RESUMO
Chimeric antigen receptor (CAR) T cells have been used to successfully treat various blood cancers, but adverse effects have limited their potential. Here, we developed chimeric adaptor proteins (CAPs) and CAR tyrosine kinases (CAR-TKs) in which the intracellular ζ T cell receptor (TCRζ) chain was replaced with intracellular protein domains to stimulate signaling downstream of the TCRζ chain. CAPs contain adaptor domains and the kinase domain of ZAP70, whereas CAR-TKs contain only ZAP70 domains. We hypothesized that CAPs and CAR-TKs would be more potent than CARs because they would bypass both the steps that define the signaling threshold of TCRζ and the inhibitory regulation of upstream molecules. CAPs were too potent and exhibited high tonic signaling in vitro. In contrast, CAR-TKs exhibited high antitumor efficacy and significantly enhanced long-term tumor clearance in leukemia-bearing NSG mice as compared with the conventional CD19-28ζ-CAR-T cells. CAR-TKs were activated in a manner independent of the kinase Lck and displayed slower phosphorylation kinetics and prolonged signaling compared with the 28ζ-CAR. Lck inhibition attenuated CAR-TK cell exhaustion and improved long-term function. The distinct signaling properties of CAR-TKs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an antitumor chimeric receptor.
Assuntos
Receptores de Antígenos de Linfócitos T , Receptores de Antígenos Quiméricos , Transdução de Sinais , Linfócitos T , Animais , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/genética , Humanos , Transdução de Sinais/imunologia , Camundongos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo , Proteína-Tirosina Quinase ZAP-70/genética , Proteína-Tirosina Quinase ZAP-70/imunologia , Imunoterapia Adotiva/métodos , Camundongos Endogâmicos NOD , Linhagem Celular Tumoral , FosforilaçãoRESUMO
BACKGROUND: One of the major challenges in chimeric antigen receptor (CAR)-T cell therapy for solid tumors is the potential for on-target off-tumor toxicity due to the expression of CAR tumor antigens in essential tissues and organs. Here, we describe a dual CAR NOT gate incorporating an inhibitory CAR (iCAR) recognizing HLA-A*02 ("A2") that enables effective treatment with a potent HER2 activating CAR (aCAR) in the context of A2 loss of heterozygosity (LOH). METHODS: A CAR-T cell screen was conducted to identify inhibitory domains derived from natural immune receptors (iDomains) to be used in a NOT gate, to kill A2- HER2+ lung cancer cell lines but spare A2+ HER2+ lung cancer cell-lines with high specificity. The extensive analysis of lead candidates included T-cell activation and killing, assays of reversibility and durability in sequential challenges, target cell specificity in mixed 3D spheroids and 2D cultures, and the characterization of CAR expression level and cell-trafficking. RESULTS: A leukocyte immunoglobulin-like receptor B1 (LIR1) iDomain iCAR was identified as most effective in regulating the cytotoxicity of a second generation HER2 aCAR. Target transfer experiments demonstrated that the 'on' and 'off' cell state of the LIR1 NOT gate CAR-T cell is both durable and reversible. Protection required iCAR signaling and was associated with reduced aCAR and iCAR surface expression. iCAR regulation was sufficient to generate high target specificity in a 3D adjacent spheroid assay designed to model the interface between clonal A2 LOH foci and normal tissue. However, we observed significant bystander killing of A2+ cells in admix culture through aCAR dependent and independent mechanisms. LIR1 NOT gate CAR-T cells conferred protection against H1703-A2+ tumors and high efficacy against H1703-A2- tumors in-vivo. We observed that the iCAR is inactive in A2+ donors due to cis-binding, but Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout of HLA-A fully restored iCAR activity. CONCLUSIONS: We have preclinically validated an iCAR NOT gate technology broadly applicable for targeting HER2 expression in the context of A2 LOH. This approach is designed to prevent off tumor toxicity while allowing highly potent antitumor activity.
Assuntos
Neoplasias Pulmonares , Linfócitos T , Humanos , Receptores de Antígenos de Linfócitos T , Complexo Ferro-Dextran/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/metabolismo , Antígenos HLA-ARESUMO
In all resolved structures of complex I, there exists a tunnel-like Q-chamber for ubiquinone binding and reduction. The entrance to the Q-chamber in ND1 subunit forms a narrow bottleneck, which is rather tight and requires thermal conformational changes for ubiquinone to get in and out of the binding chamber. The substitution of alanine with threonine at the bottleneck (AlaThr MUT), associated with 3460/ND1 mtDNA mutation in human complex I, is implicated in Leber's Hereditary Optic Neuropathy (LHON). Here, we show the AlaThr MUT further narrows the Q-chamber entrance cross-section area by almost 30%, increasing the activation free energy barrier of quinone passage by approximately 5 kJ mol-1. This severely disrupts quinone binding and reduction as quinone passage through the bottleneck is slowed down almost tenfold. Our estimate of the increase in free energy barrier is entirely due to the bottleneck narrowing, leading to a reduction of the transition state entropy between WT and MUT, and thus more difficult quinone passage. Additionally, we investigate details of possible water exchange between the Q-chamber and membrane. We find water exchange is dynamic in WT but may be severely slowed in MUT. We propose that LHON symptoms caused by 3460/ND1 mtDNA mutation are due to slowed quinone binding. This leads to an increased production of reactive oxidative species due to upstream electron backup at the FMN site of complex I, thus resulting in a mt bioenergetic defect.
Assuntos
Complexo I de Transporte de Elétrons , Atrofia Óptica Hereditária de Leber , Humanos , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , DNA Mitocondrial/genética , Ubiquinona/metabolismo , Mitocôndrias/metabolismo , Mutação , Atrofia Óptica Hereditária de Leber/genética , Água/metabolismoRESUMO
The E3 ubiquitin ligase Ube3a is biallelically expressed in neural progenitors and glial cells, suggesting that UBE3A gain-of-function mutations might cause neurodevelopmental disorders irrespective of parent of origin. Here, we engineered a mouse line that harbors an autism-linked UBE3AT485A (T503A in mouse) gain-of-function mutation and evaluated phenotypes in animals that inherited the mutant allele paternally, maternally, or from both parents. We find that paternally and maternally expressed UBE3AT503A results in elevated UBE3A activity in neural progenitors and glial cells. Expression of UBE3AT503A from the maternal allele, but not the paternal one, leads to a persistent elevation of UBE3A activity in neurons. Mutant mice display behavioral phenotypes that differ by parent of origin. Expression of UBE3AT503A, irrespective of its parent of origin, promotes transient embryonic expansion of Zcchc12 lineage interneurons. Phenotypes of Ube3aT503A mice are distinct from Angelman syndrome model mice. Our study has clinical implications for a growing number of disease-linked UBE3A gain-of-function mutations.
Assuntos
Síndrome de Angelman , Transtorno Autístico , Animais , Camundongos , Transtorno Autístico/genética , Modelos Animais de Doenças , Mutação com Ganho de Função , Interneurônios/metabolismo , Herança Materna , Fenótipo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The commonalities and differences in cell-type-specific pathways that lead to Alzheimer disease (AD) and Parkinson disease (PD) remain unknown. Here, we performed a single-nucleus transcriptome comparison of control, AD and PD striata. We describe three astrocyte subpopulations shared across different brain regions and evolutionarily conserved between humans and mice. We reveal common features between AD and PD astrocytes and regional differences that contribute toward amyloid pathology and neurodegeneration. In contrast, we found that transcriptomic changes in microglia are largely unique to each disorder. Our analysis identified a population of activated microglia that shared molecular signatures with murine disease-associated microglia (DAM) as well as disease-associated and regional differences in microglia transcriptomic changes linking microglia to disease-specific amyloid pathology, tauopathy and neuronal death. Finally, we delineate undescribed subpopulations of medium spiny neurons (MSNs) in the striatum and provide neuronal transcriptomic profiles suggesting disease-specific changes and selective neuronal vulnerability.
Assuntos
Doença de Alzheimer , Doença de Parkinson , Humanos , Camundongos , Animais , Doença de Alzheimer/genética , Doença de Parkinson/genética , Transcriptoma/genética , Encéfalo/metabolismo , Microglia/metabolismo , Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismoRESUMO
The increased use of sequencing in medicine has identified millions of coding variants in the human genome. Many of these variants occur in genes associated with neurodevelopmental disorders, but the functional significance of the vast majority of variants remains unknown. The present protocol describes the study of variants for Ube3a, a gene that encodes an E3 ubiquitin ligase linked to both autism and Angelman syndrome. Duplication or triplication of Ube3a is strongly linked to autism, whereas its deletion causes Angelman syndrome. Thus, understanding the valence of changes in UBE3A protein activity is important for clinical outcomes. Here, a rapid, cell-based method that pairs Ube3a variants with a Wnt pathway reporter is described. This simple assay is scalable and can be used to determine the valence and magnitude of activity changes in any Ube3a variant. Moreover, the facility of this method allows the generation of a wealth of structure-function information, which can be used to gain deep insights into the enzymatic mechanisms of UBE3A.
Assuntos
Síndrome de Angelman , Transtorno Autístico , Humanos , Síndrome de Angelman/genética , Síndrome de Angelman/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt , Expressão GênicaRESUMO
The mechanisms that underlie the extensive phenotypic diversity in genetic disorders are poorly understood. Here, we develop a large-scale assay to characterize the functional valence (gain or loss-of-function) of missense variants identified in UBE3A, the gene whose loss-of-function causes the neurodevelopmental disorder Angelman syndrome. We identify numerous gain-of-function variants including a hyperactivating Q588E mutation that strikingly increases UBE3A activity above wild-type UBE3A levels. Mice carrying the Q588E mutation exhibit aberrant early-life motor and communication deficits, and individuals possessing hyperactivating UBE3A variants exhibit affected phenotypes that are distinguishable from Angelman syndrome. Additional structure-function analysis reveals that Q588 forms a regulatory site in UBE3A that is conserved among HECT domain ubiquitin ligases and perturbed in various neurodevelopmental disorders. Together, our study indicates that excessive UBE3A activity increases the risk for neurodevelopmental pathology and suggests that functional variant analysis can help delineate mechanistic subtypes in monogenic disorders.
Assuntos
Síndrome de Angelman/genética , Transtornos do Neurodesenvolvimento/genética , Ubiquitina-Proteína Ligases/genética , Animais , Modelos Animais de Doenças , Ensaios Enzimáticos , Mutação com Ganho de Função , Células HEK293 , Humanos , Mutação com Perda de Função , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , Fenótipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Relação Estrutura-Atividade , Ubiquitina-Proteína Ligases/metabolismoRESUMO
We designed variant human TCRs composed of the full-length TCRα/ß or extracellular and transmembrane domains of the associated CD3 subunits fused to polypeptides derived from proteins thought to either enhance or inhibit normal T cell function. First, we showed that the C termini of both the TCR α- and ß-chains can accommodate specific additional sequences, without abrogating complex formation or acute sensitivity of the receptor. Replacement of ITAMs with ITIM-containing intracellular domains inverted the TCR signal (i.e., created a ligand-dependent inhibitory receptor). The normal signaling function of the CD3 complex was transferable to the TCR by eliminating all CD3 ITAMs and grafting three to six ITAMs onto the C termini of the α/ß-chains, with no effect on acute sensitivity. The observation that TCR variants of such diverse C-terminal composition can fold and function as signaling receptors demonstrates substantial structural and functional malleability of TCRs. These results add to knowledge about TCR structure-function with regard to acute signaling and may provide a route to use TCRs in different ways for T cell therapy.
Assuntos
Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Proteínas de Transporte/metabolismo , Humanos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
Cell therapy is poised to play a larger role in medicine, most notably for immuno-oncology. Despite the recent success of CAR-T therapeutics in the treatment of blood tumors and the rapid progress toward improved versions of both CAR- and TCR-Ts, important analytical aspects of preclinical development and manufacturing of engineered T cells remain immature. One limiting factor is the absence of robust multivariate assays to disentangle key parameters related to function of engineered effector cells, especially in the peptide-MHC (pMHC) target realm, the natural ligand for TCRs. Here we describe an imaging-based primary T cell assay that addresses several of these limitations. To our knowledge, this assay is the first quantitative, high-content assay that separates the key functional parameters of time- and antigen-dependent T cell proliferation from cytotoxicity. We show that the assay sheds light on relevant biology of CAR- and TCR-T cells, including response kinetics and the influence of effector:target ratio.
Assuntos
Imunoensaio/métodos , Linfócitos T/imunologia , Linhagem Celular , Proliferação de Células , Citotoxicidade Imunológica , Humanos , Cinética , Análise Multivariada , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/citologiaRESUMO
Engagement of the T cell receptor (TCR) by stimulatory ligand results in the rapid formation of microclusters at sites of T cell activation. Whereas microclusters have been studied extensively using confocal microscopy, the spatial and kinetic relationships of their signaling components have not been well characterized due to limits in image resolution and acquisition speed. Here we show, using TIRF-SIM to examine the organization of microclusters at sub-diffraction resolution, the presence of two spatially distinct domains composed of ZAP70-bound TCR and LAT-associated signaling complex. Kinetic analysis of microcluster assembly reveal surprising delays between the stepwise recruitment of ZAP70 and signaling proteins to the TCR, as well as distinct patterns in their disassociation. These delays are regulated by intracellular calcium flux downstream of T cell activation. Our results reveal novel insights into the spatial and kinetic regulation of TCR microcluster formation and T cell activation.
Assuntos
Cálcio/metabolismo , Ativação Linfocitária/fisiologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Proteína-Tirosina Quinase ZAP-70/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cálcio/imunologia , Retroalimentação Fisiológica , Técnicas de Inativação de Genes , Humanos , Processamento de Imagem Assistida por Computador , Microscopia Intravital/métodos , Células Jurkat , Cinética , Leucócitos Mononucleares , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Cultura Primária de Células , Domínios Proteicos/fisiologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Proteína-Tirosina Quinase ZAP-70/imunologiaRESUMO
The relative importance of plasma membrane-localized LAT versus vesicular LAT for microcluster formation and T-cell receptor (TCR) activation is unclear. Here, we show the sequence of events in LAT microcluster formation and vesicle delivery, using lattice light sheet microscopy to image a T cell from the earliest point of activation. A kinetic lag occurs between LAT microcluster formation and vesicular pool recruitment to the synapse. Correlative 3D light and electron microscopy show an absence of vesicles at microclusters at early times, but an abundance of vesicles as activation proceeds. Using TIRF-SIM to look at the activated T-cell surface with high resolution, we capture directed vesicle movement between microclusters on microtubules. We propose a model in which cell surface LAT is recruited rapidly and phosphorylated at sites of T-cell activation, while the vesicular pool is subsequently recruited and dynamically interacts with microclusters.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Membrana Celular/imunologia , Vesículas Citoplasmáticas/imunologia , Ativação Linfocitária/genética , Proteínas de Membrana/genética , Microtúbulos/imunologia , Receptores de Antígenos de Linfócitos T/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Membrana Celular/ultraestrutura , Vesículas Citoplasmáticas/ultraestrutura , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/imunologia , Regulação da Expressão Gênica , Genes Reporter , Humanos , Sinapses Imunológicas/metabolismo , Sinapses Imunológicas/ultraestrutura , Células Jurkat , Proteínas de Membrana/imunologia , Microscopia de Fluorescência , Microtúbulos/ultraestrutura , Fosforilação , Proteínas R-SNARE/genética , Proteínas R-SNARE/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Imagem com Lapso de Tempo , Proteína-Tirosina Quinase ZAP-70/genética , Proteína-Tirosina Quinase ZAP-70/imunologiaRESUMO
Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.
Assuntos
Imunofluorescência/métodos , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Linfócitos T/citologia , Corantes Fluorescentes/química , Humanos , Células Jurkat , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Linfócitos T/metabolismoRESUMO
UBE3A is a HECT domain E3 ubiquitin ligase whose dysfunction is linked to autism, Angelman syndrome, and cancer. Recently, we characterized a de novo autism-linked UBE3A mutant (UBE3AT485A) that disrupts phosphorylation control of UBE3A activity. Through quantitative proteomics and reporter assays, we found that the UBE3AT485A protein ubiquitinates multiple proteasome subunits, reduces proteasome subunit abundance and activity, stabilizes nuclear ß-catenin, and stimulates canonical Wnt signaling more effectively than wild-type UBE3A. We also found that UBE3AT485A activates Wnt signaling to a greater extent in cells with low levels of ongoing Wnt signaling, suggesting that cells with low basal Wnt activity are particularly vulnerable to UBE3AT485A mutation. Ligase-dead UBE3A did not stimulate Wnt pathway activation. Overexpression of several proteasome subunits reversed the effect of UBE3AT485A on Wnt signaling. We also observed that subunits that interact with UBE3A and affect Wnt signaling are located along one side of the 19S regulatory particle, indicating a previously unrecognized spatial organization to the proteasome. Altogether, our findings indicate that UBE3A regulates Wnt signaling in a cell context-dependent manner and that an autism-linked mutation exacerbates these signaling effects. Our study has broad implications for human disorders associated with UBE3A gain or loss of function and suggests that dysfunctional UBE3A might affect additional proteins and pathways that are sensitive to proteasome activity.
Assuntos
Transtorno Autístico/metabolismo , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Células HEK293 , Humanos , Complexo de Endopeptidases do Proteassoma/genéticaRESUMO
Single-molecule localization microscopy (SMLM) comprises methods that produce super-resolution images from molecular locations of single molecules. These techniques mathematically determine the center of a diffraction-limited spot produced by a fluorescent molecule, which represents the most likely location of the molecule. Only a small cohort of well-separated molecules is visualized in a single image, and then many images are obtained from a single sample. The localizations from all the images are combined to produce a super-resolution picture of the sample. Here we describe the application of two methods, photoactivation localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM), to the study of signaling microclusters in T cells.
Assuntos
Imagem Molecular/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Humanos , Células JurkatRESUMO
The adapter molecule linker for activation of T cells (LAT) plays a crucial role in forming signaling complexes induced by stimulation of the T cell receptor (TCR). These multi-molecular complexes are dynamic structures that activate highly regulated signaling pathways. Previously, we have demonstrated nanoscale structure in LAT-based complexes where the adapter SLP-76 (also known as LCP2) localizes to the periphery of LAT clusters. In this study, we show that initially LAT and SLP-76 are randomly dispersed throughout the clusters that form upon TCR engagement. The segregation of LAT and SLP-76 develops near the end of the spreading process. The local concentration of LAT also increases at the same time. Both changes require TCR activation and an intact actin cytoskeleton. These results demonstrate that the nanoscale organization of LAT-based signaling complexes is dynamic and indicates that different kinds of LAT-based complexes appear at different times during T cell activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Membrana/metabolismo , Nanoestruturas/química , Fosfoproteínas/metabolismo , Transdução de Sinais , Citoesqueleto de Actina/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células Jurkat , Ativação Linfocitária , Fluidez de Membrana , Microscopia , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Actin assembly and inward flow in the plane of the immunological synapse (IS) drives the centralization of T cell receptor microclusters (TCR MCs) and the integrin leukocyte functional antigen 1 (LFA-1). Using structured-illumination microscopy (SIM), we show that actin arcs populating the medial, lamella-like region of the IS arise from linear actin filaments generated by one or more formins present at the IS distal edge. After traversing the outer, Arp2/3-generated, lamellipodia-like region of the IS, these linear filaments are organized by myosin II into antiparallel concentric arcs. Three-dimensional SIM shows that active LFA-1 often aligns with arcs, whereas TCR MCs commonly reside between arcs, and total internal reflection fluorescence SIM shows TCR MCs being swept inward by arcs. Consistently, disrupting actin arc formation via formin inhibition results in less centralized TCR MCs, missegregated integrin clusters, decreased T-B cell adhesion, and diminished TCR signaling. Together, our results define the origin, organization, and functional significance of a major actomyosin contractile structure at the IS that directly propels TCR MC transport.
Assuntos
Actomiosina/metabolismo , Movimento Celular , Sinapses Imunológicas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Agregação Celular , Proteínas Fetais , Fluorescência , Forminas , Humanos , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Proteínas dos Microfilamentos , Microscopia , Miosina Tipo II/metabolismo , Proteínas Nucleares , Linfócitos T/metabolismoRESUMO
Investigation of heterogeneous cellular structures using single-molecule localization microscopy has been limited by poorly defined localization accuracy and inadequate multiplexing capacity. Using fluorescent nanodiamonds as fiducial markers, we define and achieve localization precision required for single-molecule accuracy in dSTORM images. Coupled with this advance, our new multiplexing strategy, madSTORM, allows accurate targeting of multiple molecules using sequential binding and elution of fluorescent antibodies. madSTORM is used on an activated T-cell to localize 25 epitopes, 14 of which are on components of the same multimolecular T-cell receptor complex. We obtain an average localization precision of 2.6 nm, alignment error of 2.0 nm, and <0.01% cross-talk. Combining these technical advances affords the ability to move beyond obtaining superresolved structures to defining spatial relationships among constituent molecules within structures. Probing the molecular topology of complex signaling cascades and other heterogeneous networks is feasible with madSTORM.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Imunofluorescência/métodos , Corantes Fluorescentes/química , Receptores de Antígenos de Linfócitos T/metabolismo , Estatística como Assunto/métodosRESUMO
Deletion of UBE3A causes the neurodevelopmental disorder Angelman syndrome (AS), while duplication or triplication of UBE3A is linked to autism. These genetic findings suggest that the ubiquitin ligase activity of UBE3A must be tightly maintained to promote normal brain development. Here, we found that protein kinase A (PKA) phosphorylates UBE3A in a region outside of the catalytic domain at residue T485 and inhibits UBE3A activity toward itself and other substrates. A de novo autism-linked missense mutation disrupts this phosphorylation site, causing enhanced UBE3A activity in vitro, enhanced substrate turnover in patient-derived cells, and excessive dendritic spine development in the brain. Our study identifies PKA as an upstream regulator of UBE3A activity and shows that an autism-linked mutation disrupts this phosphorylation control. Moreover, our findings implicate excessive UBE3A activity and the resulting synaptic dysfunction to autism pathogenesis.
Assuntos
Síndrome de Angelman/genética , Transtorno Autístico/genética , Mutação de Sentido Incorreto , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Síndrome de Angelman/metabolismo , Animais , Transtorno Autístico/metabolismo , Encéfalo/patologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Espinhas Dendríticas/patologia , Embrião de Mamíferos/metabolismo , Estabilidade Enzimática , Feminino , Humanos , Camundongos Endogâmicos C57BL , Mutagênese , Fosforilação , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Optogenetic control of endogenous signaling can be an important tool for probing cell behavior. Using the photoresponse of the LOV2 domain of Avena sativa phototropin 1, we developed analogues of kinase inhibitors whose activity is light dependent. Inhibitory peptides were appended to the Jα helix, where they potently inhibited kinases in the light but were sterically blocked from kinase interaction in the dark. Photoactivatable inhibitors for cyclic-AMP dependent kinase (PKA) and myosin light chain kinase (MLCK) are described, together with studies that shed light on proper positioning of the peptides in the LOV domain. These inhibitors altered endogenous signaling in living cells and produced light-dependent changes in cell morphodynamics.