1. Préparation des solutions et des matériaux Ces étapes doivent être réalisées plusieurs jours à l&#39;avance. <ol><li> Lamelles de verre. Lamelles ont été nettoyés par immersion pendant 10 minutes dans une solution de détergent Linbro 7X: l&#39;eau, mélangée à un 1: 3 et chauffée, sous agitation, jusqu&#39;à ce qu&#39;il dégage. Lamelles ont été rincés abondamment à l&#39;eau déminéralisée, puis cuite au four à 450 ° C pendant 6 heures. Chargement des lamelles en racks coloration céramique (voir réactifs) ont simplifié ce processus. </li><li> Solution de protéines pour l&#39;estampage. Fibronectine Reconstituer en suivant les instructions des fabricants d&#39;une solution stock de 1 mg / ml de concentration. </li></ol> 2. Cast timbres du maître topologique Ces étapes peuvent être réalisées plusieurs jours à l&#39;avance. Timbres magasin pattern-up dans un plat couvert, comme une boîte de culture tissulaire. <ol><li> Enlever la poussière détacher de maître en utilisant un flux de comprimé, air filtré ou gaz inerte. </li><li> Placez le maître, côté à motifs en place, dans le fond d&#39;un plat en plastique juste plus grand que le maître. 60 - ou 100-mm boîtes de culture tissulaire sont bien adaptés à cet effet. </li><li> Dans un tube à centrifuger en polystyrène de 50 ml, mélanger les composants Sylgard dans un rapport de l&#39;agent de durcissement: la base d&#39;élastomère de 1: 10 en poids. Mélanger soigneusement en utilisant un dispositif en plastique, comme une pipette jetable. Préparer au moins 0,2 ml d&#39;élastomère par centimètre carré de surface plat. </li><li> Centrifuger le tube de 50 ml à 300 g pendant 5 min pour éliminer les bulles d&#39;air. </li><li> Verser l&#39;élastomère sur le maître, puis placer dans un dessiccateur sous vide, pendant 30 minutes. </li><li> Cure de l&#39;élastomère dans un four à 65 ° C pendant au moins 2 heures. Durcissement à température élevée et pour des résultats parfois plus en plus rigide élastomère. Laissez refroidir à température ambiante. </li><li> Séparer la feuille de timbres du maître. </li></ol> 3. Impression par microcontact de la fibronectine sur le verre <ol><li> Découpez un timbre unique. Timbres mesurant 4 mm x 4 mm à 1 cm X 1 cm dans la zone et 1 - 2 mm d&#39;épaisseur sont les plus faciles pour commencer. Côté tendance Placer sur une lame de verre ou un plat en plastique. </li><li> Placez timbre dans un nettoyeur à plasma, et le processus, sous vide, pendant 30 secondes. Un Harrick scientifique Plasma cleaner (voir équipement), fixé à son cadre le plus élevé de sortie sera de rendre la surface hydrophile PDMS. Plus conséquent fois dans la fissuration de l&#39;élastomère. </li><li> Diluer la solution de fibronectine à l&#39;eau déminéralisée à une concentration d&#39;emboutissage de 50 pg / ml. </li><li> Placer une petite goutte (10 - 50 pi) de l&#39;estampage solution sur le timbre. Elle se propage à travers la surface hydrophile. Ajoutez seulement assez de solution que la chute couvre le timbre, mais ne fonctionne pas sur les bords. Laissez absorber des protéines pour éradiquer pendant 5 minutes. </li><li> L&#39;utilisation d&#39;un Kimwipe ® ou d&#39;autres tissus de papier propre, mèche pied la plupart de la solution de protéines à partir du timbre, sans toucher la région à motifs. </li><li> Sécher la solution restante de la marque sous un courant d&#39;eau propre, sec, gaz inerte, tel que l&#39;azote. </li><li> En utilisant une pince à épiler, retirez le tampon de la lame de verre, inverser, et mettre en contact avec la lamelle de verre nettoyée (la surface à motifs). Placez un poids dessus pour promouvoir un bon contact. Le poids spécifique qui fournit la meilleure structuration dépend de la taille d&#39;un timbre et le modèle; commencer avec un poids de 5 g, et d&#39;ajuster entre estampées. Laisser timbre en contact avec la surface pendant 1 minutes. </li><li> Soigneusement démonter la pile, et le cachet distinct de lamelle. </li><li> Vigoureusement rincer la lamelle à motifs en PBS, puis de l&#39;eau déminéralisée, pour éliminer les protéines qui ne sont pas adsorbés à la surface. Lamelle à sec sous un courant d&#39;azote. </li></ol> Les résultats représentatifs Impression par microcontact est un puissant processus de molécules sur les surfaces de motifs. Ce processus a la possibilité de créer des fonctions avec des dimensions allant de quelques dizaines de micromètres à quelques centaines de nanomètres; dans la Fig. 1A, les logos sur la gauche sont chacun 200 um de hauteur, tandis que les taches vertes illustré dans la figure. 1B sont une um de diamètre et espacés à intervalles de 4 um, mesurée de centre à centre. Fig. 1B illustre également une propriété de la puissante impression par microcontact, à savoir qu&#39;il est et de processus additif. Plusieurs séries d&#39;impression par microcontact peut être appliquée à une surface unique de créer des systèmes multicomposants 3. <img src="/files/ftp_upload/old/1065_2008_11_24_3/Figure_a.jpg" alt="Figure A" /><img src="/files/ftp_upload/old/1065_2008_11_24_4/FIGURE_B.jpg" alt="Figure B" />

<ol>
	<li>Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+control+of+cell+life+and+death.">Geometric control of cell life and death.</a> Science. 276, 1425-1425 (1997).</li><li>Kumar, A., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+Gold+Having+Micrometer+to+Centimeter+Dimensions+can+be+Formed+Through+a+Combination+of+Stamping+with+an+Elastomeric+Stamp+and+an+Alkanethiol+"Ink"+Followed+by+Chemical+Etching.">Features of Gold Having Micrometer to Centimeter Dimensions can be Formed Through a Combination of Stamping with an Elastomeric Stamp and an Alkanethiol "Ink" Followed by Chemical Etching.</a> Applied Physics Letters. 63, 4-4 (1993).</li><li>St. John, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+Glial+Cell+Attachment+to+Microcontact-printed+Surfaces.">Preferential Glial Cell Attachment to Microcontact-printed Surfaces.</a> Journal of Neuroscience Methods. 75, 171-171 (1997).</li><li>Kam, L., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+to+protein-micropatterned-supported+lipid+bilayer+membranes.">Cell adhesion to protein-micropatterned-supported lipid bilayer membranes.</a> Journal of Biomedical Materials Research. 55, 487-487 (2001).</li><li>Kung, L. A., Kam, L., Hovis, J. S., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+Hybrid+Surfaces+of+Proteins+and+Supported+Lipid+Bilayers.">Patterning Hybrid Surfaces of Proteins and Supported Lipid Bilayers.</a> Langmuir. 16, 6773-6773 (2000).</li><li>Shen, K., Thomas, V. K., Dustin, M. L., Kam, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+of+costimulatory+ligands+enhances+CD4++T+cell+function.">Micropatterning of costimulatory ligands enhances CD4+ T cell function.</a> Proceedings of the National Academy of Sciences. 105, 7791-7791 (2008).</li><li>Shi, P., Shen, K., Kam, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+presentation+of+L1+and+N-cadherin+in+multicomponent,+microscale+patterns+differentially+direct+neuron+function+in+vitro.">Local presentation of L1 and N-cadherin in multicomponent, microscale patterns differentially direct neuron function in vitro.</a> Developmental Neurobiology. 67, 1765-1765 (2007).</li></ol>

The authors have nothing to disclose.

Le processus d&#39;impression par microcontact est conceptuellement simple et très robuste, ayant été appliqué à la structuration un large éventail de molécules sur une variété de substrats. Cependant, ce processus reste quelque chose d&#39;un art. La géométrie spécifique de la structure à créer, protéine à motifs, le poids appliqué, et le revêtement / conditions d&#39;emboutissage tous affecter la qualité d&#39;emboutissage. Par exemple, trop peu de poids, appliquée à grands traits, aboutit souvent à des lacunes dans ...

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		<th>Catalogue Number</th>
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<td>Plasma Cleaner</td>
<td>&nbsp;</td>
<td>Harrick Scientific Products, Inc.</td>
<td>PDC-32G</td>
<td>&nbsp;</td>
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<td>Desiccator</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>5315-0150</td>
<td>&nbsp;</td>
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<td>PBS</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>10010-072</td>
<td>&nbsp;</td>
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<td>Protein labeling kit</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>A30006</td>
<td>&nbsp;</td>
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<td>Fibronectin </td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F2006</td>
<td>&nbsp;</td>
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<td>Staining rack</td>
<td>Reagent</td>
<td>Thomas Scientific</td>
<td>8542E40</td>
<td>&nbsp;</td>
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<td>Coverslips</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>12-544-12</td>
<td>&nbsp;</td>
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<td>Sylgard 184</td>
<td>Reagent</td>
<td>Ellsworth Adhesives</td>
<td>184 Sil Elast Kit</td>
<td>&nbsp;</td>
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<td>Diffraction Grating</td>
<td>Reagent</td>
<td>Edmund Scientific</td>
<td>3040267</td>
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microcontact printing of proteins for cell biology

La capacité de protéines modèle et d&#39;autres biomolécules sur des substrats est important pour saisir la complexité spatiale de l&#39;environnement extracellulaire. Développement de l&#39;impression par microcontact par le groupe Whitesides (<a href="http://gmwgroup.harvard.edu/"&gt; Http://gmwgroup.harvard.edu/</a&gt;) Dans le milieu des années 1990 a révolutionné ce domaine en faisant de la microélectronique / techniques de microfabrication accessibles aux laboratoires axés sur les sciences de la vie. Implémentations initiales de cette méthode utilisée polydiméthylsiloxane (PDMS) timbres pour créer des modèles de produits chimiques sur des surfaces fonctionnalisées matériels<sup&gt; 1</sup&gt;. Depuis lors, une série d&#39;approches innovantes ont été développées pour les molécules autre motif, y compris les protéines<sup&gt; 2</sup&gt;. Cette vidéo montre le processus de base de création des timbres de PDMS et les utilise pour des protéines motif, que ces mesures sont difficiles à exprimer avec précision des mots. Nous nous concentrons sur la structuration de la fibronectine de la matrice extracellulaire de protéines sur des lamelles de verre comme un exemple précis de structuration. Un élément important du processus d&#39;impression par microcontact est un maître topologique, à partir de laquelle les timbres sont exprimés; les régions soulevé et abaissé du maître sont reflétées dans le timbre et définissent le modèle final. Typiquement, un maître est constitué d&#39;un wafer de silicium recouvert de résine photosensible et ensuite modelé par photolithographie, comme c&#39;est le cas ici. Création de master comportant un modèle spécifique nécessite un équipement spécialisé, et il est préférable d&#39;aborder en consultation avec un centre de fabrication ou de l&#39;installation. Cependant, presque tout substrat avec la topologie peut être utilisé comme un maître, comme des réseaux de diffraction en plastique (voir Réactifs pour un exemple), et ces maîtres fortuite de fournir facilement disponibles, les modèles simples. Ce protocole commence au point d&#39;avoir un maître dans la main.

Watch this Scientific Journal Video about Microcontact Printing of Proteins for Cell Biology at JoVE.com

Microcontact Printing of Proteins for Cell Biology

Impression par microcontact est largement utilisé pour les protéines et autres molécules tendance sur les surfaces de matériaux. Nous démontrons les étapes de base de ce processus, l&#39;estampage modèles de la fibronectine sur le verre.

Impression par microcontact de protéines pour la biologie cellulaire

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

microcontact-printing-of-proteins-for-cell-biology

Research

JoVE Journal

Biology

20.0K vues.  Columbia University. Impression par microcontact est largement utilisé pour les protéines et autres molécules tendance sur les surfaces de matériaux. Nous démontrons les étapes de base de ce processus, l'estampage modèles de la fibronectine sur le verre.

Vidéo: Microcontact Printing of Proteins for Cell Biology

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

Un dispositif microfluidique Dégradé génératrices de biologie cellulaire

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Recherche

Biologie

Micro-scale Engineering for Cell Biology

Micro-échelle d&#39;ingénierie de biologie cellulaire

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye  DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan  DIGE technology and analysis of protein expression changes using DeCyder  2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan&#x2122; DIGE technology.

Étiquetage sélective des protéines de surface cellulaire utilisant CyDye DIGE Fluor Colorants Minimal

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of ...

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

L&#39;évaluation des essais de cristallisation bidimensionnelle de protéines membranaires petites pour les études en biologie structurale par cristallographie électronique

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

Marquage fluorescent du COS-7 exprimant des protéines de fusion SNAP-tag pour imagerie de cellules vivantes

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Pull-down of Calmodulin-binding Proteins

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca2+ (Ca2+-CaM) as opposed to its Ca2+-free state (ApoCaM)3.
While most target proteins bind Ca2+-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin4, neurogranin (Ng)5, and certain myosins6. These proteins have been shown to play important roles in presynaptic function7, postsynaptic function8, and muscle contraction9, respectively. Their ability to bind and release CaM in the absence or presence of Ca2+ is pivotal in their function. In contrast, many proteins only bind Ca2+-CaM and require this binding for their activation. Examples include myosin light chain kinase10, Ca2+/CaM-dependent kinases (CaMKs)11 and phosphatases (e.g. calcineurin)12, and spectrin kinase13, which have a variety of direct and downstream effects14.
The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca2+-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca2+-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.
Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca2+, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca2+ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca2+/CaM signaling and proteins that interact with CaM.

Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

Pull-down des protéines se liant à la calmoduline

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Mise à jour de levure-système à deux hybrides pour identifier les protéines qui interagissent avec le facteur de croissance progranuline

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Deux et trois dimensions imagerie de cellules vivantes de protéines endommagent l&#39;ADN de réponse

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Suivi cellule à l&#39;aide protéines photoconvertible cours de développement poisson zèbre

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

L&#39;utilisation de chémostats dans microbienne biologie des systèmes

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Impression par microcontact de protéines pour la biologie cellulaire

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Un dispositif microfluidique Dégradé génératrices de biologie cellulaire

Micro-échelle d'ingénierie de biologie cellulaire

Étiquetage sélective des protéines de surface cellulaire utilisant CyDye DIGE Fluor Colorants Minimal

L'évaluation des essais de cristallisation bidimensionnelle de protéines membranaires petites pour les études en biologie structurale par cristallographie électronique

Marquage fluorescent du COS-7 exprimant des protéines de fusion SNAP-tag pour imagerie de cellules vivantes

Pull-down des protéines se liant à la calmoduline

Mise à jour de levure-système à deux hybrides pour identifier les protéines qui interagissent avec le facteur de croissance progranuline

Deux et trois dimensions imagerie de cellules vivantes de protéines endommagent l'ADN de réponse

Suivi cellule à l'aide protéines photoconvertible cours de développement poisson zèbre

L'utilisation de chémostats dans microbienne biologie des systèmes