Département de la Défense W81XWH-05-1-0005, W81XWH-06-1-0495, W81XWH-08-1-0029 et W81XWH-08-1-0356

1. Préparer la TPS protéine de fusion <ol><li> Stocks de glycérol de bactéries compétentes JM109 contenant le premier N-terminale 90 acides aminés du domaine rhotekin Rho contraignant (RBD) sous-cloné dans le vecteur BamH1/EcoR1 pGEX3x sont faites. Garantir un rendement élevé les étapes suivantes doivent être effectuées pour chaque expérience. Dans notre expérience, fraîchement préparé TPS-RBD est la clé à un test d&#39;activation GTPase robuste et précis. </li><li> Ajouter 50 uL (approximatif, ne pas décongeler) du stock de glycérol à 50 mL de LB ampères et de croître à 37 ° C pendant la nuit avec agitation. </li><li> Diluer à 1:10 dans 500 ml LB-amp et de grandir pendant 1 h. </li><li> Provoquer la TPS production de protéines avec 0,1 mM d&#39;IPTG pendant 2 heures. </li><li> Répartir uniformément dans les bouteilles à centrifuger et centrifuger, Sorval GSA3 rotor à 5000 tours à 4 ° C pendant 20 min. </li><li> Resuspendre le culot dans des bouteilles de centrifugeuse, 10 ml de tampon de lyse *. * Tampon de lyse bactérienne: 20% de saccharose, glycérol 10%, 50 mM Tris pH 8,0, 0,2 mM Na 2 S 2 O 5, 2 mM MgCl2, 2 mM de DTT, Ajouter fraîche PMSF, benzamide, l&#39;aprotinine et leupeptine. </li><li> Soniquer 2-3 min (cela dépend du type de sonicateur. Nous utilisons un modèle de Fischer Sonic Dismembranator 500 fixé à la marque 6, cycle de 50%). </li><li> Centrifugeuse, Sorvall SS34, 20 min 10 000 rpm à 4 ° C. A ce point une petite aliquote peut être souscrite et gérée par SDS-PAGE. Le gel peut être coloré au bleu de Coomassie et d&#39;une bande doit être apparente à 46 kDa. </li><li> Enlever le surnageant et ajouter 1 mL de 50% glutatione-Sépharose 4B boue. </li><li> Incuber pendant 30 min à 4 ° C avec rotation. Laver 3 fois avec un tampon de lyse. </li><li> Remettre les billes GST-RBD/glutatione-Sepharose à une suspension de 50% (environ 1 mL) avec la TPS poissons tampon **. ** La TPS-Fish Tampon: 10% glycérol, 50 mM Tris pH 7,4, NaCl 100 mM, 1% NP-4, 2 mM MgCl 2, Ajouter fraîche PMSF, benzamide, l&#39;aprotinine et leupeptine </li></ol> 2. Fusion GST pull down test <ol><li> Utilisez 5-10 x 10 6 cellules / test </li><li> Laver les cellules une fois en PBS glacé, gardez les plaques sur la glace (nous utilisons un plat en verre plat rempli de glace) et Lyse avec 0,5 mL TPS poissons tampon. </li><li> Incuber 5 min sur la glace, les cellules de récolte à l&#39;aide d&#39;un policier en caoutchouc ou scrapper plat, transfert à microcentrifugeuse tube et centrifuger 5 min 20000 rpm à 4 ° C. </li><li> Transférer le surnageant dans un tube de micro frais. Prenez 50 ul pour déterminer totale Rho (c&#39;est à dire le PIB + GTP lié). </li><li> Ajouter 0,5 ml GST-RBD/glutatione-Sepharose perles et incuber à 4 ° C, nuit à la rotation. </li><li> Lavez 6x avec TPS-poissons tampon. </li><li> Remettre en suspension dans 40 perles tampon d&#39;échantillon 2x-uL Laemelli À ce stade de la concentration de l&#39;aliquote Rho protéines totales prise en 2.4 devrait être déterminée et 30 ug préparé pour être exécuté sur le gel avec l&#39;échantillon d&#39;activation correspondant. </li><li> Faire bouillir les échantillons à 90 ° C pendant 5 min, centrifuger brièvement et prendre surnageant (c&#39;est à dire éviter les perles) charger et d&#39;exécuter sur une pente de 12 puits 4-20% gel SDS-PAGE. </li><li> Exécuter un gel sans volts supérieure à 150, le transfert à la membrane en utilisant du tampon Towbin et constante de courant de 100 W. </li><li> Bloc membrane avec 5% de lait / TBST solution et sonde avec RhoC-specfic anticorps. </li></ol> 3. Les résultats représentatifs De bons résultats devraient produire une bande unique à environ 22 kDa. Mauvais résultats de produire des bandes multiples ou bruit de fond élevé. Ceci est révélateur de la dégradation des protéines, le lavage incomplet des échantillons ou l&#39;utilisation de l&#39;ancienne TPS-RBD. <img src="/files/ftp_upload/2083/2083fig1.jpg" alt="Figure 1" /> Figure 1. Trois lignées cellulaires séparées exprimant différents niveaux de RhoC GTPase sont présentés pour démontrer de faibles niveaux de RhoC actif et total, des niveaux élevés de RhoC active et totale ou ne RhoC. Le blot a ensuite été déshabillé et sondé avec un anticorps de la gène de l&#39;actine maison gardant pour servir de contrôler le chargement de la protéine RhoC total. <img src="/files/ftp_upload/2083/2083fig2.jpg" alt="Figure 2" /> Figure 2. Bandes multiples ou les résultats de fond élevé de dégradation des protéines, le lavage incomplet des échantillons ou l&#39;utilisation de l&#39;ancienne TPS-RBD.

<ol>
	<li>van Golen, K. L., Wu, Z. F., Qiao, X. T., Bao, L. W., Merajver, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase,+a+novel+transforming+oncogene+for+human+mammary+epithelial+cells+that+partially+recapitulates+the+inflammatory+breast+cancer+phenotype.">RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype.</a> Cancer Res. 60 (20), 5832-5838 (2000).</li><li>van Golen, K. L., Wu, Z. F., Qiao, X. T., Bao, L., Merajver, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase+overexpression+modulates+induction+of+angiogenic+factors+in+breast+cells.">RhoC GTPase overexpression modulates induction of angiogenic factors in breast cells.</a> Neoplasia. 2 (5), 418-425 (2000).</li><li>Golen, K. L. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversion+of+RhoC+GTPase-induced+inflammatory+breast+cancer+phenotype+by+treatment+with+a+farnesyl+transferase+inhibitor.">Reversion of RhoC GTPase-induced inflammatory breast cancer phenotype by treatment with a farnesyl transferase inhibitor.</a> Mol Cancer Ther. 1 (8), 575-583 (2002).</li><li>Golen, K. L. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen+activated+protein+kinase+pathway+is+involved+in+RhoC+GTPase+induced+motility,+invasion+and+angiogenesis+in+inflammatory+breast+cancer.">Mitogen activated protein kinase pathway is involved in RhoC GTPase induced motility, invasion and angiogenesis in inflammatory breast cancer.</a> Clin Exp Metastasis. 19 (4), 301-311 (2002).</li><li>Yao, H., Dashner, E. J., van Golen, C. M., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase+is+required+for+PC-3+prostate+cancer+cell+invasion+but+not+motility.">RhoC GTPase is required for PC-3 prostate cancer cell invasion but not motility.</a> Oncogene. 25 (16), 2285-2296 (2006).</li><li>Hall, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+Collagen+receptor+(alpha2beta1)+signaling+promotes+prostate+cancer+cell+invasion+through+RhoC+GTPase.">Type I Collagen receptor (alpha2beta1) signaling promotes prostate cancer cell invasion through RhoC GTPase.</a> Neoplasia. 10 (8), 797-803 (2008).</li><li>Sequeira, L., Dubyk, C. W., Riesenberger, T. A., Cooper, C. R., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+GTPases+in+PC-3+prostate+cancer+cell+morphology,+invasion+and+tumor+cell+diapadesis.">Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell diapadesis.</a> Clin Exp Metastasis. 25, 569-579 (2008).</li><li>van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL2+Induces+Prostate+Cancer+Transendothelial+Cell+Migration+via+Activation+of+the+Small+GTPase+Rac.">CCL2 Induces Prostate Cancer Transendothelial Cell Migration via Activation of the Small GTPase Rac.</a> J Cell Biochem. 104, 1587-1597 (2008).</li><li>Chatterjee, M., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Int J Cancer. , (2010).</li><li>Sander, E. E., ten Klooster, J. P., van Delft, S., van der Kammen, R. A., Collard, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac+downregulates+Rho+activity:+reciprocal+balance+between+both+GTPases+determines+cellular+morphology+and+migratory+behavior.">Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior.</a> J Cell Biol. 147 (5), 1009-1022 (1999).</li></ol>

L&#39;étape la plus critique de la procédure est la nouvelle génération de TPS-RBD. Le défaut de porter une attention particulière à cette étape est la raison principale de l&#39;échec. Toutes les étapes, de génération de la TPS-RBD (1.2) à l&#39;incubation pendant la nuit avec le lysat cellulaire (2,5), devrait être fait en une seule journée. Une autre étape cruciale est de rendre certains qu&#39;il ya suffisamment de cellules pour produire les lysats cellulaires. Ceci est d&#39;une importance particuli...

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<td>Gluthione-sepharose 4B beads</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>17-0756-01</td>
<td>&nbsp;</td>
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<td>Criterion 4-20% Tris-HCl gels</td>
<td>&nbsp;</td>
<td>BioRad</td>
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rhoc gtpase activation assay

RhoC GTPase présente une homologie de 91% à RhoA GTPase. En raison de sa prévalence dans les cellules, de nombreux réactifs et des techniques de RhoA GTPase ont été développés. Toutefois, RhoC GTPase est exprimé dans les cellules cancéreuses métastatiques à des niveaux relativement bas. Par conséquent, quelques RhoC réactifs spécifiques ont été développés. Nous avons adapté un test d&#39;activation de Rho GTPase pour détecter RhoC. Cette technique utilise un TPS-Rho protéine de liaison de fusion de domaine à sortir actifs GTPase RhoC. En outre, nous pouvons récolter des protéines totales au début de l&#39;essai pour déterminer les niveaux du total (GTP et le PIB lié) GTPase RhoC. Ceci permet pour la détermination de la GTPase RhoC actifs par rapport au total dans la cellule. Plusieurs versions commerciales de cette procédure ont été développés cependant, les kits commerciaux sont optimisés pour RhoA GTPase et typiquement ne fonctionne pas bien pour RhoC GTPase. Pièces de l&#39;essai ont été modifiés ainsi que le développement d&#39;un anticorps spécifique RhoC.

Watch this Scientific Journal Video about RhoC GTPase Activation Assay at JoVE.com

RhoC GTPase Activation Assay

Ce protocole utilise un menu déroulant d&#39;essai pour déterminer les niveaux de GTPase RhoC actifs dans les cellules.

Assay RhoC activation GTPase

RhoC GTPase has 91% homology to RhoA GTPase.  Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed.  ...

Research

JoVE Journal

Biology

12.3K vues.  University of Delaware. Ce protocole utilise un menu déroulant d'essai pour déterminer les niveaux de GTPase RhoC actifs dans les cellules.

Vidéo: RhoC GTPase Activation Assay

Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques.   In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.  Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells.  The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells.  Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy.  Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmunity, clearance of infection, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.

Interview: présentation des antigènes glycolipides par CD1d et le potentiel thérapeutique de l&#39;activation des cellules NKT

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious ...

Recherche

Biologie

Recordings of Neural Circuit Activation in Freely Behaving Animals

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. Traditional non-invasive methods require at least partially restrained research subjects, and they only allow identification of large numbers of simultaneously activated neurons. On the other hand, small ensembles of neurons or individual neurons can only be measured using single-cell recordings obtained from largely reduced preparations. Since the expression of natural behavior is limited in restrained and dissected animals, the underlying neural mechanisms that control such behavior are difficult to identify. 
Here, I present a non-invasive physiological technique that allows measuring neural circuit activation in freely behaving animals. Using a pair of wire electrodes inside a water-filled chamber, the bath electrodes record neural and muscular field potentials generated by juvenile crayfish during natural or experimentally evoked escape responses. The primary escape responses of crayfish are mediated by three different types of tail-flips which move the animals away from the point of stimulation. Each type of tail-flip is controlled by its own neural circuit; the two fastest and most powerful escape responses require activation of different sets of large &#8220;command&#8221; neurons. In combination with behavioral observations, the bath electrode recordings allow unambiguous identification of these neurons and the associated neural circuits. Thus activity of neural circuitry underlying naturally occurring behavior can be measured in unrestrained animals and in different behavioral contexts.

Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed videography.

Enregistrements de l&#39;activation des circuits neuronaux dans les Librement Behaving Animaux

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. ...

Aortic Ring Assay

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis assays involve the study of individual endothelial cells in culture conditions (1). The aortic ring assay is an angiogenesis model that is based on organ culture. In this assay, angiogenic vessels grow from a segment of the aorta (modified from (2)). Briefly, mouse thoracic aorta is excised, the fat layer and adventitia are removed, and rings approximately 1 mm in length are prepared. Individual rings are then embedded in a small solid dome of basement matrix extract (BME), cast inside individual wells of a 48-well plate. Angiogenic factors and inhibitors of angiogenesis can be directly added to the rings, and a mixed co-culture of aortic rings and other cell types can be employed for the study of paracrine angiogenic effects. Sprouting is observed by inspection under a stereomicroscope over a period of 6-12 days. Due to the large variation caused by the irregularities in the aortic segments, experimentation in 6-plicates is strongly advised. Neovessel outgrowth is monitored throughout the experiment and imaged using phase microscopy, and supernatants are collected for measurement of relevant angiogenic and anti-angiogenic factors, cell death markers and nitrite.

Angiogenesis, the sprouting of blood vessels from pre-existing vasculature, is associated with both natural and pathological processes. Here we demonstrate an aortic ring assay that allows angiogenic potentiators and inhibitors to be directly added to aortic rings in culture. Sprouting and neovessel outgrowth can be determined by inspecting the aortic rings over a period of 6-12 days.

Assay anneau aortique

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

L&#39;isolement et la In vitro Activation des Caenorhabditis elegans Sperme

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Test clonogénique: cellules adhérentes

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

L&#39;activation de l&#39;apoptose par micro-injection cytoplasmique du cytochrome C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Intracellular Refolding Assay

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding1 and have a crucial role in promoting cell survival upon stress insults like heat shock2, nutrient starvation and exposure to chemicals/poisons3. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells4 as well as neurodegeneration5. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy7 and neurodegenerative disorders9. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones10,11. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene 11 (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45&deg;C for 30 minutes. Upon recovery at 37&deg;C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).

In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.

Assay Repliement intracellulaire

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

Amide hydrogène / deutérium Analyse Bourse &amp; MALDI-TOF spectrométrie de masse de PAK2 activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

Une seule molécule d&#39;imagerie du règlement Gene<em&gt; In vivo</em&gt; Utilisation de l&#39;activation par clivage cotraductionnelle (COTRAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...