Nuestro trabajo es apoyado por becas de NIH / NIGMS, NSF, USDA / Nifa, y BARD a VC.

Fondo Simplástica transporte de macromoléculas a través de conexiones intercelulares de plantas, los plasmodesmos, es de interés para muchos patólogos de plantas y biólogos. Por ejemplo, varias proteínas virales son conocidas para regular plasmodesmal límites de exclusión de tamaño para permitir el movimiento viral 1-3. Además, algunas proteínas endógenas, entre ellos importantes reguladores de desarrollo, se supone que se mueven de célula a célula, presumiblemente a través de los plasmodesmos, que la función no de células de forma autónoma 4. Por lo tanto, una metodología fiable para identificar y visualizar transporte macromolecular entre las células vegetales es muy solicitado. 1) Cultivar las plantas de destino Para la eficiencia de transformación de alto, las plantas sanas, robustas se debe utilizar. 1. Plantas de Arabidopsis Crecer una planta de Arabidopsis en Pro-Mix BX en una olla (10 cm x 10 cm x 10 cm) en una cámara de ambiente controlado con un fotoperiodo corto (8 horas de 130 a 150 μE m -2 s -1 luz a 23 ° C/16 h oscuridad a 20 ° C) y 40-65% de humedad relativa durante 6 a 8 semanas 5. Fertilizar vez en cuando con los productos disponibles comercialmente como se describe 5. Hojas con el mayor tamaño de 15 mm x 35 mm (incluye la medición de la longitud del pecíolo) son seleccionados para los experimentos. 2. N. benthamiana y N. tabacum Crecer una planta en el Pro-Mix BX en una olla (20 cm x 20 cm x 20 cm) en una cámara de ambiente controlado con un fotoperíodo largo (16 horas de 130 a 150 μE m -2 s -1 luz a 23 ° C / 8 h oscuridad a 20 ° C) y 40-65% de humedad relativa durante 7 a 10 semanas. Fertilizar vez en cuando con los productos disponibles comercialmente como se describe. Hojas con un tamaño mayor de 50 mm x 70 mm para N. benthamiana mm o 100 x 125 mm para N. tabacum (estas medidas no incluyen la longitud del pecíolo) son seleccionados para los experimentos. 2) Preparación del cartucho de pistola de genes con el ADN recubierto de oro micropartículas El protocolo para esta etapa experimental se ha descrito en detalle previamente 6. Es muy importante usar el ADN y plásmido purificado a altas concentraciones (~ 1 mg / l) para obtener la mayor eficiencia de la transformación de la facilidad de análisis de microscopía confocal en las últimas etapas del experimento. Para este ensayo, es importante cerciorarse de que el cartucho preparado no transformar dos o más celdas adyacentes al mismo tiempo a altas frecuencias, debido a que el número de células de la asociación con un grupo de la señal fluorescente se utiliza como un indicador del grado de transporte simplástica (es decir, , una sola célula que contiene la señal indica que no hay movimiento, mientras que un grupo de señales multicelulares indica el movimiento). La calidad de los cartuchos cada uno se puede comprobar mediante el análisis de la expresión de la proteína de 16 a 20 horas después del bombardeo. Nuestro protocolo de 6 cartuchos que produce rendimientos grupos multicelulares única expresión de &lt;3% de todos los eventos de expresión en este período de tiempo, por lo tanto adecuado para este experimento. 3) Entrega biolística del ADN-micropartículas recubiertas <ol><li> Quite las hojas de la misma etapa de desarrollo (es decir, con el mismo tamaño y edad, ver paso 1) con una navaja afilada e inmediatamente ponerlos a los lados abaxial hacia arriba sobre una superficie plana de espuma de poliestireno. El lado abaxial de la hoja representa un mejor sustrato para el bombardeo, debido a su baja densidad de tricomas y más delgada cutícula. Hojas de Arabidopsis, debido a su tamaño relativamente pequeño, debe ser cubierto con un pedazo de malla ventana de la pantalla, y la malla se fija con chinchetas a la superficie de espuma de poliestireno. El mantenimiento de hojas planas aumenta la eficiencia de la entrega de las partículas y reduce al mínimo el daño a los tejidos durante microbombardment. </li><li> Inserte el cartucho con el ADN de micropartículas recubiertas (preparado en el paso 1) en la pistola. De Arabidopsis, el rodaje se lleva a cabo a presiones de 80 a 110 psi para el 1-m micropartículas y 140-160 psi para micropartículas de 0,6 micras. Para N. benthamiana y el tabaco, el rodaje se lleva a cabo a presiones de 100 a 120 psi para el 1-m micropartículas y 160-180 psi de 0,6 micras micropartículas. De Arabidopsis, por lo general utilizan un cartucho por hoja, ya que cada hoja tiene superficie suficiente sólo para un disparo que se extiende micropartículas en un área de 10-12 mm de diámetro. Con mayor N. benthamiana y las hojas de tabaco, los bombardeos múltiples de la misma hoja son posibles, sin embargo, deben hacerse en las posiciones simétricas a cada lado de la nervadura de las áreas de destino de la hoja en la misma etapa de desarrollo (ver Discusión). </li><li> Quitar las hojas de la superficie de espuma de poliestireno y colocarlos ino una placa de Petri en 3 capas de papel de filtro Whatman mojado, sellar la placa de Petri con Parafilm y se incuban a temperatura ambiente durante 36-48 horas para permitir la expresión de los transgenes entrega y el potencial de célula a célula movimiento de sus productos proteicos . </li></ol> 4) Imagen de la expresión de la proteína La señal de fluorescencia de las proteínas expresadas de forma transitoria marcado es visualizado por microscopía confocal. Se debe tener cuidado para encontrar los ajustes óptimos de microscopía para la detección de cada proteína de la prueba. Por ejemplo, las proteínas que muestran la acumulación intracelular limitada con la intensidad de la señal es débil, como la localización de la proteína plasmodesmal del mosaico del tabaco movimiento del virus (TMV MP) 1-3,6, deben ser observados bajo un lente objetivo de 40X, lo que proporciona una mayor resolución y sensibilidad, mientras que las proteínas que muestran la distribución citoplasmática con la intensidad de señal fuerte, como libre de YFP, se puede visualizar con un lente objetivo de 10X con función de zoom de imagen confocal de más rápido (ver Figura 1). Transporte simplástica se deduce de la aparición de grupos multicelulares que contienen la señal fluorescente. El número de estas agrupaciones y el número de células en cada grupo es indicativo de la magnitud del transporte de célula a célula. Para obtener datos fiables, por lo menos 100 grupos de expresión deben ser registrados por cada sistema experimental. Por ejemplo, si el movimiento de célula a célula de una proteína se compara en dos fondos genéticos diferentes (por ejemplo, de tipo salvaje y las plantas transgénicas), de un total de 200 grupos de expresión deben ser registrados. Es importante destacar que, los experimentos, cuyos resultados deben ser comparados entre sí, deben llevarse a cabo de forma simultánea. 5. Resultados representante La siguiente figura ilustra experimentos representante para la detección de las proteínas de transporte simplástica marcado con fluorescencia. Los grupos A y B muestran las imágenes típicas que se obtienen confocal microbombardment siguiente de N. benthamiana con una hoja de TMV-MP-YFP expresar construir. En el panel A movimiento, simplásica de TMV MP-YFP se observa sobre la base de la aparición de grupos multicelulares de la señal de YFP. No todos transitoriamente expresó TMV MP-YFP es capaz de moverse como se evidencia por una sola célula de la señal en algunos microbombardments (panel B). Estadísticamente, en &lt;40% de los racimos de la señal en cuenta, TMV MP-YFP no es capaz de moverse entre las celdas, mientras que en&gt; 60% de los racimos, la proteína se mueve entre 2-5 células, con la difusión de dos células es el más frecuente (ca. 50% ~ de los casos) (fig. 1C). Las proteínas con un tamaño molecular relativamente pequeño, sin actividad de los movimientos innatos pueden difundirse a través de PD para pasar de célula a célula. Por ejemplo, YFP gratuita o 1xYFP (unos 27 kDa, panel D), los diferenciales entre varias células en el 30% de los grupos de contado (paneles C). Esta difusión no específica no está presente para un dimmer YFP traslación, o 2xYFP (54 kDa, panel E), que es comparable en tamaño a la proteína de fusión TMV MP-YFP (57 kDa), y que es completamente células autónomas (Figura 1C). <img src="/files/ftp_upload/2208/2208fig1.jpg" alt="Figura 1" /> Figura 1. Típicos resultados obtenidos en el ensayo de transporte simplástica en N. benthamiana hoja de tejidos. (A, B) La visualización de TMV MP-YFP. (C) La cuantificación de grupos de la señal. 1xYFP y 2xYFP, sin YFP y traslacional YFP dímero, respectivamente. (D) Visualización de 1xYFP. (E) Visualización de 2xYFP. En microfotografías, los paneles de la izquierda muestra la señal de YFP y los paneles de la derecha muestran imágenes fusionadas de YFP (en verde) y la autofluorescencia del cloroplasto (en blanco) señales. Las imágenes son solo las secciones confocal. Los asteriscos en las micrografías muestran las células epidérmicas que muestra la señal de YFP. Barras = 50 micras.

<ol>
	<li>Waigmann, E., Ueki, S., Trutnyeva, K., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ins+and+outs+of+non-destructive+cell-to-cell+and+systemic+movement+of+plant+viruses.+.">The ins and outs of non-destructive cell-to-cell and systemic movement of plant viruses. .</a> Crit Rev Plant Sci. 23, 195-250 (2004).</li><li>Lucas, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+viral+movement+proteins:+agents+for+cell-to-cell+trafficking+of+viral+genomes.">Plant viral movement proteins: agents for cell-to-cell trafficking of viral genomes.</a> Virology. 344, 169-184 (2006).</li><li>Epel, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+viruses+spread+by+diffusion+on+ER-associated+movement-protein-rafts+through+plasmodesmata+gated+by+viral+induced+host+beta-1,3-glucanases.">Plant viruses spread by diffusion on ER-associated movement-protein-rafts through plasmodesmata gated by viral induced host beta-1,3-glucanases.</a> Semin Cell Dev Biol. 20, 1074-1081 (2009).</li><li>Zambryski, P. C., Crawford, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodesmata:+gatekeepers+for+cell-to-cell+transport+of+developmental+signals+in+plants.">Plasmodesmata: gatekeepers for cell-to-cell transport of developmental signals in plants.</a> Annu Rev Cell Dev Biol. 16, 393-421 (2000).</li><li>Rivero-Lepinckas, L., Crist, D., Scholl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+plants+and+presercation+of+seeds.">Growth of plants and presercation of seeds.</a> Methods Mol Biol. 323, 3-12 (2006).</li><li>Ueki, S., Lacroix, B., Krichevsky, A., Lazarowitz, S. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple,+but+not+branched,+plasmodesmata+allow+the+nonspecific+trafficking+of+proteins+in+developing+tobacco+leaves.">Simple, but not branched, plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves.</a> Cell. 97, 743-754 (1999).</li><li>Imlau, A., Truernit, E., Sauer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+and+long-distance+trafficking+of+the+green+fluorescent+protein+in+the+phloem+and+symplastic+unloading+of+the+protein+into+sink+tissues.">Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues.</a> Plant Cell. 11, 309-322 (1999).</li><li>Gisel, A., Barella, S., Hempel, F. D., Zambryski, P. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-targeted+and+targeted+protein+movement+through+plasmodesmata+in+leaves+in+different+developmental+and+physiological+states.">Non-targeted and targeted protein movement through plasmodesmata in leaves in different developmental and physiological states.</a> Plant Physiol. 125, 1802-1812 (2001).</li><li>Roberts, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phloem+unloading+in+sink+leaves+of+Nicotiana+benthamiana:+comparison+of+a+fluorescent+solute+with+a+fluorescent+virus.">Phloem unloading in sink leaves of Nicotiana benthamiana: comparison of a fluorescent solute with a fluorescent virus.</a> Plant Cell. 9, 1381-1396 (1997).</li><li>Guenoune-Gelbart, D., Elbaum, M., Sagi, G., Levy, A., Epel, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+mosaic+virus+(TMV)+replicase+and+movement+protein+function+synergistically+in+facilitating+TMV+spread+by+lateral+diffusion+in+the+plasmodesmal+desmotubule+of+Nicotiana+benthamiana.">Tobacco mosaic virus (TMV) replicase and movement protein function synergistically in facilitating TMV spread by lateral diffusion in the plasmodesmal desmotubule of Nicotiana benthamiana.</a> Mol Plant Microbe Interact. 21, 335-345 (2008).</li></ol>

La clave para el éxito del ensayo de transporte simplástica es la obtención de la eficiencia de transformación de alto, que permite la producción de racimos de señal estadísticamente significativa y detectable fácilmente. Esto puede lograrse utilizando las hojas cosechadas de plantas sanas, robustas, y la preparación de partículas de oro recubiertas por una preparación de ADN puro y concentrado. Utilizando las hojas en la misma etapa de crecimiento también es vital para la fiabil...

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<td>Gold microparticles, 1.0 &mu;m in diameter</td>
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a cell-to-cell macromolecular transport assay in planta utilizing biolistic bombardment

A continuación, presentamos un protocolo sencillo y rápido para detectar y evaluar la magnitud de la célula a célula de transporte macromolecular en la planta. En este protocolo, marcado con fluorescencia de proteínas de interés se expresa de forma transitoria en el tejido vegetal después de la entrega biolística de su ADN que codifica para la construcción. La distribución intra e intercelulares de la proteína marcada se analiza por microscopía confocal. Se describe en detalle esta tecnología, ofreciendo paso a paso a protocolos de ensayo y evaluar la magnitud del transporte de proteínas simplástica en tres especies de plantas, Arabidopsis thaliana, Nicotiana benthamiana y N. tabacum (tabaco).

Watch this Scientific Journal Video about A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment at JoVE.com

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

El tráfico de macromoléculas entre las células vegetales se pueden evaluar de forma transitoria que expresa una proteína fluorescente con etiquetas de interés y el análisis de su distribución intra e intercelulares mediante microscopia confocal.

Una célula a célula macromoleculares de transporte de ensayo En la Planta La utilización de bombardeo biolística

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

a-cell-to-cell-macromolecular-transport-assay-planta-utilizing

Research

JoVE Journal

Biology

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

11.8K vistas.  State University of New York at Stony Brook. El tráfico de macromoléculas entre las células vegetales se pueden evaluar de forma transitoria que expresa una proteína fluorescente con etiquetas de interés y el análisis de su distribución intra e intercelulares mediante microscopia confocal.

Video: A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

Generación de transgénicos C. elegans Por la transformación biolística

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Investigación

Biología

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Recolección y enfriamiento Cryo-cristales de proteínas de membrana cultivadas en mesofases lipídicas para la Determinación de la estructura por cristalografía macromolecular

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

Análisis in vitro de PDZ-dependientes de complejos macromoleculares de señalización CFTR

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

El cultivo<em&gt; Caenorhabditis elegans</em &gt; En axénico Liquid Media y Creación de transgénicos Worms por bombardeo de micropartículas

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

Open Source de análisis de contenidos de alta Utilizando microscopía de fluorescencia automatizado de imágenes por vida

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Optimization of Crystal Growth for Neutron Macromolecular Crystallography

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX beamlines having been built and increased availability of structure refinement software. However, the neutron sources currently available for NMX are significantly weaker than equivalent sources for X-ray crystallography. Despite advances in this field, significantly larger crystals will always be required for neutron diffraction studies, particularly with the tendency to study ever-larger macromolecules and complexes. Further improvements in methods and instrumentation suited to growing larger crystals are therefore necessary for the use of NMX to expand.
In this work, we introduce rational strategies and a crystal growth bench (OptiCrys) developed in our laboratory that combines real-time observation through a microscope-mounted video camera with precise automated control of crystallization solutions (e.g., precipitant concentration, pH, additive, temperature). We then demonstrate how this control of temperature and chemical composition facilitates the search for optimal crystallization conditions using model soluble proteins. Thorough knowledge of the crystallization phase diagram is crucial for selecting the starting position and the kinetic path for any crystallization experiment. We show how a rational approach can control the size and number of crystals generated based on knowledge of multidimensional phase diagrams.

Structural studies of biomacromolecules by crystallography require high-quality crystals. Here we demonstrate a protocol that can be used by OptiCrys (a fully automated instrument developed in our lab) and/or microdialysis buttons for growing large high-quality crystals based on knowledge of the crystallization phase diagram.

Optimización del crecimiento del cristal para la cristalografía macromolecular de neutrones

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX ...

Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample chamber-based setups, which are specifically designed to preserve the three-dimensional integrity of the specimen and usually feature sample rotation, are the best choice in developmental biology. For instance, they have been used to document the entire embryonic morphogenesis of the fruit fly Drosophila melanogaster and the red flour beetle Tribolium castaneum. However, many available live imaging protocols provide only experimental frameworks for single embryos. Especially for comparative studies, such approaches are inconvenient, since sequentially imaged specimens are affected by ambient variance. Further, this limits the number of specimens that can be assayed within a given time. We provide an experimental framework for simultaneous live imaging that increases the throughput in sample chamber-based setups and thus ensures&#160;similar ambient conditions for all specimens. Firstly, we provide a calibration guideline for light sheet fluorescence microscopes. Secondly, we propose a mounting method for multiple embryos that is compatible with sample rotation. Thirdly, we provide exemplary three-dimensional live imaging datasets of Drosophila, for which we juxtapose three transgenic lines with fluorescently labeled nuclei, as well as of Tribolium, for which we compare the performance of three transgenic sublines that carry the same transgene, but at different genomic locations. Our protocol is specifically designed for comparative studies as it pro-actively addresses ambient variance, which is always present in sequential live imaging. This is especially important for quantitative analyses and characterization of aberrational phenotypes, which result e.g., from knockout experiments. Further, it increases the overall throughput, which is highly convenient when access to light sheet&#160;fluorescence microscopes is limited. Finally, the proposed mounting method can be adapted for other insect species and further model organisms, e.g., zebrafish, with basically no optimization effort.

Light sheet-based fluorescence microscopy is the most valuable tool in developmental biology. A major issue in comparative studies is ambient variance. Our protocol describes an experimental framework for simultaneous live imaging of multiple specimens and, therefore, addresses this issue pro-actively.

Imágenes simultáneas en vivo de múltiples embriones de insectos en microscopios de fluorescencia de hoja de luz basados en cámaras de muestra

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample ...

Investigación del mecanismo de transporte de zinc en células de mamíferos con el ensayo FluoZin-3

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

Autor destacado: Explorando la regulación celular del zinc a través de la funcionalidad de ZnT1 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

Caracterización de transportadores de zinc en mamíferos mediante un ensayo de transporte de zinc in vitro

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...