos , Estudios Transversales , Factores de Crecimiento de Fibroblastos/metabolismo , Riñón/metabolismo , Insuficiencia Renal Crónica/complicacionesRESUMEN
Autosomal dominant mutations in FAM111A are causative for Kenny-Caffey syndrome type 2. Patients with Kenny-Caffey syndrome suffer from severe growth retardation, skeletal dysplasia, hypoparathyroidism, hypocalcaemia, hyperphosphataemia and hypomagnesaemia. While recent studies have reported FAM111A to function in antiviral response and DNA replication, its role in regulating electrolyte homeostasis remains unknown. In this study, we assessed the role of FAM111A in the regulation of serum electrolyte balance using a Fam111a knockout (Fam111a-/-) C57BL/6 N mouse model. Fam111a-/- mice displayed normal weight and serum parathyroid hormone (PTH) concentration and exhibited unaltered magnesium, calcium and phosphate levels in serum and 24-hour urine. Expression of calciotropic (including Cabp28k, Trpv5, Klotho and Cyp24a1), magnesiotropic (including Trpm6, Trpm7, Cnnm2 and Cnnm4) and phosphotropic (Slc20a1, Slc20a2, Slc34a1 and Slc34a3) genes in the kidneys, duodenum and colon were not affected by Fam111a depletion. Only Slc34a2 expression was significantly upregulated in the duodenum, but not in the colon. Analysis of femurs showed unaffected bone morphology and density in Fam111a-/- mice. Kidney and parathyroid histology were also normal in Fam111a-/- mice. In conclusion, our study is the first to characterise the function of FAM111A in vivo and we report that mice lacking FAM111A exhibit normal electrolyte homeostasis on a standard diet.
Asunto(s)
Hiperostosis Cortical Congénita , Hipocalcemia , Serina Proteasas , Canales Catiónicos TRPM , Animales , Humanos , Ratones , Calcio/metabolismo , Electrólitos/metabolismo , Hiperostosis Cortical Congénita/genética , Hipocalcemia/genética , Magnesio/metabolismo , Ratones Endogámicos C57BL , Hormona Paratiroidea/metabolismo , Receptores Virales , Serina Proteasas/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismo , Canales Catiónicos TRPM/metabolismo , Equilibrio HidroelectrolíticoRESUMEN
T cells depend on the phosphatase CD45 to initiate T cell receptor signaling. Although the critical role of CD45 in T cells is established, the mechanisms controlling function and localization in the membrane are not well understood. Moreover, the regulation of specific CD45 isoforms in T cell signaling remains unresolved. By using unbiased mass spectrometry, we identify the tetraspanin CD53 as a partner of CD45 and show that CD53 controls CD45 function and T cell activation. CD53-negative T cells (Cd53-/-) exhibit substantial proliferation defects, and Cd53-/- mice show impaired tumor rejection and reduced IFNγ-producing T cells compared with wild-type mice. Investigation into the mechanism reveals that CD53 is required for CD45RO expression and mobility. In addition, CD53 is shown to stabilize CD45 on the membrane and is required for optimal phosphatase activity and subsequent Lck activation. Together, our findings reveal CD53 as a regulator of CD45 activity required for T cell immunity.
Asunto(s)
Linfocitos T , Tetraspanina 25 , Animales , Movimiento Celular/inmunología , Antígenos Comunes de Leucocito/inmunología , Activación de Linfocitos , Ratones , Isoformas de Proteínas , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal , Linfocitos T/inmunología , Tetraspanina 25/inmunologíaRESUMEN
Platelets play a key role in the development, progression and resolution of the inflammatory response during sterile inflammation and infection, although the mechanism is not well understood. Here we show that platelet CLEC-2 reduces tissue inflammation by regulating inflammatory macrophage activation and trafficking from the inflamed tissues. The immune regulatory function of CLEC-2 depends on the expression of its ligand, podoplanin, upregulated on inflammatory macrophages and is independent of platelet activation and secretion. Mechanistically, platelet CLEC-2 and also recombinant CLEC-2-Fc accelerates actin rearrangement and macrophage migration by increasing the expression of podoplanin and CD44, and their interaction with the ERM proteins. During ongoing inflammation, induced by lipopolysaccharide, treatment with rCLEC-2-Fc induces the rapid emigration of peritoneal inflammatory macrophages to mesenteric lymph nodes, thus reducing the accumulation of inflammatory macrophages in the inflamed peritoneum. This is associated with a significant decrease in pro-inflammatory cytokine, TNF-α and an increase in levels of immunosuppressive, IL-10 in the peritoneum. Increased podoplanin expression and actin remodelling favour macrophage migration towards CCL21, a soluble ligand for podoplanin and chemoattractant secreted by lymph node lymphatic endothelial cells. Macrophage efflux to draining lymph nodes induces T cell priming. In conclusion, we show that platelet CLEC-2 reduces the inflammatory phenotype of macrophages and their accumulation, leading to diminished tissue inflammation. These immunomodulatory functions of CLEC-2 are a novel strategy to reduce tissue inflammation and could be therapeutically exploited through rCLEC-2-Fc, to limit the progression to chronic inflammation.
Asunto(s)
Plaquetas/metabolismo , Movimiento Celular , Lectinas Tipo C/metabolismo , Activación de Macrófagos , Macrófagos Peritoneales/metabolismo , Peritonitis/metabolismo , Animales , Plaquetas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Mediadores de Inflamación/metabolismo , Lectinas Tipo C/genética , Lipopolisacáridos , Macrófagos Peritoneales/inmunología , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Peritonitis/genética , Peritonitis/inmunología , Fagocitosis , Fenotipo , Células RAW 264.7 , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Collagen has been proposed to bind to a unique epitope in dimeric glycoprotein VI (GPVI) and the number of GPVI dimers has been reported to increase upon platelet activation. However, in contrast, the crystal structure of GPVI in complex with collagen-related peptide (CRP) showed binding distinct from the site of dimerization. Further fibrinogen has been reported to bind to monomeric but not dimeric GPVI. In the present study, we have used the advanced fluorescence microscopy techniques of single-molecule microscopy, fluorescence correlation spectroscopy (FCS) and bioluminescence resonance energy transfer (BRET), and mutagenesis studies in a transfected cell line model to show that GPVI is expressed as a mixture of monomers and dimers and that dimerization through the D2 domain is not critical for activation. As many of these techniques cannot be applied to platelets to resolve this issue, due to the high density of GPVI and its anucleate nature, we used Förster resonance energy transfer (FRET) to show that endogenous GPVI is at least partially expressed as a dimer on resting and activated platelet membranes. We propose that GPVI may be expressed as a monomer on the cell surface and it forms dimers in the membrane through diffusion, giving rise to a mixture of monomers and dimers. We speculate that the formation of dimers facilitates ligand binding through avidity.
Asunto(s)
Plaquetas/metabolismo , Membrana Celular/metabolismo , Colágeno/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Sitios de Unión , Transferencia de Energía por Resonancia de Bioluminiscencia , Células HEK293 , Humanos , Ligandos , Microscopía Fluorescente , Mutación , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Imagen Individual de Molécula , Espectrometría de Fluorescencia , Relación Estructura-ActividadRESUMEN
Tumor vasculature is in general highly heterogeneous. This characteristic is most prominent in high-grade gliomas, which present with areas of angiogenic growth, next to large areas of diffuse infiltrative growth in which tumor cells thrive on pre-existent brain vasculature. This limits the effectiveness of anti-angiogenic compounds as these will not affect more matured and co-opted vessels. Therefore, additional destruction of existing tumor vasculature may be a promising alternative avenue to effectively deprive tumors from blood. This approach requires the identification of novel tumor vascular targeting agents, which have broad tumor vessel specificities, ie are not restricted to newly formed vessels. Here, we describe the generation of a phage library displaying nanobodies that were cloned from lymphocytes of a Llama which had been immunized with clinical glioma tissue. In vivo biopanning with this library in the orthotopic glioma xenograft models E98 and E434 resulted in the selection of various nanobodies which specifically recognized glioma vessels in corresponding glioma xenografts. Importantly, also nanobodies were isolated which discriminated incorporated pre-existent vessels in highly infiltrative cerebral E434 xenografts from normal brain vessels. Our results suggest that the generation of nanobody-displaying immune phage libraries and subsequent in vivo biopanning in appropriate animal models is a promising approach for the identification of novel vascular targeting agents.
Asunto(s)
Anticuerpos/inmunología , Anticuerpos/aislamiento & purificación , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/inmunología , Glioblastoma/irrigación sanguínea , Glioblastoma/inmunología , Nanoestructuras , Animales , Afinidad de Anticuerpos , Formación de Anticuerpos , Vasos Sanguíneos/inmunología , Camélidos del Nuevo Mundo , Línea Celular Tumoral , Humanos , Inmunohistoquímica , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Biblioteca de Péptidos , Trasplante HeterólogoRESUMEN
BACKGROUND: Reorganization of the actin cytoskeleton is required for proper functioning of platelets following activation in response to vascular damage. Formins are a family of proteins that regulate actin polymerization and cytoskeletal organization via a number of domains including the FH2 domain. However, the role of formins in platelet spreading has not been studied in detail. OBJECTIVES: Several formin proteins are expressed in platelets so we used an inhibitor of FH2 domains (SMIFH2) to uncover the role of these proteins in platelet spreading and in maintenance of resting platelet shape. METHODS: Washed human and mouse platelets were treated with various concentrations of SMIFH2 and the effects on platelet spreading, platelet size, platelet cytoskeletal dynamics, and organization were analyzed using fluorescence and electron microscopy. RESULTS: Pretreatment with SMIFH2 completely blocks platelet spreading in both mouse and human platelets through effects on the organization and dynamics of actin and microtubules. However, platelet aggregation and secretion are unaffected. SMIFH2 also caused a decrease in resting platelet size and disrupted the balance of tubulin post-translational modification. CONCLUSIONS: These data therefore demonstrated an important role for formin-mediated actin polymerization in platelet spreading and highlighted the importance of formins in cross-talk between the actin and tubulin cytoskeletons.
Asunto(s)
Plaquetas , Citoesqueleto , Citoesqueleto de Actina , Actinas , Animales , Forminas , RatonesRESUMEN
Dendritic cells (DCs), which are essential for initiating immune responses, are comprised of different subsets. Tetraspanins organize dendritic cell membranes by facilitating protein-protein interactions within the so called tetraspanin web. In this study we analyzed expression of the complete tetraspanin superfamily in primary murine (CD4+, CD8+, pDC) and human DC subsets (CD1c+, CD141+, pDC) at the transcriptome and proteome level. Different RNA and protein expression profiles for the tetraspanin genes across human and murine DC subsets were identified. Although RNA expression levels of CD37 and CD82 were not significantly different between human DC subsets, CD9 RNA was highly expressed in pDCs, while CD9 protein expression was lower. This indicates that relative RNA and protein expression levels are not always in agreement. Both murine CD8α+ DCs and its regarded human counterpart, CD141+ DCs, displayed relatively high protein levels of CD81. CD53 protein was highly expressed on human pDCs in contrast to the relatively low protein expression of most other tetraspanins. This study demonstrates that tetraspanins are differentially expressed by human and murine DC subsets which provides a valuable resource that will aid the understanding of tetraspanin function in DC biology.
Asunto(s)
Células Dendríticas/metabolismo , Tetraspaninas/metabolismo , Animales , Antígenos de Neoplasias/genética , Células Cultivadas , Citometría de Flujo , Humanos , Proteína Kangai-1/genética , Leucocitos Mononucleares/metabolismo , Ratones , Unión Proteica , ARN Mensajero/genética , Tetraspanina 25/genética , Tetraspanina 28/genética , Tetraspaninas/genéticaRESUMEN
Activation of B cells by the binding of antigens to the B cell receptor (BCR) requires the protein kinase C (PKC) family member PKCß. Because PKCs must translocate to the plasma membrane to become activated, we investigated the mechanisms regulating their spatial distribution in mouse and human B cells. Through live-cell imaging, we showed that BCR-stimulated production of the second messenger diacylglycerol (DAG) resulted in the translocation of PKCß from the cytosol to plasma membrane regions containing the tetraspanin protein CD53. CD53 was specifically enriched at sites of BCR signaling, suggesting that BCR-dependent PKC signaling was initiated at these tetraspanin microdomains. Fluorescence lifetime imaging microscopy studies confirmed the molecular recruitment of PKC to CD53-containing microdomains, which required the amino terminus of CD53. Furthermore, we showed that Cd53-deficient B cells were defective in the phosphorylation of PKC substrates. Consistent with this finding, PKC recruitment to the plasma membrane was impaired in both mouse and human CD53-deficient B cells compared to that in their wild-type counterparts. These data suggest that CD53 promotes BCR-dependent PKC signaling by recruiting PKC to the plasma membrane so that it can phosphorylate its substrates and that tetraspanin-containing microdomains can act as signaling hotspots in the plasma membrane.