Biz Dr teşekkür ederim. Erez Eliyahu, Daniel Melamed, Ophry Pines ve bu protokolün kurulması sırasında yardım ve yorumlar için Doron Rapaport. Bu çalışma ISF (hibe sayısı 1193-1109) tarafından finanse edilmektedir.

1.. Mitokondri Arıtma Hücre pelet tartılır. Yaklaşık 0.6 g 100 mi hücrelerden elde edilir. <ol><li> 30 ° C&#39;de 600 = 1-1,5 OD maya hücrelerinin 100-150 ml büyümek Mitokondri için zenginleştirmek için, bu tür galaktoz bazlı bir büyüme ortamı olarak bir nonfermentable büyüme ortamı, üzerinde C °. </li><li> Santrifüj hücreleri oda sıcaklığında 5 dakika boyunca 3000 x g ve supernatant atın. </li><li> Yine iki kere damıtılmış su ve santrifüj ile pelet yıkayın. Süpernatant atın. </li><li> Hücrelerin 0.5 gramı başına 1 ml DTT tampon içinde pelletini. Böylece, hidroliz geliştirilmesi, hücre duvarı içinde disülfid bağları kırmak amacıyla bir indirgeme maddesi ile hücreleri tedavi etmek için önemlidir. </li><li> 30 ° C&#39;de 10 dakika boyunca inkübe hücreleri Nazik sallayarak C °. Öte yandan, hücreler 1 gramı başına 6 mg ağırlığında ve askiyle sonuçlanır ki askiyle sonuçlanır ki Tamponu içinde askıya. </li><li> 5 dk a boyunca 3.000 xg&#39;de Santrifüj hücrelerit oda sıcaklığı ve süpernatant atın. </li><li> Hücrelerin 0.15 g başına 1 mi askiyle sonuçlanır ki Tampon pelet tekrar. Vorteks. </li><li> 990 ul su içindeki hücrelerin 10 ul alikotunun OD 600 ölçün. </li><li> Β-1 ,3-glukan bağları glikoz polimerler, hidrolize ve sferoplastlar oluşturmak için, hücrelere askiyle sonuçlanır ki (adım 1.5) ilave edin. Bu aşama, sferoplastlar lizis önlemek amacıyla bir izotonik çözelti içinde tutulmalıdır. </li><li> Hafif sallama ile 30 ° C (askiyle sonuçlanır ki optimal aktivitesi için) 15 dakika boyunca inkübe hücreleri. Hücre duvarı ve sferoplastlar nesil hidroliz doğrulamak için, 990 ul su ile hücrelerin 10 ul karıştırın. Spheroplastlar nedeniyle osmotik farkı lize beklenmektedir ve OD 600 aşamasında, en azından 1.9 belirlenen değerden daha düşük 10 misli olmalıdır. Aksi takdirde, başka bir 15 dakika boyunca inkübasyon askiyle sonuçlanır ki bu devam eder. </li><li> Oda sıcaklığında 5 dakika boyunca 3000 x g&#39;de santrifüjleyin hücreleri. Dikkatle diskart süpernatan, pelet olarak kararsız olabilir. </li><li> Askiyle sonuçlanır ki Tampon ile spheroplastlar yıkayın ve süpernatant atın. </li><li> 100 ml Kurtarma ortamı ile spheroplastlar süspanse edin. Bir Erlenmeyer şişeye spheroplastlar aktarın ve 30 ° C&#39;de 1 saat boyunca inkübe Çalkalanarak C °. Askiyle sonuçlanır ki tedavi çeviri sırasında tutuklandı ve mRNA lokalizasyon (Şekil 1B) bozulur çünkü bu kurtarma adım gereklidir. </li><li> Bir soğutulmuş 50 ml konik tüp, 0.1 mg / ml cHx ve transfer spheroplastlar ekleyin. CHX ilave mRNA&#39;larla ribozomlarla &#39;dernek dondurmak için önemlidir. Böylece, mitokondri ile ribozomlar-bağımlı mRNA ilişki korunur. </li><li> Santrifüj sferoplastlar &#39;ı içerir, 4 ° C&#39;de 5 dakika boyunca 3000 x g ve supernatant atın. </li><li> Soğuk Mannitol Tampon ile iki kez yıkayın. </li><li> 4 ml soğuk tampon ile Mannitol spheroplastlar yeniden süspanse edin ve sıkı geçme ile donatılmış 15 ml kapasiteli bir Dounce homojenizatör aktarınpestle. Yavaşça 15 darbelerle spheroplastlar kırmak ve bir 13 ml tüp lizat aktarın. </li><li> Çekirdek ve kesintisiz hücrelerini pellet haline getirmek üzere, 4 ° C&#39;de 6 dakika boyunca 1500 x g&#39;de lizat santrifüj. </li><li> Dikkatle yeni bir tüp süpernatant aktarın. Bölünmemiş bir örnek (&quot;Toplam&quot; örneği) olarak bir yana numunenin 1 ml (% 25) olarak ayarlayın. Yeni bir tüpe aktarın Bu örneğin 50 ml ve western blot analizi için 4X LSB 15 ml ekleyin. &quot;Toplam&quot; numunenin geri kalanından Çökelti RNA (Protokol 3, RNA çökeltme bakınız). </li><li> 4 ° C&#39;de 10 dakika boyunca 10,000 x g&#39;de santrifüje süpernatan Mitokondri pelet için C °. </li><li> Yeni bir tüp süpernatant (~ 3 ml) aktarın ve buz üzerinde tutmak. Bu &quot;Sitosolik&quot; bölümüdür. Yeni bir tüpe aktarın Bu örneğin 50 ml ve western blot analizi için LSB 4x 15 ml ekleyin. &quot;Sitosolik&quot; numunenin geri kalanından Çökelti RNA (Protocol 3, RNA yağış). </li><li> 4 ° C&#39;de 10 dakika boyunca 10,000 x g&#39;de tekrar 3 ml Tampon Mannitol ve santrifüj ile pelet yıkayın </li><li> 3 ml mannitol Tampon ile pelletini. Bu örnek, mitokondri içerir ve &quot;Mitokondri&quot; fraksiyonu olarak adlandırılır. Yeni bir tüpe aktarın Bu örneğin 50 ml ve western blot analizi için LSB 4x 15 ml ekleyin. </li></ol> 2. RNA Ekstraksiyon <ol><li> Her bir örnek 8 M guanidinyum-HCl, bir hacim ve% 100 etanolden iki hacmin ekle. Vorteks ve -20 ° C&#39;de en az 2 saat boyunca inkübe ° C. </li><li> 4 ° C&#39;de 20 dakika boyunca 10,000 x g&#39;de santrifüjleyin örnekleri ° C. Süpernatant atın. Pelet kararsız olabilir dikkatli olun. </li><li> % 70 EtOH ile pelet yıkayın. </li><li> RNase-içermeyen su içinde 400 ml pelet yeniden süspanse edin ve yeni bir Eppendorf tüpüne örnek aktarın. </li><li> 3 M sodyum Etilasetat 0.1 hacim ilave edilerek RNA Çökeltie pH 5.2 ve% 100 EtOH iki hacimleri. Vorteks ve -20 ° C&#39;de en az 2 saat boyunca inkübe ° C. </li><li> 4 ° C&#39;de 20 dakika boyunca 20,000 x g&#39;de santrifüjleyin örnekleri Süpernatantı atın ve% 70 EtOH ile yıkayın. </li><li> Hava pelet kuru ve RNase-barındırmayan su ile yeniden süspanse edin. -80 ° C&#39;de saklayın RNA numuneleri ° C. Örnekler Northern analizinde 19 ya da mikro-dizi analizi 18 (Protokol 3 bak) için kullanılabilir. </li></ol> 3. Mikrodizi Analizi RNA hazırlanması <ol><li> RNase-içermeyen su içinde 650 ml aşama 2.2 &#39;den mRNA yeniden süspanse edin. </li><li> Kuvvetli bir şekilde kloroform (5:1, pH 4.7) ve vorteks: herhangi bir kalıntı DNA ya da proteinleri çıkarmak için, eşit asidik fenol hacmi (650 mi) ilave edin. 2 dakika boyunca maksimum hızda santrifüj. </li><li> Yeni bir tüpe, üst faz (sulu faz) 500 ml aktarın. DNA&#39;yı içerdiği gibi, interfazda almaktan kaçının. Ayrıca, ters transkripsiyon reaksiyonu inhibe fenol, alma dikkat edin. &lt;/ Li&gt; <li> RNA örnek ters transkriptazın bir inhibitörü olan heparin önemli bir miktarda içerir. Böylece, RT-PCR veya mikroarray etiketleme için kaldırılması gerekmektedir. LiCI çökeltme yoluyla heparin çıkarmak: 2 M bir son konsantrasyona kadar LiCI ekleyin ve -20 ° C&#39;de gece boyunca inkübe örnekleri ° C. </li><li> 4 ° C&#39;de örnekleri Çözülme ° C ve 4 ° C&#39;de 20 dakika boyunca 20,000 xg&#39;de santrifüj </li><li> Dikkatlice süpernatant atın ve% 80 etanol ile pelet yıkayın. Aşama 3.5 de tarif edildiği gibi tekrar santrifüj. </li><li> Süpernatantı, kuru hava atın ve RNase-içermeyen su içinde 150 ml pelet tekrar süspansiyon. </li><li> Herhangi bir artık LiCI kaldırmak için, 3 M sodyum asetat pH 5.3 ve 0.1 hacim% 100 etanol içinde 3 hacim ile tekrar çökeltilmesi. -20 ° C de inkübe En az 2 saat boyunca ° C. </li><li> 4 ° C&#39;de 20 dakika boyunca en yüksek hızda santrifüje ° C. % 80 etanol ve kuru hava ile dikkatlice şeffaf pelet yıkayın. </li><li> Süspanse25 ml RNase içermeyen su ile pelet ve -80 ° C&#39;de örnekleri tutmak ° C. </li><li> 18,20 açıklanan RNA etiketleme ve melezleme için adımları izleyin. </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>

Yazarlar herhangi bir mali çıkarlarını beyan.

Görüntülemede Teknolojik gelişmeler mRNA yerelleştirme incelemek için yüksek çözünürlüklü araçları vermiştir. Bugün, bir msn zaman ölçeği 23-26 hatta tek bir mRNA molekülünün hareketini ölçebilirsiniz. Bununla birlikte, geleneksel biyokimyasal yaklaşımlar, yukarıda tarif edilen gibi, aynı zamanda bilgi ve bazı durumlarda tercih edilir. Biyokimyasal izolasyon mRNA ve proteinlerin büyük bir repertuar saflaştırılmasını sağlar ve genom çalışmaları için bu nedenle tercih ...

Ökaryotik hücreler, belirli işlevleri olan farklı bölümlerinde düzenlenmektedir. Bu fonksiyonu yerine getirmek için, her bir bölme bu etkinlik için temel olan protein benzersiz bir dizi içerir. Bu proteinler bölmesini hangi yaklaşımı sayesinde mümkün olan bir mekanizma lokalize çeviri 1,2 gereğidir. Bu işlemde, protein ribozomlar ve orada bulunan mRNA tarafından hedefe sentezlenir. Lokalize çeviri muhtemel avantajları protein hedeflemesi verimliliği artmış arasında protein chaperones ihtiyacının azalması ve site-spesifik düzenleme mekanizmalarını sağlayarak. Ayrıca, lokalize mRNAlarının ve ribozomlar genel çeviri inhibe hücresel stres, durumlarında çeviri makine tenha bir rezervuar olabilir. Mitokondri son yıllarda lokalize çeviri incelemek için merkezi bir model oldu. En mitokondri proteinleri, sitosol tercüme çekirdeğinde, kodlanmıştırve organel içine ithal. Kanıtlar çeşitli çizgiler, bu proteinler çoğu yerel için bir süreç yoluyla üretilmiş olduğunu göstermektedir. Başlangıçta, elektron mikroskobu ve biyokimyasal bölme çalışmaları mitokondri 3-5 ile ilişkili ribozomlan algılandı. Sonra sadece bir cotranslational şekilde 6,7 ithal edilecek bulundu belirli mRNA&#39;lardan çalışmalardan in vivo doğrulanmıştır Bu çalışmalar. Mitokondri ile ilişkili mRNA genom çalışmalar mRNA önemli bir kısmı mitokondri civarı 8-10 lokalize olduğunu ortaya çıkardı. Bu mRNA bazıları ayrıca, balık veya MTAG 9,11 olarak in vivo floresan yöntemler ile karakterize edilmiştir. Bu derneğin bir düz ileri yorumlama bu mRNAlarının lokalize çeviri için şablon olarak hizmet olmasıdır. Bu mRNAları mitokondri yaklaşım hangi mekanizmalar bilinmemektedir. Noncoding etki (en significantly 3 &#39;UTRs) mitokondri 12 mRNA birlikte dahil olmak gösterilmiştir. Bu etki kendi taşıma aracılık eden RNA bağlayıcı proteinlere bir bağlama alanı olarak hizmet etmek olasıdır. Mayada çalışmalar proteinlerin PUM ailesinin (Puf3) üyesi mitokondri 8,13 ile mRNA ilişki desteklediğini göstermiştir. Diğer aile üyelerinin fonksiyonları dayanmaktadır Puf3 için mantıklı bir rolü, mRNA rota 14 tr ise çeviri inhibe etmektir. Bu durumda, mRNA kodlayıcı olmayan bölgeler ile etkileşime RNA bağlayıcı protein tarafından, bir çevrilmemiş durumu nakledilebilir. Seçenek olarak ise, işin büyük bir gövde, protein sentez edilirken taşıma cereyan ettiğini ortaya çıkarmaktadır. Özellikle de, çeviri inhibitörleri mRNA&#39;lar ilişki 8,13 etkilediği gösterilmiştir. Ayrıca, bu tür Ağustos olarak tercüme özellikleri, mitokondriyal hedefleme sekansı (MTS) ya da ORF bölgeler lokalizasyon 8,11,15 yardımcı gösterilmiştir. Hsp70-family protein chmitokondri dış zar üzerinde aperone ve protein reseptör daha da kodlanmış protein özellikleri mRNA lokalizasyonu 16 için önemli olduğunu ima, mRNA ilişki destek gösterilmiştir. Bu, ribozom ortaya çıkan protein mitokondri 17 mRNA ribozom-protein kompleksi hedefleme için tanıma elemanı olarak hizmet ettiği bir modelle tutarlıdır. Mitokondri yakın olarak lokalize için elektron mikroskopisi (ribozomlar görselleştirmek için) 3, FISH 9, (spesifik mRNA tespit etmek için), yeşil RNA 11 ve biyokimyasal fraksiyonasyon (RNA ve hem ribozomlar tespit etmek için) 10,18 dahil olmak üzere çeşitli yöntemler ile araştırılmıştır. Eski yöntemler, canlı ortam içinde lokalizasyonu tespit etmek ve taşıma dinamikleri görselleştirme izin verirken, ikinci tek bir deneyde bir çok farklı mRNA tespiti sağlar. Ayrıca, biyokimyasal ayırma kodlayıcı veya kodlayıcı olmayan bir etki için al olması gerekmezolarak girilir, bu nedenle belirli bir rolleri değerlendirilebilir. Biyokimyasal ayırma başarılı bir şekilde bir çok farklı hücre bölmelerinin izole edilmesi için, uzun yıllardır kullanılmaktadır. Onun ilke ve sınırlamaları iyi kurulmuş, ve kolayca farklı bir amaç için mevcut protokolleri değiştirebilirsiniz. Gerekli enstrümantasyon, bu nedenle genellikle hücre içi lokalizasyonu eğitim için tercih edilen ilk yöntemdir, birçok laboratuvar standarttır. Biz, ribozomlar mitokondri ile ilişkili ise mRNA izolasyonu için optimize edilmiş bir protokol açıklar. Bu protokol, bu nedenle mitokondri yakın lokalize çeviri katılan faktörleri incelemek için en uygunudur.

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			<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>
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			<td height="21" style="height:21px;">BactoPeptone</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<td height="21" style="height:21px;">Dithiothreitol (DTT)</td>
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			<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>
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		<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<td height="21" style="height:21px;">0.1 mg/mL Cycloheximide (CHX)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
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			<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>
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			<td height="21" style="height:21px;">5 mM MgAc</td>
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			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:216px;">100 mM KCl</td>
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			<td height="21" style="height:21px;width:216px;">0.5 mg/mL Heparin</td>
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			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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			<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>
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			<td height="21" style="height:21px;">SDS</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
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			<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>
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			<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

Mitokondriyal proteinlerin çoğu çekirdeğine kodlanmış ve organel içine ithal edilecek ihtiyacı vardır. Protein, mitokondri yakın sentezlenir Alma oluşabilir. Bu olasılığı için destek mitokondriyal proteinleri kodlayan birçok mRNA&#39;lar mitokondri civarı lokalize edilmesi gösterilmiştir edildiği son çalışmalar, türetilmiştir. Birlikte dış zar ile ribozomların &#39;derneği önceki gösterileri ile, bu sonuçlar yerelleştirilmiş bir çeviri sürecini öneririz. Bu tür lokalize çeviri, ithalat verimliliğini artırmak benzersiz düzenleme siteleri sağlamak ve ektopik ifade vakalarını minimize edebilir. Çeşitli yöntemler lokalize çeviri aracılık faktörler ve unsurları karakterize etmek için kullanılmıştır. Bunlar arasında standart diferansiyel santrifüjle hücre içi fraksiyonlarının. Bu protokol, tek bir prosedür olarak mRNA, ribozomlar ve proteinlerinin izolasyonu gibi bir avantajı vardır. Bunlar daha sonra çeşitli moleküler ve bi karakterize edilebilirochemical yöntemleri. Ayrıca, transkriptomik ve proteomik yöntemleri böylece genom çapında derin bir bakış açısı sağlar, elde edilen malzemeye uygulanabilir. Bu çalışmalar için bir model organizma olarak maya kullanımı hızı, maliyetler ve basitlik avantajları vardır. Ayrıca, gelişmiş bir genetik araçları ve mevcut silme suşları aday faktörlerin incelenmesini kolaylaştırmak.

Bu protokol, sitosolik bileşenlerden bir mitokondri içeren fraksiyonu ayrılmasını sağlar. Başarısını test etmek için en iyi yolu, farklı izolasyon adımlardan örnekler kuzey analizi ve batı analizi (Şekil 1) yapmaktır. Izolasyon kalitesi için üç parametre, bu analizlerden elde edilir. İlk olarak, örneklerde, RNA ya da protein bozulmamış olsun - bu analizlerde ayrı bantlar olarak tespit edilecektir. Parçalanma olaylar ilave, kısa bantlar veya smear görünümünü neden olacakt?...

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

Mitokondriyal proteinleri kodlayan birçok mRNA&#39;lar mitokondri dış membran ile ilişkilidir. Biz ilişkili mRNA&#39;lardan ve ribozomlarla maya mitokondri izolasyonu amaçlayan bir hücre altı bölme prosedürü tarif. Bu protokol, mRNA lokalizasyonu mekanizmaları ve mitokondri yakın olarak lokalize çeviri ortaya çıkarmak için çeşitli koşullar altında yetiştirilen hücreler uygulanabilir.

Lokalize Çeviri Mekanizmaları Eğitim için Maya Mitokondri ile mRNAlarının Associated İzolasyonu

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 Görüntüleme.  Technion - Israel Institute of Technology.  Mitokondriyal proteinleri kodlayan birçok mRNA'lar mitokondri dış membran ile ilişkilidir. Biz ilişkili mRNA'lardan ve ribozomlarla maya mitokondri izolasyonu amaçlayan bir hücre altı bölme prosedürü tarif. Bu protokol, mRNA lokalizasyonu mekanizmaları ve mitokondri yakın olarak lokalize çeviri ortaya çıkarmak için çeşitli koşullar altında yetiştirilen hücreler uygulanabilir. 

Video: 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.

Kertenkeleler Hareket Hız ve Yön Visual Hassasiyet Test

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

Biyoloji

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.

Maya hücreleri Mitokondri saflaştırılması

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.

İzolasyon ve Arıtma Drosophila</emManyetik Boncuk Sıralama> Periferik Nöronlar

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.

Memeli hücrelerinde endoplazmik retikulum lokalize mRNA&#39;ların görselleştirilmesi

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.

Tek Mitokondriyal süperoksit Bozulmamış Kalp Flaşlar veya konfokal Görüntüleme<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.

Kullanılarak hücresiz Deneyi<em&gt; Xenopus laevis</em&gt; Embriyo Nükleer Boyut Yönetmeliğin Mekanizmaları Eğitim için ayıklar

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.

3D-FRAP Mikroskopisi ile Gap Birleşimine Bağlı MiRNA Transferinin Analizi

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.

Mitokondrinin Termojenik Kapasitesini İncelemek için Yama-Kelepçe Tekniğinin Kullanımı

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.

Kemirgenlerin Kafalarında Tanımlanmış İvmelenmeler Oluşturmak için Pasif Kafa Hareketinin Uygulanması

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.

Respirometrik Testler için Fare İskelet Kasından Mitokondri İzolasyonu

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 ...