RESUMEN
The evolution of flight in feathered dinosaurs and early birds over millions of years required flight feathers whose architecture features hierarchical branches. While barb-based feather forms were investigated, feather shafts and vanes are understudied. Here, we take a multi-disciplinary approach to study their molecular control and bio-architectural organizations. In rachidial ridges, epidermal progenitors generate cortex and medullary keratinocytes, guided by Bmp and transforming growth factor ß (TGF-ß) signaling that convert rachides into adaptable bilayer composite beams. In barb ridges, epidermal progenitors generate cylindrical, plate-, or hooklet-shaped barbule cells that form fluffy branches or pennaceous vanes, mediated by asymmetric cell junction and keratin expression. Transcriptome analyses and functional studies show anterior-posterior Wnt2b signaling within the dermal papilla controls barbule cell fates with spatiotemporal collinearity. Quantitative bio-physical analyses of feathers from birds with different flight characteristics and feathers in Burmese amber reveal how multi-dimensional functionality can be achieved and may inspire future composite material designs. VIDEO ABSTRACT.
Asunto(s)
Adaptación Fisiológica , Plumas/anatomía & histología , Plumas/fisiología , Vuelo Animal/fisiología , Animales , Evolución Biológica , Aves/anatomía & histología , Moléculas de Adhesión Celular/metabolismo , Citoesqueleto/metabolismo , Dermis/anatomía & histología , Células Madre/citología , Factores de Tiempo , Transcriptoma/genética , Vía de Señalización Wnt/genéticaRESUMEN
Drug resistance and relapse remain key challenges in pancreatic cancer. Here, we have used RNA sequencing (RNA-seq), chromatin immunoprecipitation (ChIP)-seq, and genome-wide CRISPR analysis to map the molecular dependencies of pancreatic cancer stem cells, highly therapy-resistant cells that preferentially drive tumorigenesis and progression. This integrated genomic approach revealed an unexpected utilization of immuno-regulatory signals by pancreatic cancer epithelial cells. In particular, the nuclear hormone receptor retinoic-acid-receptor-related orphan receptor gamma (RORγ), known to drive inflammation and T cell differentiation, was upregulated during pancreatic cancer progression, and its genetic or pharmacologic inhibition led to a striking defect in pancreatic cancer growth and a marked improvement in survival. Further, a large-scale retrospective analysis in patients revealed that RORγ expression may predict pancreatic cancer aggressiveness, as it positively correlated with advanced disease and metastasis. Collectively, these data identify an orthogonal co-option of immuno-regulatory signals by pancreatic cancer stem cells, suggesting that autoimmune drugs should be evaluated as novel treatment strategies for pancreatic cancer patients.
Asunto(s)
Adenocarcinoma/patología , Células Madre Neoplásicas/metabolismo , Neoplasias Pancreáticas/patología , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Epigénesis Genética , Biblioteca de Genes , Humanos , Ratones , Ratones Noqueados , Ratones SCID , Células Madre Neoplásicas/citología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Interleucina-10/antagonistas & inhibidores , Receptores de Interleucina-10/genética , Receptores de Interleucina-10/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transcriptoma , Células Tumorales CultivadasRESUMEN
Migration of leukocytes from the skin to lymph nodes (LNs) via afferent lymphatic vessels (LVs) is pivotal for adaptive immune responses1,2. Circadian rhythms have emerged as important regulators of leukocyte trafficking to LNs via the blood3,4. Here, we demonstrate that dendritic cells (DCs) have a circadian migration pattern into LVs, which peaks during the rest phase in mice. This migration pattern is determined by rhythmic gradients in the expression of the chemokine CCL21 and of adhesion molecules in both mice and humans. Chronopharmacological targeting of the involved factors abrogates circadian migration of DCs. We identify cell-intrinsic circadian oscillations in skin lymphatic endothelial cells (LECs) and DCs that cogovern these rhythms, as their genetic disruption in either cell type ablates circadian trafficking. These observations indicate that circadian clocks control the infiltration of DCs into skin lymphatics, a process that is essential for many adaptive immune responses and relevant for vaccination and immunotherapies.
Asunto(s)
Inmunidad Adaptativa , Quimiotaxis , Relojes Circadianos , Células Dendríticas/inmunología , Ganglios Linfáticos/inmunología , Vasos Linfáticos/inmunología , Piel/inmunología , Anciano , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Quimiocina CCL21/genética , Quimiocina CCL21/metabolismo , Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Péptidos y Proteínas de Señalización del Ritmo Circadiano/metabolismo , Células Dendríticas/metabolismo , Femenino , Humanos , Ganglios Linfáticos/metabolismo , Vasos Linfáticos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Piel/metabolismo , Factores de TiempoRESUMEN
CRELD1 is a pivotal factor for heart development, the function of which is unknown in adult life. We here provide evidence that CRELD1 is an important gatekeeper of immune system homeostasis. Exploiting expression variance in large human cohorts contrasting individuals with the lowest and highest CRELD1 expression levels revealed strong phenotypic, functional and transcriptional differences, including reduced CD4+ T cell numbers. These findings were validated in T cell-specific Creld1-deficient mice. Loss of Creld1 was associated with simultaneous overactivation and increased apoptosis, resulting in a net loss of T cells with age. Creld1 was transcriptionally and functionally linked to Wnt signaling. Collectively, gene expression variance in large human cohorts combined with murine genetic models, transcriptomics and functional testing defines CRELD1 as an important modulator of immune homeostasis.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Homeostasis , Sistema Inmunológico/inmunología , Sistema Inmunológico/metabolismo , Inmunomodulación , Animales , Moléculas de Adhesión Celular/genética , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Proteínas de la Matriz Extracelular/genética , Expresión Génica , Técnicas de Inactivación de Genes , Homeostasis/inmunología , Humanos , Inmunosenescencia , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Ratones , Linfocitos T/inmunología , Linfocitos T/metabolismo , Vía de Señalización WntRESUMEN
Benanti et al. report that Burkholderia pseudomallei and Burkholderia mallei bacteria express proteins that mimic Ena/Vasp family proteins to polymerize actin, thereby inducing actin-based motility. Thus, bacteria can use the various cellular actin polymerization mechanisms for intra- and inter-cellular dissemination.
Asunto(s)
Actinas/metabolismo , Infecciones por Burkholderia/microbiología , Burkholderia/fisiología , Burkholderia/patogenicidad , Moléculas de Adhesión Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Animales , HumanosRESUMEN
Burkholderia pseudomallei and B. mallei are bacterial pathogens that cause melioidosis and glanders, whereas their close relative B. thailandensis is non-pathogenic. All use the trimeric autotransporter BimA to facilitate actin-based motility, host cell fusion, and dissemination. Here, we show that BimA orthologs mimic different host actin-polymerizing proteins. B. thailandensis BimA activates the host Arp2/3 complex. In contrast, B. pseudomallei and B. mallei BimA mimic host Ena/VASP actin polymerases in their ability to nucleate, elongate, and bundle filaments by associating with barbed ends, as well as in their use of WH2 motifs and oligomerization for activity. Mechanistic differences among BimA orthologs resulted in distinct actin filament organization and motility parameters, which affected the efficiency of cell fusion during infection. Our results identify bacterial Ena/VASP mimics and reveal that pathogens imitate the full spectrum of host actin-polymerizing pathways, suggesting that mimicry of different polymerization mechanisms influences key parameters of infection.
Asunto(s)
Actinas/metabolismo , Infecciones por Burkholderia/microbiología , Burkholderia/fisiología , Burkholderia/patogenicidad , Moléculas de Adhesión Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Secuencia de Aminoácidos , Animales , Burkholderia/clasificación , Burkholderia/enzimología , Células COS , Fusión Celular , Línea Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Humanos , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
The protocadherin Fat is known as a tumor suppressor regulating growth in Drosophila and for its conserved function during planar cell polarity establishment. McNeill and colleagues now identify an unsuspected role for a C-terminal proteolytic product of Fat in mitochondria: regulating the electron transport machinery and metabolism.
Asunto(s)
Cadherinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Mitocondrias/metabolismo , Animales , HumanosRESUMEN
Fat (Ft) cadherins are enormous cell adhesion molecules that function at the cell surface to regulate the tumor-suppressive Hippo signaling pathway and planar cell polarity (PCP) tissue organization. Mutations in Ft cadherins are found in a variety of tumors, and it is presumed that this is due to defects in either Hippo signaling or PCP. Here, we show Drosophila Ft functions in mitochondria to directly regulate mitochondrial electron transport chain integrity and promote oxidative phosphorylation. Proteolytic cleavage releases a soluble 68 kDa fragment (Ft(mito)) that is imported into mitochondria. Ft(mito) binds directly to NADH dehydrogenase ubiquinone flavoprotein 2 (Ndufv2), a core component of complex I, stabilizing the holoenzyme. Loss of Ft leads to loss of complex I activity, increases in reactive oxygen species, and a switch to aerobic glycolysis. Defects in mitochondrial activity in ft mutants are independent of Hippo and PCP signaling and are reminiscent of the Warburg effect.
Asunto(s)
Cadherinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Mitocondrias/metabolismo , Secuencia de Aminoácidos , Animales , Moléculas de Adhesión Celular/química , Polaridad Celular , Proteínas de Drosophila/química , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Ojo/crecimiento & desarrollo , Genes Supresores de Tumor , Humanos , MAP Quinasa Quinasa 4/metabolismo , Datos de Secuencia Molecular , Transporte de Proteínas , Especies Reactivas de Oxígeno/metabolismo , Alas de Animales/crecimiento & desarrolloRESUMEN
Cell adhesion molecules are ubiquitous in multicellular organisms, specifying precise cell-cell interactions in processes as diverse as tissue development, immune cell trafficking and the wiring of the nervous system1-4. Here we show that a wide array of synthetic cell adhesion molecules can be generated by combining orthogonal extracellular interactions with intracellular domains from native adhesion molecules, such as cadherins and integrins. The resulting molecules yield customized cell-cell interactions with adhesion properties that are similar to native interactions. The identity of the intracellular domain of the synthetic cell adhesion molecules specifies interface morphology and mechanics, whereas diverse homotypic or heterotypic extracellular interaction domains independently specify the connectivity between cells. This toolkit of orthogonal adhesion molecules enables the rationally programmed assembly of multicellular architectures, as well as systematic remodelling of native tissues. The modularity of synthetic cell adhesion molecules provides fundamental insights into how distinct classes of cell-cell interfaces may have evolved. Overall, these tools offer powerful abilities for cell and tissue engineering and for systematically studying multicellular organization.
Asunto(s)
Moléculas de Adhesión Celular , Comunicación Celular , Biología Sintética , Cadherinas/química , Adhesión Celular , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Integrinas/química , Biología Sintética/métodos , Dominios Proteicos , Sitios de Unión , Ingeniería CelularRESUMEN
How are E3 ubiquitin ligases configured to match substrate quaternary structures? Here, by studying the yeast GID complex (mutation of which causes deficiency in glucose-induced degradation of gluconeogenic enzymes), we discover supramolecular chelate assembly as an E3 ligase strategy for targeting an oligomeric substrate. Cryoelectron microscopy (cryo-EM) structures show that, to bind the tetrameric substrate fructose-1,6-bisphosphatase (Fbp1), two minimally functional GID E3s assemble into the 20-protein Chelator-GIDSR4, which resembles an organometallic supramolecular chelate. The Chelator-GIDSR4 assembly avidly binds multiple Fbp1 degrons so that multiple Fbp1 protomers are simultaneously ubiquitylated at lysines near the allosteric and substrate binding sites. Importantly, key structural and biochemical features, including capacity for supramolecular assembly, are preserved in the human ortholog, the CTLH E3. Based on our integrative structural, biochemical, and cell biological data, we propose that higher-order E3 ligase assembly generally enables multipronged targeting, capable of simultaneously incapacitating multiple protomers and functionalities of oligomeric substrates.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Moléculas de Adhesión Celular/química , Fructosa-Bifosfatasa/química , Péptidos y Proteínas de Señalización Intracelular/química , Complejos Multienzimáticos/química , Proteínas de Saccharomyces cerevisiae/química , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitina/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Sitios de Unión , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Microscopía por Crioelectrón , Fructosa-Bifosfatasa/genética , Fructosa-Bifosfatasa/metabolismo , Expresión Génica , Gluconeogénesis/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células K562 , Cinética , Modelos Moleculares , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Células Sf9 , Spodoptera , Homología Estructural de Proteína , Especificidad por Sustrato , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , UbiquitinaciónRESUMEN
The key sites within the gastrointestinal (GI) tract where T cells mediate effector responses and the impact of these responses on intestinal stem cells (ISCs) remain unclear. Using experimental bone marrow transplantation to model immune-mediated GI damage and 3D imaging to analyze T cell localization, we found that the ISC compartment is the primary intestinal site targeted by T cells after transplantation. Recruitment to the crypt base region resulted in direct T cell engagement with the stem cell compartment and loss of crypt base columnar ISCs, which expressed both MHC classes I and II. Vasculature expressing the adhesion molecule MAdCAM-1 clustered near the crypt base, preferentially regulating crypt compartment invasion and ISC reduction without affecting T cell migration to villi. These findings indicate that allogeneic T cells rapidly access the stem cell niche after transplantation, and this targeted recruitment to the stem cell compartment results in ISC loss during immune-mediated GI damage.
Asunto(s)
Células Madre Adultas/inmunología , Trasplante de Médula Ósea , Mucosa Intestinal/inmunología , Nicho de Células Madre/inmunología , Linfocitos T/inmunología , Animales , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular , Citotoxicidad Inmunológica , Femenino , Humanos , Imagenología Tridimensional , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Modelos Animales , Mucoproteínas , Trasplante HomólogoRESUMEN
The functions of Nr4a1-dependent Ly6C(low) monocytes remain enigmatic. We show that they are enriched within capillaries and scavenge microparticles from their lumenal side in a steady state. In the kidney cortex, perturbation of homeostasis by a TLR7-dependent nucleic acid "danger" signal, which may signify viral infection or local cell death, triggers Gαi-dependent intravascular retention of Ly6C(low) monocytes by the endothelium. Then, monocytes recruit neutrophils in a TLR7-dependent manner to mediate focal necrosis of endothelial cells, whereas the monocytes remove cellular debris. Prevention of Ly6C(low) monocyte development, crawling, or retention in Nr4a1(-/-), Itgal(-/-), and Tlr7(host-/-BM+/+) and Cx3cr1(-/-) mice, respectively, abolished neutrophil recruitment and endothelial killing. Prevention of neutrophil recruitment in Tlr7(host+/+BM-/-) mice or by neutrophil depletion also abolished endothelial cell necrosis. Therefore, Ly6C(low) monocytes are intravascular housekeepers that orchestrate the necrosis by neutrophils of endothelial cells that signal a local threat sensed via TLR7 followed by the in situ phagocytosis of cellular debris.
Asunto(s)
Células Endoteliales/metabolismo , Monitorización Inmunológica , Monocitos/inmunología , Animales , Moléculas de Adhesión Celular/metabolismo , Micropartículas Derivadas de Células , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Humanos , Inflamación , Molécula 1 de Adhesión Intercelular/metabolismo , Riñón/irrigación sanguínea , Riñón/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Ratones , Monocitos/metabolismo , Neutrófilos/inmunología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Receptores de Quimiocina/metabolismoRESUMEN
Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.
Asunto(s)
Moléculas de Adhesión Celular/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas Receptoras/química , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/química , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Humanos , Ratones , Modelos Moleculares , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/química , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/antagonistas & inhibidores , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Receptores de la Familia Eph/antagonistas & inhibidores , Receptores de la Familia Eph/química , Receptores de la Familia Eph/genética , Receptores de la Familia Eph/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Spodoptera , Homología Estructural de Proteína , Especificidad por SustratoRESUMEN
The ability of neurites of the same neuron to avoid each other (self-avoidance) is a conserved feature in both invertebrates and vertebrates. The key to self-avoidance is the generation of a unique subset of cell-surface proteins in individual neurons engaging in isoform-specific homophilic interactions that drive neurite repulsion rather than adhesion. Among these cell-surface proteins are fly Dscam1 and vertebrate clustered protocadherins (cPcdhs), as well as the recently characterized shortened Dscam (sDscam) in the Chelicerata. Herein, we review recent advances in our understanding of how cPcdh, Dscam, and sDscam cell-surface recognition codes are expressed and translated into cellular functions essential for neural wiring.
Asunto(s)
Moléculas de Adhesión Celular , Proteínas de Drosophila , Protocadherinas , Animales , Moléculas de Adhesión Celular/metabolismo , Comunicación Celular , Proteínas de Drosophila/metabolismo , Neuronas/metabolismo , Isoformas de Proteínas/metabolismo , Invertebrados , VertebradosRESUMEN
Cell-cell fusion is indispensable for creating life and building syncytial tissues and organs. Ever since the discovery of cell-cell fusion, how cells join together to form zygotes and multinucleated syncytia has remained a fundamental question in cell and developmental biology. In the past two decades, Drosophila myoblast fusion has been used as a powerful genetic model to unravel mechanisms underlying cell-cell fusion in vivo. Many evolutionarily conserved fusion-promoting factors have been identified and so has a surprising and conserved cellular mechanism. In this review, we revisit key findings in Drosophila myoblast fusion and highlight the critical roles of cellular invasion and resistance in driving cell membrane fusion.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/citología , Mioblastos/citología , Actinas/metabolismo , Actomiosina/metabolismo , Animales , Moléculas de Adhesión Celular/metabolismo , Fusión Celular , Drosophila/embriología , Drosophila/fisiología , Proteínas de Drosophila/genética , Embrión no Mamífero/citología , Membrana Dobles de Lípidos/metabolismo , Músculos/citología , Músculos/embriología , Mioblastos/fisiología , Pupa/citologíaRESUMEN
The number of leukocytes present in circulation varies throughout the day, reflecting bone marrow output and emigration from blood into tissues. Using an organism-wide circadian screening approach, we detected oscillations in pro-migratory factors that were distinct for specific vascular beds and individual leukocyte subsets. This rhythmic molecular signature governed time-of-day-dependent homing behavior of leukocyte subsets to specific organs. Ablation of BMAL1, a transcription factor central to circadian clock function, in endothelial cells or leukocyte subsets demonstrated that rhythmic recruitment is dependent on both microenvironmental and cell-autonomous oscillations. These oscillatory patterns defined leukocyte trafficking in both homeostasis and inflammation and determined detectable tumor burden in blood cancer models. Rhythms in the expression of pro-migratory factors and migration capacities were preserved in human primary leukocytes. The definition of spatial and temporal expression profiles of pro-migratory factors guiding leukocyte migration patterns to organs provides a resource for the further study of the impact of circadian rhythms in immunity.
Asunto(s)
Movimiento Celular/inmunología , Ritmo Circadiano/inmunología , Regulación de la Expresión Génica/inmunología , Leucocitos/inmunología , Factores de Transcripción/inmunología , Adulto , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular/genética , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Femenino , Perfilación de la Expresión Génica , Homeostasis/genética , Homeostasis/inmunología , Humanos , Leucocitos/citología , Leucocitos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Persona de Mediana Edad , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Store operated calcium entry (SOCE) is a principal cellular process by which cells regulate basal calcium, refill intracellular Ca(2+) stores, and execute a wide range of specialized activities. STIM and Orai proteins have been identified as the essential components enabling the reconstitution of Ca(2+) release-activated Ca(2+) (CRAC) channels that mediate SOCE. Here, we report the molecular identification of SARAF as a negative regulator of SOCE. Using heterologous expression, RNAi-mediated silencing and site directed mutagenesis combined with electrophysiological, biochemical and imaging techniques we show that SARAF is an endoplasmic reticulum membrane resident protein that associates with STIM to facilitate slow Ca(2+)-dependent inactivation of SOCE. SARAF plays a key role in shaping cytosolic Ca(2+) signals and determining the content of the major intracellular Ca(2+) stores, a role that is likely to be important in protecting cells from Ca(2+) overfilling.
Asunto(s)
Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Señalización del Calcio , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Membrana Celular/metabolismo , Citosol/metabolismo , Citometría de Flujo , Humanos , Proteínas Sensoras del Calcio Intracelular , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Molécula de Interacción Estromal 1 , Molécula de Interacción Estromal 2RESUMEN
SARS-CoV-2 infection-which involves both cell attachment and membrane fusion-relies on the angiotensin-converting enzyme 2 (ACE2) receptor, which is paradoxically found at low levels in the respiratory tract1-3, suggesting that there may be additional mechanisms facilitating infection. Here we show that C-type lectin receptors, DC-SIGN, L-SIGN and the sialic acid-binding immunoglobulin-like lectin 1 (SIGLEC1) function as attachment receptors by enhancing ACE2-mediated infection and modulating the neutralizing activity of different classes of spike-specific antibodies. Antibodies to the amino-terminal domain or to the conserved site at the base of the receptor-binding domain, while poorly neutralizing infection of ACE2-overexpressing cells, effectively block lectin-facilitated infection. Conversely, antibodies to the receptor binding motif, while potently neutralizing infection of ACE2-overexpressing cells, poorly neutralize infection of cells expressing DC-SIGN or L-SIGN and trigger fusogenic rearrangement of the spike, promoting cell-to-cell fusion. Collectively, these findings identify a lectin-dependent pathway that enhances ACE2-dependent infection by SARS-CoV-2 and reveal distinct mechanisms of neutralization by different classes of spike-specific antibodies.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Lectinas/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Moléculas de Adhesión Celular/metabolismo , Fusión Celular , Línea Celular , Cricetinae , Femenino , Humanos , Lectinas/inmunología , Lectinas Tipo C/metabolismo , Fusión de Membrana , Receptores de Superficie Celular/metabolismo , SARS-CoV-2/inmunología , Lectina 1 Similar a Ig de Unión al Ácido Siálico/metabolismo , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
In the polarity/protrusion model of growth cone repulsion from UNC-6/netrin, UNC-6 first polarizes the growth cone of the VD motor neuron axon via the UNC-5 receptor, and then regulates protrusion asymmetrically across the growth cone based on this polarity. UNC-6 stimulates protrusion dorsally through the UNC-40/DCC receptor, and inhibits protrusion ventrally through UNC-5, resulting in net dorsal growth. Previous studies showed that UNC-5 inhibits growth cone protrusion via the flavin monooxygenases and potential destabilization of F-actin, and via UNC-33/CRMP and restriction of microtubule plus-end entry into the growth cone. We show that UNC-5 inhibits protrusion through a third mechanism involving TOM-1/tomosyn. A short isoform of TOM-1 inhibited protrusion downstream of UNC-5, and a long isoform had a pro-protrusive role. TOM-1/tomosyn inhibits formation of the SNARE complex. We show that UNC-64/syntaxin is required for growth cone protrusion, consistent with a role of TOM-1 in inhibiting vesicle fusion. Our results are consistent with a model whereby UNC-5 utilizes TOM-1 to inhibit vesicle fusion, resulting in inhibited growth cone protrusion, possibly by preventing the growth cone plasma membrane addition required for protrusion.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/metabolismo , Conos de Crecimiento/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Axones/metabolismo , Netrinas/metabolismo , Proteínas Portadoras/metabolismo , Receptores de Netrina/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Moléculas de Adhesión Celular/metabolismoRESUMEN
HSV infects keratinocytes in the epidermis of skin via nectin-1. We established a human foreskin explant infection model to investigate HSV entry and spread. HSV1 entry could only be achieved by the topical application of virus via high density microarray projections (HD-MAPs) to the epidermis, which penetrated beyond one third of its thickness, simulating in vivo microtrauma. Rapid lateral spread of HSV1 to a mean of 13 keratinocytes wide occurred after 24 hours and free virus particles were observed between keratinocytes, consistent with an intercellular route of spread. Nectin-1 staining was markedly decreased in foci of infection in the epidermis and in the human keratinocyte HaCaT cell line. Nectin-1 was redistributed, at the protein level, in adjacent uninfected cells surrounding infection, inducible by CCL3, IL-8 (or CXCL8), and possibly CXCL10 and IL-6, thus facilitating spread. These findings provide the first insights into HSV1 entry and spread in human inner foreskin in situ.