RESUMEN
Integrins link the extracellular environment to the actin cytoskeleton in cell migration and adhesiveness. Rapid coordination between events outside and inside the cell is essential. Single-molecule fluorescence dynamics show that ligand binding to the bent-closed integrin conformation, which predominates on cell surfaces, is followed within milliseconds by two concerted changes, leg extension and headpiece opening, to give the high-affinity integrin conformation. The extended-closed integrin conformation is not an intermediate but can be directly accessed from the extended-open conformation and provides a pathway for ligand dissociation. In contrast to ligand, talin, which links the integrin ß-subunit cytoplasmic domain to the actin cytoskeleton, modestly stabilizes but does not induce extension or opening. Integrin activation is thus initiated by outside-in signaling and followed by inside-out signaling. Our results further imply that talin binding is insufficient for inside-out integrin activation and that tensile force transmission through the ligand-integrin-talin-actin cytoskeleton complex is required.
Asunto(s)
Integrinas , Talina , Animales , Humanos , Ratones , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/química , Adhesión Celular , Células CHO , Cricetulus , Integrinas/metabolismo , Integrinas/química , Ligandos , Unión Proteica , Conformación Proteica , Transducción de Señal , Imagen Individual de Molécula , Talina/metabolismo , Talina/químicaRESUMEN
Small molecule chaperones have been exploited as therapeutics for the hundreds of diseases caused by protein misfolding. The most successful examples are the CFTR correctors, which transformed cystic fibrosis therapy. These molecules revert folding defects of the ΔF508 mutant and are widely used to treat patients. To investigate the molecular mechanism of their action, we determined cryo-electron microscopy structures of CFTR in complex with the FDA-approved correctors lumacaftor or tezacaftor. Both drugs insert into a hydrophobic pocket in the first transmembrane domain (TMD1), linking together four helices that are thermodynamically unstable. Mutating residues at the binding site rendered ΔF508-CFTR insensitive to lumacaftor and tezacaftor, underscoring the functional significance of the structural discovery. These results support a mechanism in which the correctors stabilize TMD1 at an early stage of biogenesis, prevent its premature degradation, and thereby allosterically rescuing many disease-causing mutations.
Asunto(s)
Aminopiridinas/metabolismo , Benzodioxoles/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Indoles/metabolismo , Pliegue de Proteína , Aminopiridinas/química , Aminopiridinas/uso terapéutico , Animales , Benzodioxoles/química , Benzodioxoles/uso terapéutico , Sitios de Unión , Células CHO , Membrana Celular/química , Membrana Celular/metabolismo , Cricetulus , Microscopía por Crioelectrón , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Indoles/química , Indoles/uso terapéutico , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/uso terapéutico , Mutación , Dominios Proteicos/genética , Células Sf9 , TransfecciónRESUMEN
Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 µg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Sitios de Unión de Anticuerpos , Células CHO , Chlorocebus aethiops , Cricetulus , Epítopos , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Transgénicos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , SARS-CoV-2/inmunología , Células VeroRESUMEN
SARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes and CD8+ T cells for optimal clinical and virological benefit. Thus, potently neutralizing mAbs utilize Fc effector functions during therapy to mitigate lung infection and disease.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Linfocitos T CD8-positivos , COVID-19 , Fragmentos Fc de Inmunoglobulinas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/uso terapéutico , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Células CHO , COVID-19/inmunología , COVID-19/terapia , Chlorocebus aethiops , Cricetulus , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , SARS-CoV-2/inmunología , Células Vero , Carga ViralRESUMEN
SARS-CoV-2 has caused over 2 million deaths in little over a year. Vaccines are being deployed at scale, aiming to generate responses against the virus spike. The scale of the pandemic and error-prone virus replication is leading to the appearance of mutant viruses and potentially escape from antibody responses. Variant B.1.1.7, now dominant in the UK, with increased transmission, harbors 9 amino acid changes in the spike, including N501Y in the ACE2 interacting surface. We examine the ability of B.1.1.7 to evade antibody responses elicited by natural SARS-CoV-2 infection or vaccination. We map the impact of N501Y by structure/function analysis of a large panel of well-characterized monoclonal antibodies. B.1.1.7 is harder to neutralize than parental virus, compromising neutralization by some members of a major class of public antibodies through light-chain contacts with residue 501. However, widespread escape from monoclonal antibodies or antibody responses generated by natural infection or vaccination was not observed.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Células CHO , COVID-19/epidemiología , Chlorocebus aethiops , Cricetulus , Células HEK293 , Humanos , Pandemias , Unión Proteica , Relación Estructura-Actividad , Células VeroRESUMEN
Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by binding of the viral Spike protein to host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. Although antibodies that block this interaction are in emergency use as early coronavirus disease 2019 (COVID-19) therapies, the precise determinants of neutralization potency remain unknown. We discovered a series of antibodies that potently block ACE2 binding but exhibit divergent neutralization efficacy against the live virus. Strikingly, these neutralizing antibodies can inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in individuals with COVID-19. As revealed by cryoelectron microscopy, multiple structures of Spike-antibody complexes have distinct binding modes that not only block ACE2 binding but also alter the Spike protein conformational cycle triggered by ACE2 binding. We show that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion with profound implications for COVID-19 pathology and immunity.
Asunto(s)
Anticuerpos Neutralizantes/química , Células Gigantes/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/metabolismo , Sitios de Unión , Células CHO , COVID-19/patología , COVID-19/virología , Cricetinae , Cricetulus , Microscopía por Crioelectrón , Células Gigantes/citología , Humanos , Fusión de Membrana , Biblioteca de Péptidos , Unión Proteica , Dominios Proteicos , Estructura Cuaternaria de Proteína , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Diseño de Fármacos , Receptor Muscarínico M1/agonistas , Anciano , Anciano de 80 o más Años , Envejecimiento/patología , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/diagnóstico por imagen , Enfermedad de Alzheimer/patología , Secuencia de Aminoácidos , Animales , Presión Sanguínea/efectos de los fármacos , Células CHO , Inhibidores de la Colinesterasa/farmacología , Cricetulus , Cristalización , Modelos Animales de Enfermedad , Perros , Donepezilo/farmacología , Electroencefalografía , Femenino , Células HEK293 , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Masculino , Ratones Endogámicos C57BL , Modelos Moleculares , Simulación de Dinámica Molecular , Degeneración Nerviosa/complicaciones , Degeneración Nerviosa/patología , Primates , Ratas , Receptor Muscarínico M1/química , Transducción de Señal , Homología Estructural de ProteínaRESUMEN
Repeated exposure to pathogens or their antigens triggers anamnestic antibody responses that are higher in magnitude and affinity than the primary response. These involve reengagement of memory B cell (MBC) clones, the diversity and specificity of which determine the breadth and effectiveness of the ensuing antibody response. Using prime-boost models in mice, we find that secondary responses are characterized by a clonality bottleneck that restricts the engagement of the large diversity of MBC clones generated by priming. Rediversification of mutated MBCs is infrequent within secondary germinal centers (GCs), which instead consist predominantly of B cells without prior GC experience or detectable clonal expansion. Few MBC clones, generally derived from higher-affinity germline precursors, account for the majority of secondary antibody responses, while most primary-derived clonal diversity is not reengaged detectably by boosting. Understanding how to counter this bottleneck may improve our ability to elicit antibodies to non-immunodominant epitopes by vaccination.
Asunto(s)
Linfocitos B/inmunología , Centro Germinal/inmunología , Memoria Inmunológica/inmunología , Inmunidad Adaptativa/inmunología , Animales , Formación de Anticuerpos/inmunología , Formación de Anticuerpos/fisiología , Antígenos/inmunología , Linfocitos B/metabolismo , Células CHO , Línea Celular , Cricetulus , Femenino , Centro Germinal/metabolismo , Humanos , Memoria Inmunológica/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos AnimalesRESUMEN
Integrin αvß8 binds with exquisite specificity to latent transforming growth factor-ß (L-TGF-ß). This binding is essential for activating L-TGF-ß presented by a variety of cell types. Inhibiting αvß8-mediated TGF-ß activation blocks immunosuppressive regulatory T cell differentiation, which is a potential therapeutic strategy in cancer. Using cryo-electron microscopy, structure-guided mutagenesis, and cell-based assays, we reveal the binding interactions between the entire αvß8 ectodomain and its intact natural ligand, L-TGF-ß, as well as two different inhibitory antibody fragments to understand the structural underpinnings of αvß8 binding specificity and TGF-ß activation. Our studies reveal a mechanism of TGF-ß activation where mature TGF-ß signals within the confines of L-TGF-ß and the release and diffusion of TGF-ß are not required. The structural details of this mechanism provide a rational basis for therapeutic strategies to inhibit αvß8-mediated L-TGF-ß activation.
Asunto(s)
Microscopía por Crioelectrón/métodos , Integrinas/química , Integrinas/metabolismo , Proteínas de Unión a TGF-beta Latente/química , Proteínas de Unión a TGF-beta Latente/metabolismo , Factor de Crecimiento Transformador beta1/química , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Anticuerpos/inmunología , Sitios de Unión , Bronquios/citología , Células CHO , Cricetulus , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Integrinas/inmunología , Activación de Linfocitos , Masculino , Visón , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Linfocitos T Reguladores/inmunologíaRESUMEN
Drugs selectively targeting CB2 hold promise for treating neurodegenerative disorders, inflammation, and pain while avoiding psychotropic side effects mediated by CB1. The mechanisms underlying CB2 activation and signaling are poorly understood but critical for drug design. Here we report the cryo-EM structure of the human CB2-Gi signaling complex bound to the agonist WIN 55,212-2. The 3D structure reveals the binding mode of WIN 55,212-2 and structural determinants for distinguishing CB2 agonists from antagonists, which are supported by a pair of rationally designed agonist and antagonist. Further structural analyses with computational docking results uncover the differences between CB2 and CB1 in receptor activation, ligand recognition, and Gi coupling. These findings are expected to facilitate rational structure-based discovery of drugs targeting the cannabinoid system.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Receptor Cannabinoide CB2/química , Transducción de Señal , Animales , Sitios de Unión , Células CHO , Agonistas de Receptores de Cannabinoides/síntesis química , Agonistas de Receptores de Cannabinoides/farmacología , Antagonistas de Receptores de Cannabinoides/síntesis química , Antagonistas de Receptores de Cannabinoides/farmacología , Cricetinae , Cricetulus , Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Unión Proteica , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/antagonistas & inhibidores , Receptor Cannabinoide CB2/metabolismo , Células Sf9 , SpodopteraRESUMEN
Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role.
Asunto(s)
Agonistas de Receptores de Cannabinoides/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Receptor Cannabinoide CB1/química , Receptor Cannabinoide CB2/química , Transducción de Señal , Regulación Alostérica , Sitio Alostérico , Animales , Células CHO , Agonistas de Receptores de Cannabinoides/química , Cannabinoides/química , Cannabinoides/farmacología , Línea Celular Tumoral , Colesterol/química , Colesterol/farmacología , Cricetinae , Cricetulus , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Simulación de Dinámica Molecular , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Células Sf9 , SpodopteraRESUMEN
Broadly neutralizing antibodies (bNAbs) represent a promising approach to prevent and treat HIV-1 infection. However, viral escape through mutation of the HIV-1 envelope glycoprotein (Env) limits clinical applications. Here we describe 1-18, a new VH1-46-encoded CD4 binding site (CD4bs) bNAb with outstanding breadth (97%) and potency (GeoMean IC50 = 0.048 µg/mL). Notably, 1-18 is not susceptible to typical CD4bs escape mutations and effectively overcomes HIV-1 resistance to other CD4bs bNAbs. Moreover, mutational antigenic profiling uncovered restricted pathways of HIV-1 escape. Of most promise for therapeutic use, even 1-18 alone fully suppressed viremia in HIV-1-infected humanized mice without selecting for resistant viral variants. A 2.5-Å cryo-EM structure of a 1-18-BG505SOSIP.664 Env complex revealed that these characteristics are likely facilitated by a heavy-chain insertion and increased inter-protomer contacts. The ability of 1-18 to effectively restrict HIV-1 escape pathways provides a new option to successfully prevent and treat HIV-1 infection.
Asunto(s)
Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Sitios de Unión , Antígenos CD4/metabolismo , Células CHO , Estudios de Cohortes , Cricetulus , Epítopos/inmunología , Femenino , Células HEK293 , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Persona de Mediana Edad , Mutación , Unión Proteica/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The hyperpolarization-activated cyclic nucleotide-gated (HCN) channel is a voltage-gated cation channel that mediates neuronal and cardiac pacemaker activity. The HCN channel exhibits reversed voltage dependence, meaning it closes with depolarization and opens with hyperpolarization. Different from Na+, Ca2+, and Kv1-Kv7 channels, the HCN channel does not have domain-swapped voltage sensors. We introduced a reversible, metal-mediated cross bridge into the voltage sensors to create the chemical equivalent of a hyperpolarized conformation and determined the structure using cryoelectron microscopy (cryo-EM). Unlike the depolarized HCN channel, the S4 helix is displaced toward the cytoplasm by two helical turns. Near the cytoplasm, the S4 helix breaks into two helices, one running parallel to the membrane surface, analogous to the S4-S5 linker of domain-swapped voltage-gated channels. These findings suggest a basis for allosteric communication between voltage sensors and the gate in this kind of channel. They also imply that voltage sensor movements are not the same in all voltage-gated channels.
Asunto(s)
Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/química , Activación del Canal Iónico , Animales , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Potenciales de la Membrana , Conformación Proteica en Hélice alfa , Células Sf9 , SpodopteraRESUMEN
In search of the molecular identities of cold-sensing receptors, we carried out an unbiased genetic screen for cold-sensing mutants in C. elegans and isolated a mutant allele of glr-3 gene that encodes a kainate-type glutamate receptor. While glutamate receptors are best known to transmit chemical synaptic signals in the CNS, we show that GLR-3 senses cold in the peripheral sensory neuron ASER to trigger cold-avoidance behavior. GLR-3 transmits cold signals via G protein signaling independently of its glutamate-gated channel function, suggesting GLR-3 as a metabotropic cold receptor. The vertebrate GLR-3 homolog GluK2 from zebrafish, mouse, and human can all function as a cold receptor in heterologous systems. Mouse DRG sensory neurons express GluK2, and GluK2 knockdown in these neurons suppresses their sensitivity to cold but not cool temperatures. Our study identifies an evolutionarily conserved cold receptor, revealing that a central chemical receptor unexpectedly functions as a thermal receptor in the periphery.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/genética , Receptores de Glutamato/fisiología , Receptores de Ácido Kaínico/fisiología , Receptores de Glutamato Metabotrópico/fisiología , Sensación Térmica/fisiología , Animales , Células CHO , Proteínas de Caenorhabditis elegans/genética , Frío , Cricetulus , Humanos , Ratones , Neuronas/metabolismo , Receptores de Glutamato/genética , Receptores de Ácido Kaínico/genética , Receptores de Glutamato Metabotrópico/genética , Sensación Térmica/genéticaRESUMEN
Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human Nav1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable â¼10 Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective Nav channel antagonists.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/ultraestructura , Péptidos/metabolismo , Venenos de Araña/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Cricetulus , Microscopía por Crioelectrón/métodos , Cristalografía por Rayos X/métodos , Células HEK293 , Humanos , Activación del Canal Iónico , Péptidos/toxicidad , Dominios Proteicos , Venenos de Araña/toxicidad , Arañas , Bloqueadores del Canal de Sodio Activado por Voltaje , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a major class of lipid-anchored plasma membrane proteins. GPI-APs form nanoclusters generated by cortical acto-myosin activity. While our understanding of the physical principles governing this process is emerging, the molecular machinery and functional relevance of GPI-AP nanoclustering are unknown. Here, we first show that a membrane receptor signaling pathway directs nanocluster formation. Arg-Gly-Asp motif-containing ligands bound to the ß1-integrin receptor activate src and focal adhesion kinases, resulting in RhoA signaling. This cascade triggers actin-nucleation via specific formins, which, along with myosin activity, drive the nanoclustering of membrane proteins with actin-binding domains. Concurrently, talin-mediated activation of the mechano-transducer vinculin is required for the coupling of the acto-myosin machinery to inner-leaflet lipids, thereby generating GPI-AP nanoclusters. Second, we show that these nanoclusters are functional; disruption of their formation either in GPI-anchor remodeling mutants or in vinculin mutants impairs cell spreading and migration, hallmarks of integrin function.
Asunto(s)
Integrina beta1/metabolismo , Mecanotransducción Celular , Microdominios de Membrana/metabolismo , Secuencias de Aminoácidos , Animales , Células CHO , Cricetulus , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Humanos , Integrina beta1/genética , Microdominios de Membrana/genética , Vinculina/genética , Vinculina/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.
Asunto(s)
Encéfalo/fisiología , Proteínas Activadoras de GTPasa/genética , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Optogenética/métodos , Ritmo Teta , Vigilia , Potenciales de Acción , Animales , Encéfalo/metabolismo , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Femenino , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Ratas , Ratas Sprague-Dawley , CarreraRESUMEN
Neutrophils display distinct gene expression patters depending on their developmental stage, activation state and tissue microenvironment. To determine the transcription factor networks that shape these responses in a mouse model, we integrated transcriptional and chromatin analyses of neutrophils during acute inflammation. We showed active chromatin remodeling at two transition stages: bone marrow-to-blood and blood-to-tissue. Analysis of differentially accessible regions revealed distinct sets of putative transcription factors associated with control of neutrophil inflammatory responses. Using ex vivo and in vivo approaches, we confirmed that RUNX1 and KLF6 modulate neutrophil maturation, whereas RELB, IRF5 and JUNB drive neutrophil effector responses and RFX2 and RELB promote survival. Interfering with neutrophil activation by targeting one of these factors, JUNB, reduced pathological inflammation in a mouse model of myocardial infarction. Therefore, our study represents a blueprint for transcriptional control of neutrophil responses in acute inflammation and opens possibilities for stage-specific therapeutic modulation of neutrophil function in disease.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Inflamación/inmunología , Neutrófilos/inmunología , Activación Transcripcional/genética , Animales , Células CHO , Línea Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Cricetulus , Femenino , Factores Reguladores del Interferón/metabolismo , Factor 6 Similar a Kruppel/metabolismo , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/inmunología , Infarto del Miocardio/patología , Factores de Transcripción del Factor Regulador X/metabolismo , Factor de Transcripción ReIB/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genéticaRESUMEN
The mechanisms underlying sterol transport in mammalian cells are poorly understood. In particular, how cholesterol internalized from HDL is made available to the cell for storage or modification is unknown. Here, we describe three ER-resident proteins (Aster-A, -B, -C) that bind cholesterol and facilitate its removal from the plasma membrane. The crystal structure of the central domain of Aster-A broadly resembles the sterol-binding fold of mammalian StARD proteins, but sequence differences in the Aster pocket result in a distinct mode of ligand binding. The Aster N-terminal GRAM domain binds phosphatidylserine and mediates Aster recruitment to plasma membrane-ER contact sites in response to cholesterol accumulation in the plasma membrane. Mice lacking Aster-B are deficient in adrenal cholesterol ester storage and steroidogenesis because of an inability to transport cholesterol from SR-BI to the ER. These findings identify a nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals.
Asunto(s)
HDL-Colesterol/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de la Membrana/ultraestructura , Células 3T3 , Animales , Transporte Biológico/fisiología , Antígenos CD36/metabolismo , Células CHO , Proteínas Portadoras/metabolismo , Línea Celular , Membrana Celular/metabolismo , Membrana Celular/fisiología , Colesterol/metabolismo , Cricetulus , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/fisiología , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Membranas Mitocondriales/metabolismo , Alineación de Secuencia , Esteroles/metabolismoRESUMEN
The Notch signaling pathway comprises multiple ligands that are used in distinct biological contexts. In principle, different ligands could activate distinct target programs in signal-receiving cells, but it is unclear how such ligand discrimination could occur. Here, we show that cells use dynamics to discriminate signaling by the ligands Dll1 and Dll4 through the Notch1 receptor. Quantitative single-cell imaging revealed that Dll1 activates Notch1 in discrete, frequency-modulated pulses that specifically upregulate the Notch target gene Hes1. By contrast, Dll4 activates Notch1 in a sustained, amplitude-modulated manner that predominantly upregulates Hey1 and HeyL. Ectopic expression of Dll1 or Dll4 in chick neural crest produced opposite effects on myogenic differentiation, showing that ligand discrimination can occur in vivo. Finally, analysis of chimeric ligands suggests that ligand-receptor clustering underlies dynamic encoding of ligand identity. The ability of the pathway to utilize ligands as distinct communication channels has implications for diverse Notch-dependent processes.