Nous remercions les Drs. Erez Eliyahu, Daniel Melamed, Ophry Pines et Doron Rapaport de l&#39;aide et des commentaires lors de la mise en place de ce protocole. Ce travail est financé par l&#39;ISF (numéro de subvention 1193-1109).

Une. Mitochondries Purification Peser le culot de cellules. Environ 0,6 g sont obtenus à partir de 100 cellules ml. <ol><li> Cultivez 100-150 ml de cellules de levure à DO 600 = 1-1,5 à 30 ° C sur un milieu de croissance non-fermentescible, comme milieu de croissance à base de galactose, de façon à enrichir pour des mitochondries. </li><li> Centrifuger les cellules à 3000 g pendant 5 min à température ambiante et jeter le surnageant. </li><li> Laver le culot avec de l&#39;eau distillée deux fois et centrifuger à nouveau. Jeter le surnageant. </li><li> Reprendre le culot dans 1 ml de tampon TNT par 0,5 g de cellules. Il est important de traiter les cellules avec un agent réducteur afin de rompre les liaisons disulfures à l&#39;intérieur de la paroi cellulaire, ce qui améliore l&#39;hydrolyse. </li><li> Incuber les cellules pendant 10 min à 30 ° C avec agitation douce. Pendant ce temps, peser zymolyase 6 mg pour 1 g de cellules et de le suspendre dans le tampon Zymolyase. </li><li> Centrifuger les cellules à 3000 g pendant 5 min untempérature t ambiante et jeter le surnageant. </li><li> Reprendre le culot dans 1 ml de tampon Zymolyase par 0,15 g de cellules. Ne pas vortex. </li><li> Mesurer la DO 600 de 10 ul aliquote de cellules dans 990 ul d&#39;eau. </li><li> Ajouter Zymolyase (étape 1.5) aux cellules d&#39;hydrolyser des polymères de glucose à des liaisons β-1 ,3-glucane et de générer des sphéroplastes. A partir de cette étape, les sphéroplastes doivent être conservés dans une solution isotonique pour éviter la lyse. </li><li> Incuber les cellules pendant 15 min à 30 ° C (pour une activité optimale de zymolyase) avec agitation douce. Pour vérifier l&#39;hydrolyse de la paroi cellulaire et la production de sphéroplastes, mélanger 10 ul de cellules avec 990 ul d&#39;eau. Sphéroplastes sont attendus à la lyse osmotique due à la différence, et la DO 600 devraient être au moins 10 fois inférieure à la valeur déterminée dans l&#39;étape 1.9. Sinon, continuez incubation avec zymolyase pendant 15 min. </li><li> Centrifuger les cellules à 3000 g pendant 5 min à température ambiante. Dis soigneusementcarte du surnageant, comme le culot peut être instable. </li><li> Laver sphéroplastes avec Zymolyase tampon et jeter le surnageant. </li><li> Reprendre sphéroplastes avec 100 ml de milieu de récupération. Transfert sphéroplastes à un Erlenmeyer et incuber pendant 1 heure à 30 ° C avec agitation. Cette étape de récupération est nécessaire, car lors de la translation de Zymolyase traitement est arrêté et la localisation de l&#39;ARNm est perturbée (figure 1B). </li><li> Ajouter 0,1 mg / ml CHX et sphéroplastes de transfert à un tube conique de 50 ml préalablement refroidi. plus de CHX est important de geler l&#39;association des ribosomes avec des ARNm. Ainsi, les ribosomes dépendantes association d&#39;ARNm avec des mitochondries est maintenue. </li><li> sphéroplastes à centrifuger à 3000 g pendant 5 min à 4 ° C et jeter le surnageant. </li><li> Laver deux fois avec le tampon mannitol froid. </li><li> Reprendre sphéroplastes avec 4 ml de tampon mannitol froid et les transférer dans un homogénéisateur Dounce de 15 ml de capacité équipé hermétiquepilon. Briser doucement sphéroplastes avec 15 coups et transférer le lysat dans un tube de 13 ml. </li><li> Centrifuger le lysat à 1500 x g pendant 6 minutes à 4 ° C pour sédimenter les noyaux et les cellules non rompues. </li><li> Soigneusement transférer le surnageant dans un nouveau tube. Mettre de côté 1 ml (25%) de l&#39;échantillon comme un échantillon non fractionnée (échantillon «Total»). Transfert de 50 ml de cet échantillon dans un nouveau tube et ajouter 15 ml de 4X LSB pour l&#39;analyse Western blot. Précipité l&#39;ARN du reste de l&#39;échantillon «Total» (voir le protocole 3, ARN précipitations). </li><li> Centrifuger le surnageant à 10000 g pendant 10 min à 4 ° C pour sédimenter les mitochondries. </li><li> Transférer le surnageant (~ 3 ml) dans un nouveau tube et le garder sur la glace. Il s&#39;agit de la fraction &quot;cytosolique&quot;. Transfert de 50 ml de cet échantillon dans un nouveau tube et ajouter 15 ml de 4x LSB pour l&#39;analyse Western blot. ARN précipité du reste de l&#39;échantillon &quot;cytosolique&quot; (Voir Protocol 3, ARN précipitations). </li><li> Laver le culot avec 3 ml de tampon mannitol et centrifuger à 10 000 g pendant 10 min à 4 ° C. </li><li> Reprendre le culot avec 3 ml de mannitol tampon. Cet exemple contient les mitochondries, et appelé fraction «mitochondries». Transfert de 50 ml de cet échantillon dans un nouveau tube et ajouter 15 ml de 4x LSB pour l&#39;analyse Western blot. </li></ol> 2. Extraction de l&#39;ARN <ol><li> Ajouter à chaque échantillon un volume de 8 M de guanidinium-HCl et deux volumes d&#39;éthanol à 100%. Vortex et incuber pendant au moins 2 heures à -20 ° C. </li><li> Centrifuger les échantillons à 10 000 g pendant 20 min à 4 ° C. Rejeter le surnageant. Soyez prudent car la pastille peut être instable. </li><li> Laver le culot avec 70% EtOH. </li><li> Reprendre le culot avec 400 ml d&#39;eau sans RNase et transférer l&#39;échantillon dans un nouveau tube Eppendorf. </li><li> Précipiter l&#39;ARN à nouveau en ajoutant 0,1 volume d&#39;acétate de 3 M de sodiume pH 5,2 et deux volumes d&#39;EtOH à 100%. Vortex et incuber pendant au moins 2 heures à -20 ° C. </li><li> Centrifuger les échantillons à 20 000 g pendant 20 min à 4 ° C. Jeter le surnageant et laver avec de l&#39;EtOH à 70%. </li><li> Air sécher le culot et remettre en suspension avec de l&#39;eau sans RNase. échantillons d&#39;ARN de magasin à -80 ° C. Les échantillons peuvent être utilisés pour l&#39;analyse du nord 19 ou analyse de puces à ADN 18 (voir protocole 3). </li></ol> 3. Préparation de l&#39;ARN pour l&#39;analyse de puces à ADN <ol><li> Reprendre l&#39;ARNm de l&#39;étape 2.2 avec 650 ml d&#39;eau sans RNase. </li><li> Pour retirer tout l&#39;ADN ou de protéines résiduel, ajouter un volume égal (650 ml) d&#39;acide phénol: chloroforme (5:1, pH 4,7) et vortexer vigoureusement. Centrifuger à vitesse maximale pendant 2 min. </li><li> Transférer 500 ml de la phase supérieure (phase aqueuse) dans un nouveau tube. Évitez de prendre de l&#39;interphase, car il contient de l&#39;ADN. Aussi soyez prudent de prendre phénol, qui peut inhiber la réaction de transcription inverse. &lt;/ Li&gt; <li> L&#39;échantillon d&#39;ARN contient une quantité importante de l&#39;héparine, qui est un puissant inhibiteur de la transcriptase inverse. Ainsi, par RT-PCR ou le marquage par puce à ADN, il doit être retiré. Retirer l&#39;héparine par la précipitation de LiCl: ajouter LiCl à une concentration finale de 2 M et incuber les échantillons pendant une nuit à -20 ° C. </li><li> Décongeler les échantillons à 4 ° C et centrifuger à 20 000 g pendant 20 min à 4 ° C. </li><li> Prenez soin de le surnageant et laver le culot avec 80% d&#39;éthanol. Centrifuger à nouveau comme décrit dans l&#39;étape 3.5. </li><li> Jeter le surnageant, l&#39;air sec et remettre en suspension le culot dans 150 ml d&#39;eau sans RNase. </li><li> Pour retirer tout résidu LiCl, précipiter à nouveau avec 0,1 volume de 3 M d&#39;acétate de sodium pH 5,3 et 3 volumes d&#39;éthanol à 100%. Incuber à -20 ° C pendant au moins 2 heures. </li><li> Centrifuger à vitesse maximale pendant 20 min à 4 ° C. Laver soigneusement la pastille transparente avec 80% d&#39;éthanol et de l&#39;air sec. </li><li> Reprendre leculot avec de l&#39;eau sans RNase 25 ml et conserver les échantillons à -80 ° C. </li><li> Suivez les étapes de l&#39;étiquetage de l&#39;ARN et hybridation décrit dans 18,20. </li></ol>

<ol>
	<li>Holt, C. E., Bullock, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+mRNA+localization+in+animal+cells+and+why+it+matters.">Subcellular mRNA localization in animal cells and why it matters.</a> Science. 326, 1212-1216 (2009).</li><li>Donnelly, C. J., Fainzilber, M., Twiss, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+communication+through+RNA+transport+and+localized+protein+synthesis.">Subcellular communication through RNA transport and localized protein synthesis.</a> Traffic. 11, 1498-1505 (2010).</li><li>Kellems, R. E., Allison, V. F., Butow, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+type+80+S+ribosomes+associated+with+yeast+mitochondria.+II.+Evidence+for+the+association+of+cytoplasmic+ribosomes+with+the+outer+mitochondrial+membrane+in+situ.">Cytoplasmic type 80 S ribosomes associated with yeast mitochondria. II. Evidence for the association of cytoplasmic ribosomes with the outer mitochondrial membrane in situ.</a> J. Biol. Chem. 249, 3297-3303 (1974).</li><li>Kellems, R. E., Butow, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+type+80+S+ribosomes+associated+with+yeast+mitochondria.+3.+Changes+in+the+amount+of+bound+ribosomes+in+response+to+changes+in+metabolic+state.">Cytoplasmic type 80 S ribosomes associated with yeast mitochondria. 3. Changes in the amount of bound ribosomes in response to changes in metabolic state.</a> J. Biol. Chem. 249, 3304-3310 (1974).</li><li>Suissa, M., Schatz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Import+of+proteins+into+mitochondria.+Translatable+mRNAs+for+imported+mitochondrial+proteins+are+present+in+free+as+well+as+mitochondria-bound+cytoplasmic+polysomes.">Import of proteins into mitochondria. Translatable mRNAs for imported mitochondrial proteins are present in free as well as mitochondria-bound cytoplasmic polysomes.</a> J. Biol. Chem. 257, 13048-13055 (1982).</li><li>Fujiki, M., Verner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+cytosolic+protein+synthesis+and+mitochondrial+protein+import+in+yeast.+Evidence+for+cotranslational+import+in+vivo.">Coupling of cytosolic protein synthesis and mitochondrial protein import in yeast. Evidence for cotranslational import in vivo.</a> J. Biol. Chem. 268, 1914-1920 (1993).</li><li>Yogev, O., Karniely, S., Pines, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation-coupled+translocation+of+yeast+fumarase+into+mitochondria+in+vivo.">Translation-coupled translocation of yeast fumarase into mitochondria in vivo.</a> J. Biol. Chem. 282, 29222-29229 (2007).</li><li>Eliyahu, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tom20+mediates+localization+of+mRNAs+to+mitochondria+in+a+translation-dependent+manner.">Tom20 mediates localization of mRNAs to mitochondria in a translation-dependent manner.</a> Mol. Cell. Biol. 30, 284-294 (2010).</li><li>Garcia, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria-associated+yeast+mRNAs+and+the+biogenesis+of+molecular+complexes.">Mitochondria-associated yeast mRNAs and the biogenesis of molecular complexes.</a> Mol. Cell. Biol. 18, 362-368 (2007).</li><li>Marc, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+mRNAs+targeted+to+yeast+mitochondria.">Genome-wide analysis of mRNAs targeted to yeast mitochondria.</a> EMBO Rep. 3, 159-164 (2002).</li><li>Gadir, N., Haim-Vilmovsky, L., Kraut-Cohen, J., Gerst, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+mRNAs+coding+for+mitochondrial+proteins+in+the+yeast+Saccharomyces+cerevisiae.">Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae.</a> RNA. 17, 1551-1565 (2011).</li><li>Margeot, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Saccharomyces+cerevisiae,+ATP2+mRNA+sorting+to+the+vicinity+of+mitochondria+is+essential+for+respiratory+function.">In Saccharomyces cerevisiae, ATP2 mRNA sorting to the vicinity of mitochondria is essential for respiratory function.</a> J EMBO. 21, 6893-6904 (2002).</li><li>Saint-Georges, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+mitochondrial+biogenesis:+a+role+for+the+PUF+RNA-binding+protein+Puf3p+in+mRNA+localization.">Yeast mitochondrial biogenesis: a role for the PUF RNA-binding protein Puf3p in mRNA localization.</a> PloS one. 3, (2008).</li><li>Quenault, T., Lithgow, T., Traven, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PUF+proteins:+repression,+activation+and+mRNA+localization.">PUF proteins: repression, activation and mRNA localization.</a> Trends Cell Biol. 21, 104-112 (2011).</li><li>Garcia, M., Delaveau, T., Goussard, S., Jacq, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+presequence+and+open+reading+frame+mediate+asymmetric+localization+of+messenger+RNA.">Mitochondrial presequence and open reading frame mediate asymmetric localization of messenger RNA.</a> EMBO Rep. 11, 285-291 (2010).</li><li>Eliyahu, E., Lesnik, C., Arava, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+chaperone+Ssa1+affects+mRNA+localization+to+the+mitochondria.">The protein chaperone Ssa1 affects mRNA localization to the mitochondria.</a> FEBS Lett. 586, 64-69 (2012).</li><li>Ahmed, A. U., Fisher, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Import+of+nuclear-encoded+mitochondrial+proteins:+a+cotranslational+perspective.">Import of nuclear-encoded mitochondrial proteins: a cotranslational perspective.</a> Int. Rev. Cell Biol. 273, 49-68 (2009).</li><li>Eliyahu, E., Melamed, D., Arava, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+RNA+extracted+from+isolated+mitochondria.">Genome-wide analysis of RNA extracted from isolated mitochondria.</a> Methods Mol. Biol. 714, 287-299 (2011).</li><li>Eldad, N., Yosefzon, Y., Arava, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+extensive+intra-molecular+associations+between+3'-UTRs+and+their+ORFs.">Identification and characterization of extensive intra-molecular associations between 3'-UTRs and their ORFs.</a> Nucleic Acids RES. 36, 6728-6738 (2008).</li><li>Melamed, D., Arava, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+mRNA+polysomal+profiles+with+spotted+DNA+microarrays.">Genome-wide analysis of mRNA polysomal profiles with spotted DNA microarrays.</a> Methods Enzymol. 431, 177-201 (2007).</li><li>Margeot, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+are+many+mRNAs+translated+to+the+vicinity+of+mitochondria:+a+role+in+protein+complex+assembly.">Why are many mRNAs translated to the vicinity of mitochondria: a role in protein complex assembly.</a> Gene. 354, 64-71 (2005).</li><li>Mathieu, L., Marsy, S., Saint-Georges, Y., Jacq, C., Dujardin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcriptome+screen+in+yeast+identifies+a+novel+assembly+factor+for+the+mitochondrial+complex+III.">A transcriptome screen in yeast identifies a novel assembly factor for the mitochondrial complex III.</a> Mitochondrion. 11, 391-396 (2011).</li><li>Hocine, S., Raymond, P., Zenklusen, D., Chao, J. A., Singer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+analysis+of+gene+expression+using+two-color+RNA+labeling+in+live+yeast.">Single-molecule analysis of gene expression using two-color RNA labeling in live yeast.</a> Nat. Methods. 10, 119-121 (2013).</li><li>Park, H. Y., Buxbaum, A. R., Singer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+mRNA+tracking+in+live+cells.">Single mRNA tracking in live cells.</a> Methods Enzymol. 472, 387-406 (2010).</li><li>Thompson, M. A., Casolari, J. M., Badieirostami, M., Brown, P. O., Moerner, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+tracking+of+single+mRNA+particles+in+Saccharomyces+cerevisiae+using+a+double-helix+point+spread+function.">Three-dimensional tracking of single mRNA particles in Saccharomyces cerevisiae using a double-helix point spread function.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 17864-17871 (2010).</li><li>Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+RNA+distribution+and+dynamics+in+living+cells.">Imaging intracellular RNA distribution and dynamics in living cells.</a> Nat. Methods. 6, 331-338 (2009).</li><li>Garcia, M., Darzacq, X., Devaux, F., Singer, R. H., Jacq, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+mitochondrial+transcriptomics.">Yeast mitochondrial transcriptomics.</a> Methods Mol. Biol. 372, 505-528 (2007).</li><li>Reinders, J., Sickmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+of+yeast+mitochondria.">Proteomics of yeast mitochondria.</a> Methods Mol. Biol. 372, 543-557 (2007).</li><li>Meisinger, C., Sommer, T., Pfanner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Saccharomcyes+cerevisiae+mitochondria+devoid+of+microsomal+and+cytosolic+contaminations.">Purification of Saccharomcyes cerevisiae mitochondria devoid of microsomal and cytosolic contaminations.</a> Anal. Biochem. 287, 339-342 (2000).</li><li>Glick, B. S., Pon, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+highly+purified+mitochondria+from+Saccharomyces+cerevisiae.">Isolation of highly purified mitochondria from Saccharomyces cerevisiae.</a> Methods Enzymol. 260, 213-223 (1995).</li><li>Boldogh, I. R., Pon, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+subfractionation+of+mitochondria+from+the+yeast+Saccharomyces+cerevisiae.">Purification and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae.</a> Methods Cell Biol. 80, 45-64 (2007).</li><li>Rieder, S. E., Emr, S. D., Bonifacino, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Isolation of subcellular fractions from the yeast Saccharomyces cerevisiae. Current protocols in cell biology / editorial board. 8, (2001).</li><li>Diekert, K., de Kroon, A. I., Kispal, G., Lill, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+mitochondria+from+the+yeast+Saccharomyces+cerevisiae.">Isolation and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae.</a> Methods Cell Biol. 65, 37-51 (2001).</li></ol>

Les auteurs déclarent aucun intérêt financier concurrents.

Les progrès technologiques en matière d&#39;imagerie ont donné des outils de haute résolution pour étudier la localisation des ARNm. Aujourd&#39;hui, on peut mesurer le mouvement même d&#39;une molécule d&#39;ARNm unique à l&#39;échelle de temps ms 23-26. Pourtant, des approches biochimiques traditionnels, comme celui décrit ci-dessus, sont également informative et sont préférables dans certains cas. L&#39;isolement biochimique permet la purification d&#39;un large répertoire d&#39;ARNm et de pr...

Les cellules eucaryotes sont organisés dans des compartiments distincts ayant des fonctions spécifiques. Pour remplir sa fonction, chaque compartiment contient un ensemble unique de protéines qui sont essentielles pour son activité. Un mécanisme possible par lequel ces protéines abordent leur compartiment est localisée par 1,2 de traduction. Dans ce procédé, la protéine est synthétisée à sa destination par les ribosomes et les ARNm qui y sont situés. Parmi les avantages probables de traduction localisée augmentent l&#39;efficacité de la protéine de ciblage, de réduction du besoin de protéines chaperons, et permettant des mécanismes de régulation spécifiques au site. En outre, les ARNm et des ribosomes localisées peuvent être un réservoir isolé de machinerie de traduction en cas de stress cellulaire, lorsque la traduction générale est inhibée. Mitochondries est devenu ces dernières années un modèle central à étudier traduction localisée. La plupart des protéines mitochondriales sont codées dans le noyau, traduites dans le cytosol,et importées dans l&#39;organite. Divers éléments de preuve indiquent que bon nombre de ces protéines sont produites par un processus de traduction locale. Initialement, les études de microscopie électronique et de fractionnement biochimique détectés ribosomes associés aux mitochondries 5.3. Ces études ont été ensuite corroborées in vivo par le travail sur des ARNm spécifiques, qui ont été trouvés à être importés que d&#39;une manière 6,7 cotraductionnelle. études de génome entier d&#39;ARNm liaison avec les mitochondries ont montré qu&#39;une fraction significative d&#39;ARNm sont localisés au voisinage de la mitochondrie 8-10. Certains de ces ARNm ont été en outre caractérisé par fluorescence in vivo méthodes, comme le poisson ou MTAG 9,11. Une interprétation straight-forward de cette association est que ces ARNm servent de modèles pour la traduction localisée. Les mécanismes par lesquels ces ARNm approchent les mitochondries ne sont pas connus. domaines non codantes (plus significantl3 &#39;UTR y) ont été présentés à participer à l&#39;ARNm association aux mitochondries 12. Ces domaines sont susceptibles de servir de site de liaison à des protéines liant l&#39;ARN qui assurent la médiation de leur transport. Les études chez la levure ont révélé qu&#39;un membre de la famille de PUM de protéines (Puf3) prend en charge l&#39;association d&#39;ARNm avec des mitochondries 8,13. Un rôle plausible pour Puf3, qui est basée sur les fonctions des autres membres de la famille, est d&#39;inhiber la traduction de l&#39;ARNm en est en route 14. Ainsi, les ARNm peuvent être transportés dans un état non traduite, par des protéines de liaison d&#39;ARN qui interagissent avec les régions non codantes. En variante, un grand nombre de travaux suggèrent que le transport a lieu pendant que la protéine est synthétisée. En particulier, les inhibiteurs de la traduction ont été montrés pour affecter ARNm association 8,13. En outre, les caractéristiques traduits tels que l&#39;AUG, la séquence de ciblage mitochondrial (MTS) ou les régions ORF sont présentés pour aider à la localisation 8,11,15. Hsp70-famille de protéines chaperone et récepteur de la protéine sur la membrane externe des mitochondries ont également été présentés à l&#39;appui de liaison de l&#39;ARNm, ce qui implique en outre que les caractéristiques-protéine codée sont importantes pour la localisation de l&#39;ARNm 16. Ceci est en accord avec un modèle dans lequel la protéine de ribosome-émergentes sert d&#39;élément de reconnaissance pour le ciblage complexe ribosome-ARNm-protéine à la mitochondrie 17. Traduction localisée près de la mitochondrie a été étudié par diverses méthodes, y compris la microscopie électronique (pour visualiser les ribosomes) 3, FISH 9, ARN vert (pour détecter les ARNm spécifiques) 11, et le fractionnement biochimique (pour détecter l&#39;ARN et des ribosomes) 10,18. Alors que les méthodes antérieures de détecter la localisation in vivo et peuvent permettre la visualisation de la dynamique de transport, celui-ci permet une détection de plusieurs ARNm différents dans une seule expérience. En outre, pour le codage de fractionnement biochimique ou non codantes domaines n&#39;ont pas besoin d&#39;être altation, par conséquent, leurs rôles spécifiques peut être évaluée. Fractionnement biochimique a été utilisée avec succès pendant de nombreuses années, pour l&#39;isolement de nombreux compartiments cellulaires différents. Ses principes et les limites sont bien établies, et on peut facilement modifier les protocoles existants à des fins différentes. L&#39;instrumentation nécessaire est la norme dans de nombreux laboratoires, il est donc généralement la première méthode de choix pour l&#39;étude de la localisation intracellulaire. Nous décrivons un protocole qui a été optimisée pour l&#39;isolement des ARNm tandis que les ribosomes sont associés aux mitochondries. Ce protocole est donc optimale pour étudier les facteurs impliqués dans la traduction localisée près de mitochondries.

<table border="0" cellpadding="0" cellspacing="0" width="969">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yeast Extract</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BactoPeptone</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Galactose</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">Do not autoclave Galactose</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Growth medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">For mitochondrial enrichment, you should use any non- fermentable carbon source, such as Galactose-based growth medium sterilized 1% Yeast Extract, 2% BactoPeptone, 2% Galactose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1 M TrisHCl pH9.4</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30 mM TrisHCl pH7.6</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol (DTT)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;10 mM DTT Buffer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">0.1 M TrisHCl pH 9.4, 10 mM Dithiothreitol (DTT). Make fresh every time</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.2 M Sorbitol</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">4 mM KH2PO4</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">16 mM K2HPO4</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.2&#956;m filter</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;">Zymolyase Buffer&#160;</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">1.2 M Sorbitol, 4 mM KH2PO4 , 16 mM K2HPO4. Filter this&#160; buffer (0.2&#956;m) and keep at room temperature for future use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Zymolyase 20T&#160;</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">20,000 U/gr</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recovery medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">Galactose-based growth medium supplemented with 1 M Sorbitol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1 mg/mL Cycloheximide (CHX)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.6 M Mannitol</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 mM MgAc</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 mM KCl</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5 mg/mL Heparin</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1 mM phenylmethanesulfonylfluoride (PMSF)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Mannitol Buffer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">0.6 M Mannitol, 30 mM TrisHCl pH7.6, 5 mM MgAc, 100 mM KCl. Add freshly: 0.5 mg/mL Heparin, 0.1 mg/mL CHX and 1mM (PMSF). Filter this buffer and use it ice-cold</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">8M Guanidinium-HCl</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;100% and 70% Ethanol (EtOH)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;3 M Sodium Acetate pH5.2</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;10 M LiCl stock solution</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 mM Tris HCl pH 6.8</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SDS</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Glycerol</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-mercaptoethanol</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromophenolblue</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">&#160;4xLSB</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">250 mM Tris HCl pH 6.8, 8% SDS, 40% Glycerol, 20% &#946;-mercaptoethanol and 0.02% Bromophenol blue</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dounce homogenizer of 15-ml capacity equipped with tight- fitting pestle</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
	</tbody>
</table>

isolation of mrnas associated with yeast mitochondria to study mechanisms of localized translation

La plupart des protéines mitochondriales sont codées dans le noyau et doivent être importées dans l&#39;organite. Importation peut se produire alors que la protéine est synthétisée à proximité des mitochondries. L&#39;assistance pour cette possibilité est fondée sur des études récentes, dans lequel de nombreux ARNm codant pour des protéines mitochondriales ont été présentées à être localisées à proximité de mitochondries. Avec des manifestations antérieures de l&#39;association des ribosomes à la membrane externe, ces résultats suggèrent un processus de traduction localisée. Cette traduction localisée peut améliorer l&#39;efficacité de l&#39;importation, fournir des sites de régulation uniques et réduire les cas de l&#39;expression ectopique. Diverses méthodes ont été utilisées pour caractériser les facteurs et les éléments qui assurent la médiation de traduction localisée. Norme d&#39;entre eux est un fractionnement sous-cellulaire par centrifugation différentielle. Ce protocole présente l&#39;avantage d&#39;isoler des ARNm, les ribosomes et les protéines en une seule procédure. Ceux-ci peuvent ensuite être caractérisés par divers moléculaire et biméthodes ochemical. En outre, la transcriptomique et la protéomique méthodes peuvent être appliquées à la matière résultante, permettre ainsi un aperçu de l&#39;ensemble du génome. L&#39;utilisation de la levure comme organisme modèle pour ces études a les avantages de rapidité, de simplicité et de coût. En outre, les outils de la génétique de pointe et des souches de suppression disponibles faciliter la vérification des facteurs de candidats.

Ce protocole permet la séparation d&#39;une fraction contenant des mitochondries, à partir des composants cytosoliques. La meilleure façon de tester son succès consiste à effectuer une analyse de Northern et une analyse Western (figure 1) à partir des échantillons des différentes étapes d&#39;isolement. Trois paramètres de la qualité de l&#39;isolation sont obtenus à partir de ces analyses. Tout d&#39;abord, si l&#39;ARN ou des protéines dans les échantillons sont intacts - ceux-ci sont d?...

Watch this Scientific Journal Video about Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation at JoVE.com

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

De nombreux ARNm codant pour des protéines mitochondriales sont associés à la membrane externe de la mitochondrie. Nous décrivons un procédé de fractionnement subcellulaire visant à l&#39;isolement des mitochondries de levure avec ses ARNm et des ribosomes associés. Ce protocole peut être appliqué à des cellules cultivées dans des conditions diverses dans le but de révéler les mécanismes de localisation des ARNm et traduction localisée près des mitochondries.

Isolement des ARNm associés aux mitochondries de levure pour étudier les mécanismes de traduction localisée

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

isolation-mrnas-associated-with-yeast-mitochondria-to-study

Research

JoVE Journal

Biology

12.8K vues.  Technion - Israel Institute of Technology.  De nombreux ARNm codant pour des protéines mitochondriales sont associés à la membrane externe de la mitochondrie. Nous décrivons un procédé de fractionnement subcellulaire visant à l'isolement des mitochondries de levure avec ses ARNm et des ribosomes associés. Ce protocole peut être appliqué à des cellules cultivées dans des conditions diverses dans le but de révéler les mécanismes de localisation des ARNm et traduction localisée près des mitochond...

Vidéo: Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test sensibilité visuelle à la vitesse et la direction du mouvement chez les lézards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Recherche

Biologie

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Purification des mitochondries de cellules de levure

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Isolement et purification de Drosophile Neurones périphériques en triant bille magnétique

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

Visualisation des ARNm localisés dans le réticulum endoplasmique des cellules de mammifères

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Imagerie confocale de simples mitochondriales superoxyde Flashs à coeur Intact ou<em&gt; In Vivo</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies.

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

Un dosage cellulaire sans l&#39;aide<em&gt; Xenopus laevis</em&gt; Embryo Extraits d&#39;étudier les mécanismes de règlement des tailles nucléaire

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Analyse du Gap Junction-dependent Transfert de miARN avec microscopie 3D-FRAP

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

The Use of the Patch-Clamp Technique to Study the Thermogenic Capacity of Mitochondria

Mitochondrial thermogenesis (also known as mitochondrial uncoupling) is one of the most promising targets for increasing energy expenditure to combat metabolic syndrome. Thermogenic tissues such as brown and beige fats develop highly specialized mitochondria for heat production. Mitochondria of other tissues, which primarily produce ATP, also convert up to 25% of the total mitochondrial energy production into heat and can, therefore, have a considerable impact on the physiology of the whole body. Mitochondrial thermogenesis is not only essential for maintaining the body temperature, but also prevents diet-induced obesity and reduces the production of reactive oxygen species (ROS) to protect cells from oxidative damage. Since mitochondrial thermogenesis is a key regulator of cellular metabolism, a mechanistic understanding of this fundamental process will help in the development of therapeutic strategies to combat many pathologies associated with mitochondrial dysfunction. Importantly, the precise molecular mechanisms that control acute activation of thermogenesis in mitochondria are poorly defined. This lack of information is largely due to a dearth of methods for the direct measurement of uncoupling proteins. The recent development of patch-clamp methodology applied to mitochondria enabled, for the first time, the direct study of the phenomenon at the origin of mitochondrial thermogenesis, H+ leak through the IMM, and the first biophysical characterization of mitochondrial transporters responsible for it, the uncoupling protein 1 (UCP1), specific of brown and beige fats, and the ADP/ATP transporter (AAC) for all other tissues. This unique approach will provide new insights into the mechanisms that control H+ leak and mitochondrial thermogenesis and how they can be targeted to combat metabolic syndrome. This paper describes the patch-clamp methodology applied to mitochondria to study their thermogenic capacity by directly measuring H+ currents through the IMM.

This method article details the main steps in measuring H+ leak across the inner mitochondrial membrane with the patch-clamp technique, a new approach to study the thermogenic capacity of mitochondria.

L’utilisation de la technique Patch-Clamp pour étudier la capacité thermogénique des mitochondries

Mitochondrial thermogenesis (also known as mitochondrial uncoupling) is one of the most promising targets for increasing energy expenditure to combat ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

Application du mouvement passif de la tête pour générer des accélérations définies à la tête des rongeurs

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Isolement des mitochondries du muscle squelettique de la souris pour les tests respirométriques

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...