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1.
Mol Syst Biol ; 11(4): 804, 2015 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-25908733

RESUMEN

Numerous molecular components have been identified that regulate the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemoattractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96-well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis phenotypes for 285 siRNA perturbations. A key finding was that the G-protein Giα subunit selectively controls the direction of migration while the receptor and Gß subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutrophils chemotax persistently in response to gradients of fMLF but only transiently in response to gradients of ATP. The method we introduce is applicable for diverse chemical cues and systematic perturbations, can be used to measure multiple cell migration and signaling parameters, and is compatible with low- and high-resolution fluorescence microscopy.


Asunto(s)
Quimiotaxis , Rayos Ultravioleta , Adenosina Trifosfato/farmacología , Línea Celular Tumoral , Factores Quimiotácticos/farmacología , Quimiotaxis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Diseño de Equipo , Fluoresceínas/análisis , Colorantes Fluorescentes/análisis , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Humanos , Procesamiento de Imagen Asistido por Computador , Leucemia Mieloide Aguda/patología , Microscopía Fluorescente/métodos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Proteínas de Neoplasias/fisiología , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Fotoquímica , ARN Interferente Pequeño/farmacología , Imagen de Lapso de Tiempo
2.
Nat Methods ; 5(5): 393-5, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18408727

RESUMEN

We present a high-throughput method that enables efficient delivery of biomolecules into cells. The device consists of an array of 96 suspended electrode pairs, where small sample volumes are top-loaded, electroporated and bottom-ejected into 96-well plates. We demonstrate the use of this suspended-drop electroporation (SDE) device to effectively introduce fluorescent dextran, small interfering RNA (siRNA) or cDNA into primary neurons, differentiated neutrophils and other cell types with conventionally low transfection rates.


Asunto(s)
Electroporación/métodos , Transfección/métodos , Células Cultivadas , ADN Complementario/genética , Dextranos/metabolismo , Electroporación/instrumentación , Silenciador del Gen , Humanos , Neuronas/metabolismo , Neutrófilos/metabolismo , ARN Interferente Pequeño/genética , Transfección/instrumentación
3.
Nat Biotechnol ; 22(4): 440-4, 2004 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15034592

RESUMEN

Chemical and biological labeling is fundamental for the elucidation of the function of proteins within biochemical cellular networks. In particular, fluorescent probes allow detection of molecular interactions, mobility and conformational changes of proteins in live cells with high temporal and spatial resolution. We present a generic method to label proteins in vivo selectively, rapidly (seconds) and reversibly, with small molecular probes that can have a wide variety of properties. These probes comprise a chromophore and a metal-ion-chelating nitrilotriacetate (NTA) moiety, which binds reversibly and specifically to engineered oligohistidine sequences in proteins of interest. We demonstrate the feasibility of the approach by binding NTA-chromophore conjugates to a representative ligand-gated ion channel and G protein-coupled receptor, each containing a polyhistidine sequence. We investigated the ionotropic 5HT(3) serotonin receptor by fluorescence measurements to characterize in vivo the probe-receptor interactions, yielding information on structure and plasma membrane distribution of the receptor.


Asunto(s)
Biotecnología/métodos , Membrana Celular/metabolismo , Técnicas Genéticas , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes , Histidina/química , Humanos , Iones , Cinética , Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Ratones , Microscopía Fluorescente , Ácido Nitrilotriacético/química , Unión Proteica , Factores de Tiempo
4.
Chemphyschem ; 8(8): 1221-7, 2007 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-17436349

RESUMEN

Sensitive live-cell fluorescence microscopy and single-molecule imaging are severely limited by rapid photobleaching of fluorescent probes. Herein, we show how to circumvent this problem using a novel, generic labeling strategy. Small nickel-nitrilotriacetate fluorescent probes are reversibly bound to oligohistidine sequences of exposed proteins on cell surfaces, permitting selective observation of the proteins by fluorescence microscopy. Photobleached probes are removed by washing and replaced by new fluorophores, thus enabling repetitive acquisition of single-molecule trajectories on the same cell and allowing variation of experimental conditions between acquisitions. This method offers free choice of fluorophores while being minimally perturbing. The strength of the method is demonstrated by labeling engineered polyhistidine sequences of the serotonin-gated 5-HT(3) receptor on the surface of live mammalian cells. Single-molecule microscopy reveals pronounced heterogeneous mobility patterns of the 5-HT(3) receptor. After activating the receptor with serotonin, the number of immobile receptors increases substantially, which might be important for receptor regulation at synapses.


Asunto(s)
Células Epiteliales/metabolismo , Histidina/química , Receptores de Serotonina 5-HT3/química , Secuencia de Aminoácidos , Línea Celular , Membrana Celular/química , Membrana Celular/metabolismo , Supervivencia Celular , Colorantes Fluorescentes , Humanos , Conformación Proteica , Receptores de Serotonina 5-HT3/metabolismo , Serotonina/metabolismo
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