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>
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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>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2262</td>
<td>&nbsp;</td>
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<td>Gold microparticles, 0.6 &mu;m in diameter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2263</td>
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<td>Spermidine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0266-1G</td>
<td>&nbsp;</td>
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<td>Tefzel tubing</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2441</td>
<td>&nbsp;</td>
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<td>Helios cartridge preparatory station</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2420</td>
<td>&nbsp;</td>
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<td>Tubing cutter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2422</td>
<td>&nbsp;</td>
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<td>Helios gene gun</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2432</td>
<td>&nbsp;</td>
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<td>Helium gas regulator</td>
<td>&nbsp;</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

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

Preparación de balas de cañón de genes y transfección biolística de neuronas en cultivo Cortar

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

Investigación

Biología

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

Ventana en un micromundo: Sistemas sencillos de microfluidos para el estudio de transporte de microbios en medios porosos

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

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 ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Mediada por Agrobacterium inducida por el virus Gene ensayo silenciamiento en algodón

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

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 ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

Métodos de estudio de la función de proteínas de transporte epitelial y expresión en nativo Intestino y Caco-2 células cultivadas en 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 (VAMP7) by total internal reflection fluorescence microscopy (TIRFM) is a straightforward way to explore secretion from the lysosomal compartment. Taking advantage of cell culture on micropatterned surfaces to normalize cell shape, a variety of statistical tools were employed to perform a spatial analysis of secretory patterns. Using Ripley&#8217;s K function and a statistical test based on the nearest neighbor distance (NND), we confirmed that secretion from lysosomes is not a random process but shows significant clustering. Of note, our analysis revealed that exocytosis events are also clustered in nonadhesion areas, indicating that adhesion molecules are not the only structures that can induce secretory hot spots at the plasma membrane. Still, we found that cell adhesion enhances clustering. In addition to precisely defined adhesive and nonadhesive areas, the circular geometry of these micropatterns allows the use of polar coordinates, simplifying analyses. We used Kernel Density Estimation (KDE) and the cumulative distribution function on polar coordinates of exocytosis events to identify enriched areas of exocytosis. In ring-shaped micropattern cells, clustering occurred at the border between the adhesive and nonadhesive areas. Our analysis illustrates how statistical tools can be employed to investigate spatial distributions of diverse biological processes.

Live imaging of lysosomal exocytosis on micropatterned cells allows a spatial quantification of this process. Morphology normalization using micropatterns is an outstanding tool to uncover general rules about the spatial distribution of cellular processes.

Cuantificación de parámetros espaciotemporales de la exocitostosis celular en células micropatr babeadas

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 ...

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.

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

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