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1.
Cell ; 187(11): 2785-2800.e16, 2024 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-38657604

RESUMEN

Natural cell death pathways such as apoptosis and pyroptosis play dual roles: they eliminate harmful cells and modulate the immune system by dampening or stimulating inflammation. Synthetic protein circuits capable of triggering specific death programs in target cells could similarly remove harmful cells while appropriately modulating immune responses. However, cells actively influence their death modes in response to natural signals, making it challenging to control death modes. Here, we introduce naturally inspired "synpoptosis" circuits that proteolytically regulate engineered executioner proteins and mammalian cell death. These circuits direct cell death modes, respond to combinations of protease inputs, and selectively eliminate target cells. Furthermore, synpoptosis circuits can be transmitted intercellularly, offering a foundation for engineering synthetic killer cells that induce desired death programs in target cells without self-destruction. Together, these results lay the groundwork for programmable control of mammalian cell death.


Asunto(s)
Muerte Celular , Humanos , Apoptosis , Caspasas/metabolismo , Células HEK293 , Proteolisis , Piroptosis/efectos de los fármacos , Biología Sintética/métodos , Células Cultivadas
2.
Cell ; 186(17): 3642-3658.e32, 2023 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-37437570

RESUMEN

A system for programmable export of RNA molecules from living cells would enable both non-destructive monitoring of cell dynamics and engineering of cells capable of delivering executable RNA programs to other cells. We developed genetically encoded cellular RNA exporters, inspired by viruses, that efficiently package and secrete cargo RNA molecules from mammalian cells within protective nanoparticles. Exporting and sequencing RNA barcodes enabled non-destructive monitoring of cell population dynamics with clonal resolution. Further, by incorporating fusogens into the nanoparticles, we demonstrated the delivery, expression, and functional activity of exported mRNA in recipient cells. We term these systems COURIER (controlled output and uptake of RNA for interrogation, expression, and regulation). COURIER enables measurement of cell dynamics and establishes a foundation for hybrid cell and gene therapies based on cell-to-cell delivery of RNA.


Asunto(s)
Técnicas Citológicas , Técnicas Genéticas , ARN , Animales , Transporte Biológico , Mamíferos/metabolismo , ARN/genética , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Virus/genética , Tipificación Molecular , Análisis de Secuencia de ARN
3.
Cell ; 170(6): 1184-1196.e24, 2017 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-28886385

RESUMEN

The bone morphogenetic protein (BMP) signaling pathway comprises multiple ligands and receptors that interact promiscuously with one another and typically appear in combinations. This feature is often explained in terms of redundancy and regulatory flexibility, but it has remained unclear what signal-processing capabilities it provides. Here, we show that the BMP pathway processes multi-ligand inputs using a specific repertoire of computations, including ratiometric sensing, balance detection, and imbalance detection. These computations operate on the relative levels of different ligands and can arise directly from competitive receptor-ligand interactions. Furthermore, cells can select different computations to perform on the same ligand combination through expression of alternative sets of receptor variants. These results provide a direct signal-processing role for promiscuous receptor-ligand interactions and establish operational principles for quantitatively controlling cells with BMP ligands. Similar principles could apply to other promiscuous signaling pathways.


Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Transducción de Señal , Animales , Línea Celular , Células Madre Embrionarias/metabolismo , Retroalimentación , Citometría de Flujo , Ligandos , Ratones , Modelos Biológicos , Células 3T3 NIH
4.
Cell ; 144(4): 577-89, 2011 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-21335239

RESUMEN

The hair follicle bulge in the epidermis associates with the arrector pili muscle (APM) that is responsible for piloerection ("goosebumps"). We show that stem cells in the bulge deposit nephronectin into the underlying basement membrane, thus regulating the adhesion of mesenchymal cells expressing the nephronectin receptor, α8ß1 integrin, to the bulge. Nephronectin induces α8 integrin-positive mesenchymal cells to upregulate smooth muscle markers. In nephronectin knockout mice, fewer arrector pili muscles form in the skin, and they attach to the follicle above the bulge, where there is compensatory upregulation of the nephronectin family member EGFL6. Deletion of α8 integrin also abolishes selective APM anchorage to the bulge. Nephronectin is a Wnt target; epidermal ß-catenin activation upregulates epidermal nephronectin and dermal α8 integrin expression. Thus, bulge stem cells, via nephronectin expression, create a smooth muscle cell niche and act as tendon cells for the APM. Our results reveal a functional role for basement membrane heterogeneity in tissue patterning. PAPERCLIP:


Asunto(s)
Membrana Basal/citología , Folículo Piloso/citología , Células Madre/metabolismo , Animales , Membrana Basal/metabolismo , Células Epidérmicas , Epidermis/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Cadenas alfa de Integrinas/metabolismo , Ratones , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
5.
Nature ; 541(7635): 107-111, 2017 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-27869821

RESUMEN

Reconstructing the lineage relationships and dynamic event histories of individual cells within their native spatial context is a long-standing challenge in biology. Many biological processes of interest occur in optically opaque or physically inaccessible contexts, necessitating approaches other than direct imaging. Here we describe a synthetic system that enables cells to record lineage information and event histories in the genome in a format that can be subsequently read out of single cells in situ. This system, termed memory by engineered mutagenesis with optical in situ readout (MEMOIR), is based on a set of barcoded recording elements termed scratchpads. The state of a given scratchpad can be irreversibly altered by CRISPR/Cas9-based targeted mutagenesis, and later read out in single cells through multiplexed single-molecule RNA fluorescence hybridization (smFISH). Using MEMOIR as a proof of principle, we engineered mouse embryonic stem cells to contain multiple scratchpads and other recording components. In these cells, scratchpads were altered in a progressive and stochastic fashion as the cells proliferated. Analysis of the final states of scratchpads in single cells in situ enabled reconstruction of lineage information from cell colonies. Combining analysis of endogenous gene expression with lineage reconstruction in the same cells further allowed inference of the dynamic rates at which embryonic stem cells switch between two gene expression states. Finally, using simulations, we show how parallel MEMOIR systems operating in the same cell could enable recording and readout of dynamic cellular event histories. MEMOIR thus provides a versatile platform for information recording and in situ, single-cell readout across diverse biological systems.


Asunto(s)
Linaje de la Célula , Perfilación de la Expresión Génica/métodos , Hibridación Fluorescente in Situ/métodos , Células Madre Embrionarias de Ratones/citología , Imagen Individual de Molécula/métodos , Análisis de la Célula Individual/métodos , Animales , Sistemas CRISPR-Cas/genética , Proliferación Celular , Simulación por Computador , Ratones , Mutagénesis , ARN/análisis
6.
Cell Syst ; 13(5): 388-407.e10, 2022 05 18.
Artículo en Inglés | MEDLINE | ID: mdl-35421361

RESUMEN

Cell-cell communication systems typically comprise families of ligand and receptor variants that function together in combinations. Pathway activation depends on the complex way in which ligands are presented extracellularly and receptors are expressed by the signal-receiving cell. To understand the combinatorial logic of such a system, we systematically measured pairwise bone morphogenetic protein (BMP) ligand interactions in cells with varying receptor expression. Ligands could be classified into equivalence groups based on their profile of positive and negative synergies with other ligands. These groups varied with receptor expression, explaining how ligands can functionally replace each other in one context but not another. Context-dependent combinatorial interactions could be explained by a biochemical model based on the competitive formation of alternative signaling complexes with distinct activities. Together, these results provide insights into the roles of BMP combinations in developmental and therapeutic contexts and establish a framework for analyzing other combinatorial, context-dependent signaling systems.


Asunto(s)
Proteínas Morfogenéticas Óseas , Transducción de Señal , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Ligandos , Lógica
7.
Cell Syst ; 13(5): 408-425.e12, 2022 05 18.
Artículo en Inglés | MEDLINE | ID: mdl-35421362

RESUMEN

In multicellular organisms, secreted ligands selectively activate, or "address," specific target cell populations to control cell fate decision-making and other processes. Key cell-cell communication pathways use multiple promiscuously interacting ligands and receptors, provoking the question of how addressing specificity can emerge from molecular promiscuity. To investigate this issue, we developed a general mathematical modeling framework based on the bone morphogenetic protein (BMP) pathway architecture. We find that promiscuously interacting ligand-receptor systems allow a small number of ligands, acting in combinations, to address a larger number of individual cell types, defined by their receptor expression profiles. Promiscuous systems outperform seemingly more specific one-to-one signaling architectures in addressing capability. Combinatorial addressing extends to groups of cell types, is robust to receptor expression noise, grows more powerful with increases in the number of receptor variants, and is maximized by specific biochemical parameter relationships. Together, these results identify design principles governing cellular addressing by ligand combinations.


Asunto(s)
Proteínas Morfogenéticas Óseas , Transducción de Señal , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Ligandos
8.
Nat Biotechnol ; 38(1): 66-75, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31740838

RESUMEN

Molecular barcoding technologies that uniquely identify single cells are hampered by limitations in barcode measurement. Readout by sequencing does not preserve the spatial organization of cells in tissues, whereas imaging methods preserve spatial structure but are less sensitive to barcode sequence. Here we introduce a system for image-based readout of short (20-base-pair) DNA barcodes. In this system, called Zombie, phage RNA polymerases transcribe engineered barcodes in fixed cells. The resulting RNA is subsequently detected by fluorescent in situ hybridization. Using competing match and mismatch probes, Zombie can accurately discriminate single-nucleotide differences in the barcodes. This method allows in situ readout of dense combinatorial barcode libraries and single-base mutations produced by CRISPR base editors without requiring barcode expression in live cells. Zombie functions across diverse contexts, including cell culture, chick embryos and adult mouse brain tissue. The ability to sensitively read out compact and diverse DNA barcodes by imaging will facilitate a broad range of barcoding and genomic recording strategies.


Asunto(s)
Emparejamiento Base/genética , Código de Barras del ADN Taxonómico/métodos , Edición Génica , Transcripción Genética , Animales , Secuencia de Bases , Encéfalo/metabolismo , Embrión de Pollo , ARN Polimerasas Dirigidas por ADN/metabolismo , Biblioteca de Genes , Células HEK293 , Humanos , Lentivirus/genética , Ratones , Nucleótidos/genética , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética
9.
Nat Biotechnol ; 38(2): 245, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31988471

RESUMEN

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

10.
Cell Syst ; 3(5): 419-433.e8, 2016 11 23.
Artículo en Inglés | MEDLINE | ID: mdl-27883889

RESUMEN

As they proliferate, living cells undergo transitions between specific molecularly and developmentally distinct states. Despite the functional centrality of these transitions in multicellular organisms, it has remained challenging to determine which transitions occur and at what rates without perturbations and cell engineering. Here, we introduce kin correlation analysis (KCA) and show that quantitative cell-state transition dynamics can be inferred, without direct observation, from the clustering of cell states on pedigrees (lineage trees). Combining KCA with pedigrees obtained from time-lapse imaging and endpoint single-molecule RNA-fluorescence in situ hybridization (RNA-FISH) measurements of gene expression, we determined the cell-state transition network of mouse embryonic stem (ES) cells. This analysis revealed that mouse ES cells exhibit stochastic and reversible transitions along a linear chain of states ranging from 2C-like to epiblast-like. Our approach is broadly applicable and may be applied to systems with irreversible transitions and non-stationary dynamics, such as in cancer and development.


Asunto(s)
Análisis de la Célula Individual , Animales , Linaje de la Célula , Células Madre Embrionarias , Regulación del Desarrollo de la Expresión Génica , Hibridación Fluorescente in Situ , Ratones , Modelos Biológicos , Células Madre Embrionarias de Ratones
11.
Aging Cell ; 13(2): 206-15, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24134630

RESUMEN

One goal of aging research is to find drugs that delay the onset of age-associated disease. Studies in invertebrates, particularly Caenorhabditis elegans, have uncovered numerous genes involved in aging, many conserved in mammals. However, which of these encode proteins suitable for drug targeting is unknown. To investigate this question, we screened a library of compounds with known mammalian pharmacology for compounds that increase C. elegans lifespan. We identified 60 compounds that increase longevity in C. elegans, 33 of which also increased resistance to oxidative stress. Many of these compounds are drugs approved for human use. Enhanced resistance to oxidative stress was associated primarily with compounds that target receptors for biogenic amines, such as dopamine or serotonin. A pharmacological network constructed with these data reveal that lifespan extension and increased stress resistance cluster together in a few pharmacological classes, most involved in intercellular signaling. These studies identify compounds that can now be explored for beneficial effects on aging in mammals, as well as tools that can be used to further investigate the mechanisms underlying aging in C. elegans.


Asunto(s)
Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/fisiología , Longevidad/efectos de los fármacos , Longevidad/fisiología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Humanos , Estrés Oxidativo/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos
12.
Development ; 134(13): 2501-9, 2007 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-17537792

RESUMEN

Development of the metanephric kidney crucially depends on proper interactions between cells and the surrounding extracellular matrix. For example, we showed previously that in the absence of alpha8beta1 integrin, invasion by the ureteric bud into the metanephric mesenchyme is inhibited, resulting in renal agenesis. Here we present genetic evidence that the extracellular matrix protein nephronectin is an essential ligand that engages alpha8beta1 integrin during early kidney development. We show that embryos lacking a functional nephronectin gene frequently display kidney agenesis or hypoplasia, which can be traced to a delay in the invasion of the metanephric mesenchyme by the ureteric bud at an early stage of kidney development. Significantly, we detected no defects in extracellular matrix organization in the nascent kidneys of the nephronectin mutants. Instead, we found that Gdnf expression was dramatically reduced in both nephronectin- and alpha8 integrin-null mutants specifically in the metanephric mesenchyme at the time of ureteric bud invasion. We show that this reduction is sufficient to explain the agenesis and hypoplasia observed in both mutants. Interestingly, the reduction in Gdnf expression is transient, and its resumption presumably enables the nephronectin-deficient ureteric buds to invade the metanephric mesenchyme and begin branching. Our results thus place nephronectin and alpha8beta1 integrin in a pathway that regulates Gdnf expression and is essential for kidney development.


Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Integrinas/metabolismo , Riñón/embriología , Riñón/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Alelos , Animales , Animales Recién Nacidos , Membrana Basal/embriología , Membrana Basal/metabolismo , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/genética , Femenino , Redes Reguladoras de Genes , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Integrinas/deficiencia , Integrinas/genética , Riñón/irrigación sanguínea , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo
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