Notre travail est soutenu par des subventions du NIH / NIGMS, NSF, USDA / NIFA et BARD à VC.

Contexte Le transport des macromolécules symplastique par des connexions intercellulaires des plantes, les plasmodesmes, est d&#39;intérêt pour les pathologistes de nombreuses plantes et des biologistes. Par exemple, plusieurs protéines virales sont connues pour réguler plasmodesmal limites d&#39;exclusion de taille pour permettre un mouvement virale 1-3. En outre, certaines protéines endogènes, parmi eux d&#39;importants régulateurs du développement, sont supposés évoluer de cellule à cellule, vraisemblablement par plasmodesmes, à la fonction des cellules non-autonome 4. Ainsi, une méthodologie fiable pour identifier et visualiser transport macromoléculaire entre les cellules végétales est très en demande. 1) Cultiver les plantes cibles Pour une efficacité de transformation élevé, sain, plantes robustes devraient être utilisés. Des plantes d&#39;Arabidopsis 1. Cultiver une plante Arabidopsis sur le Pro-Mix BX dans un pot (10 cm x 10 cm x 10 cm) dans une chambre à environnement contrôlé avec une photopériode courte (8 heures de 130-150 uE m -2 s -1 à 23 ° de lumière C/16 h obscurité à 20 ° C) et humidité relative de 40 à 65% 6 à 8 semaines 5. Les fertiliser parfois avec des produits disponibles dans le commerce tel que décrit 5. Feuilles avec la plus grande taille de 15 mm x 35 mm (la mesure de la longueur du pétiole comprend) sont sélectionnés pour les expériences. 2. N. benthamiana et N. tabacum Cultivez une usine sur le Pro-Mix BX dans un pot (20 cm x 20 cm x 20 cm) dans une chambre à environnement contrôlé avec une photopériode longue (16 heures de 130-150 uE m -2 s -1 légers à 23 ° C / 8 h obscurité à 20 ° C) et humidité relative de 40-65% 7 à 10 semaines. Les fertiliser parfois avec des produits disponibles dans le commerce que de décrire. Feuilles de taille supérieure à 50 mm x 70 mm pour N. benthamiana mm ou 100 x 125 mm pour N. tabacum (ces mesures ne comprennent pas la longueur du pétiole) sont sélectionnés pour les expériences. 2) Préparation de la cartouche de pistolet à gènes enrobées d&#39;ADN Or microparticules Le protocole de cette démarche expérimentale a été décrite en détail précédemment 6. Il est très important de bien l&#39;utiliser ADN plasmidique purifié à des concentrations élevées (~ 1 ug / ul) pour obtenir le meilleur rendement de transformation de la facilité d&#39;analyse par microscopie confocale pendant les derniers stades de l&#39;expérimentation. Pour ce dosage, il est important de s&#39;assurer que la cartouche ne se transforme pas préparés deux ou plusieurs cellules adjacentes simultanément à haute fréquence, car le nombre de cellules avec un cluster associant signal fluorescent est utilisé comme un indicateur de l&#39;étendue des transports symplastique (ie , une seule cellule contenant le signal indique l&#39;absence de mouvement, alors que le signal d&#39;une grappe multicellulaire indique le mouvement). La qualité de la cartouche de chacun peut être vérifiée par l&#39;analyse de l&#39;expression de la protéine de 16 à 20 heures après le bombardement. Notre protocole de 6 cartouches produit que le rendement des grappes d&#39;expression multicellulaire seulement dans &lt;3% de tous les événements d&#39;expression dans ce laps de temps, donc adaptée à cette expérience. 3) La livraison de l&#39;ADN Biolistic microparticules tapissées <ol><li> Retirez les feuilles de la même étape de développement (c&#39;est à dire avec la taille et de même âge, voir étape 1) avec une lame de rasoir et les placent immédiatement avec les côtés abaxiale face sur une surface plane en styromousse. Le côté abaxiale de la feuille représente un meilleur substrat pour le bombardement en raison de sa faible densité de trichomes et plus mince cuticule. Feuilles d&#39;Arabidopsis, en raison de leur taille relativement petite, devrait être couvert avec un morceau de grillage moustiquaire, et le maillage, puis fixé avec des punaises à la surface de styromousse. Maintenir feuilles plates augmente l&#39;efficacité de la livraison de particules et réduit les dommages aux tissus au cours microbombardment. </li><li> Insérez la cartouche avec de l&#39;ADN microparticules tapissées (préparé à l&#39;étape 1) dans le pistolet. Pour Arabidopsis, le tir est effectué à des pressions de 80-110 psi pour le 1-um microparticules, et 140 à 160 psi pour 0,6 um microparticules. Pour N. benthamiana et le tabac, le tir est effectué à des pressions de 100-120 psi pour le 1-um microparticules, et 160 à 180 psi pour 0,6 um microparticules. Pour Arabidopsis, nous avons l&#39;habitude d&#39;utiliser une cartouche par feuille, parce que chaque feuille a une surface suffisante pour un seul coup de feu qui se propage microparticules sur une superficie de 10-12 mm de diamètre. Avec de plus grandes N. benthamiana et les feuilles de tabac, les bombardements multiples de la même feuille sont possibles, cependant, ils doivent être fait à des positions symétriques de chaque côté de la nervure centrale pour cibler les zones de feuilles au même stade de développement (voir discussion). </li><li> Retirer les feuilles de la surface de styromousse et placez-les ipas une boîte de Pétri sur 3 couches de papier filtre Whatman mouillés, sceller la boîte de Pétri avec du Parafilm, et il incuber à température ambiante pendant 36-48 heures pour permettre l&#39;expression des transgènes livré et le potentiel de cellule à cellule-circulation de leurs produits protéiques . </li></ol> 4) Imagerie d&#39;expression de protéines Le signal de fluorescence des protéines exprimées transitoirement étiqueté est visualisé par microscopie confocale. Des précautions doivent être prises pour trouver les réglages optimaux pour la détection de la microscopie de chaque protéine testée. Par exemple, les protéines qui montrent limitée accumulation intracellulaire avec des intensités de signal faible, tels que la localisation de la protéine plasmodesmal la mosaïque du tabac mouvement du virus (TMV MP) 1-3,6, doit être observé sous un objectif de 40X, ce qui offre une meilleure résolution et sensibilité, tandis que les protéines qui montrent la distribution cytoplasmique avec des intensités signal fort, comme sans YFP, peuvent être visualisées sous un objectif 10X avec fonction zoom confocal pour l&#39;imagerie rapide (voir Figure 1). Transports symplastique est déduite de l&#39;apparition de grappes multicellulaire qui contiennent le signal fluorescent. Le nombre de ces pôles et le nombre de cellules dans chaque grappe est révélatrice de l&#39;ampleur de la cellule à cellule de transport. Pour obtenir des données fiables, au moins 100 groupes d&#39;expression doivent être enregistrées par chaque système expérimental. Par exemple, si le mouvement de cellule à cellule d&#39;une protéine est comparée dans deux contextes génétiques différents (par exemple, de type sauvage et des plantes transgéniques), d&#39;un total de 200 grappes d&#39;expression doivent être enregistrées. Surtout, des expériences, dont les résultats doivent être comparés les uns aux autres, doivent être menées simultanément. 5. Les résultats représentatifs La figure ci-dessous illustre expériences représentatives pour la détection du transport des protéines par fluorescence symplastique marqués. Les panneaux A et B montrent les images typiques confocale qui sont obtenus à la suite d&#39;une microbombardment N. feuilles benthamiana avec une TMV MP-YFP-exprimant la construction. Dans le panneau, un mouvement, symplastique de TMV MP-YFP est observée basée sur l&#39;apparition de grappes multicellulaire du signal YFP. Pas tous transitoirement exprimé TMV MP-YFP est capable de se déplacer comme en témoigne une seule cellule du signal dans certains microbombardments (panneau B). Statistiquement, dans &lt;40% de l&#39;amas de signal compté, TMV MP-YFP est incapable de se déplacer entre les cellules alors que dans&gt; 60% des grappes, la protéine se déplace entre 2-5 cellules, avec la diffusion de deux cellules étant les plus fréquents (ca. ~ 50% des cas) (figure 1C). Les protéines avec la taille moléculaire relativement faible sans activité mouvement inné peuvent diffuser à travers PD de se déplacer de cellule à cellule. Par exemple, YFP libre, ou 1xYFP (environ 27 kDa, partie D), les écarts entre plusieurs cellules dans 30% des glomérules comptés (panneaux C). Cette diffusion non-spécifique n&#39;a pas lieu pour un gradateur YFP translationnelle, ou 2xYFP (54 kDa, panneau E), ce qui est comparable en taille à la protéine de fusion TMV MP-YFP (57 kDa), et qui est complètement cellulaire autonome (figure 1C). <img src="/files/ftp_upload/2208/2208fig1.jpg" alt="Figure 1" /> Résultats de la figure 1. Typiques obtenus à partir de l&#39;essai de transport symplastique dans N. benthamiana tissus foliaires. (A, B) Visualisation de TMV MP-YFP. (C) Quantification des pôles du signal. 1xYFP et 2xYFP, sans YFP et translationnelle YFP dimère, respectivement. (D) Visualisation des 1xYFP. (E) Visualisation des 2xYFP. En micrographies, les panneaux de gauche montrent le signal YFP et les panneaux de droite montrent des images fusionnées YFP (en vert) et autofluorescence chloroplaste (en blanc) des signaux. Les images sont simples sections confocale. Les astérisques dans les micrographies montrent les cellules de l&#39;épiderme montrant le signal YFP. Bars = 50 um.

<ol>
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La clé de la réussite de l&#39;essai de transport symplastique est d&#39;obtenir une efficacité de transformation élevé, ce qui permet la production de grappes de signal statistiquement significative et facilement détectable. Ceci peut être réalisé en utilisant les feuilles récoltées à partir plants sains et robustes, et la préparation de particules d&#39;or recouvertes par une préparation d&#39;ADN pur et concentré. Utiliser les feuilles au même stade de croissance est égal...

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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>
<td>&nbsp;</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

Ici, nous présentons un protocole simple et rapide pour détecter et évaluer l&#39;étendue de la cellule à cellule transport macromoléculaire in planta. Dans ce protocole, une fluorescence taggés-protéine d&#39;intérêt est transitoirement exprimé dans les tissus végétaux suivant la livraison de ses biolistique d&#39;ADN codant pour construire. La distribution intra-et intercellulaires de la protéine marquée est ensuite analysée par microscopie confocale. Nous décrivons en détail cette technologie, offrant, étape par étape, des protocoles d&#39;essai et d&#39;évaluer l&#39;ampleur du transport des protéines symplastique en trois espèces de plantes, Arabidopsis thaliana, Nicotiana benthamiana et N. tabacum (tabac).

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

Le trafic entre les cellules végétales macromoléculaire peut être évaluée par transitoirement exprimant une protéine fluorescente-marqués d&#39;intérêt et d&#39;analyser sa distribution intra-et intercellulaires par microscopie confocale.

Une cellule à cellule essai de transport macromoléculaire In planta Utilisant bombardement biolistique

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 ...

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Research

JoVE Journal

Biology

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

11.8K vues.  State University of New York at Stony Brook. Le trafic entre les cellules végétales macromoléculaire peut être évaluée par transitoirement exprimant une protéine fluorescente-marqués d'intérêt et d'analyser sa distribution intra-et intercellulaires par microscopie confocale.

Vidéo: 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.

Génération de souris transgéniques C. elegans Par transformation biolistique

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 ...

Recherche

Biologie

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.

Cristaux de récolte et de Cryo-refroidissement de protéines membranaires cultivées dans des mésophases lipidiques pour la détermination de la structure par cristallographie macromoléculaire

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.

L&#39;analyse in vitro de PDZ-dépendants complexes macromoléculaires CFTR de signalisation

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.

Culture<em&gt; Caenorhabditis elegans</em &gt; En Axenic Liquid Media et création d&#39;transgéniques Worms par bombardement de microparticules

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 Content Haute Analyse Utilisant automatisé Fluorescence Lifetime Imaging Microscopy

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.

Optimisation de la croissance cristalline pour la cristallographie macromoléculaire neutronique

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.

Imagerie en direct simultanée d’embryons d’insectes multiples dans des microscopes à fluorescence à feuille de lumière à base de chambre d’échantillonnage

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

Étude du mécanisme de transport du zinc dans les cellules de mammifères avec le dosage de 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.

Pleins feux sur l’auteur : Explorer la régulation cellulaire du zinc grâce à la fonctionnalité 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.

Caractérisation des transporteurs de zinc chez les mammifères à l’aide d’un test in vitro de transport du zinc

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