Yazarlar, Şekil hazırlanması ile yardım için Rene Scriwanek ederiz. FR ALW Açık Programı (821.02.017 ve 822.02.014), DFG-NWO işbirliği (DN82-303) ve ZonMW VICI (016.130.606) hibeler, ECHO (700.59.003) tarafından desteklenmektedir.

1.. Sferoplast Hazırlık <ol><li> Deneyin tasarımı ile belirlenen uygun bir ortam, 10 ml, 30 ° C&#39;de gece boyunca maya inkübe edin. </li><li> Bir gün sonra, bir fotometre kullanılarak 600 nm (OD 600) de, kültürün optik yoğunluğu ölçmek bir OD 0,2-0,4 600 aynı kültür ortamı içinde seyreltilir ve hücrelere tasarımında sürece üstel büyüme fazına büyüyebilir deneyi farklı bir durum gerektirir. Kültür OD 1-2 600 olduğunda Hücreler üslü faza içindedir. </li><li> 50 ml&#39;lik bir tüp içinde 5 dakika boyunca 3500 x g&#39;de santrifüj ile hücreler 10 OD 600 eşdeğer toplayın. Santrifüj işleminden sonra, süpernatant atın. </li><li> 100 mM PIPES (pH 9.6), 5 ml, 10 mM ditiyotreitol ve 10 dakika boyunca 30 ° C&#39;de inkübe hücre pelletini. </li><li> 5 dakika boyunca 3500 x g&#39;de santrifüj ile tekrar hücreleri toplamak. Süpernatant atın. </li><li> Hücrelerin tekrar in, 1 M sorbitol ve litik enzimin, 5 mg ihtiva eden (Deneyin tasarımı ile belirlenir) ve yumuşak 30 dakika boyunca çalkalama ile 30 ° C&#39;de inkübe karışımı ortam 5 ml. </li><li> Spheroplastlar karşılık gelen pelet fraksiyonu toplamak için 5 dakika boyunca 300 x g&#39;de hücre süspansiyonu santrifüj. Süpernatant atın. </li><li> Buz gibi soğuk 960 ul ortam içerisinde yeniden süspanse spheroplastlar, 1 M sorbitol ihtiva eden (ortam Deneyin tasarımı ile belirlenir) ve bir buz soğuk 2 ml mikrosantrifüj tüpü içine karışımı aktarın. </li></ol> 2.. Nanogold Alımı <ol><li> Bir pipet kullanarak, yumuşak su 40 ul içinde yeniden süspansiyon haline getirildi pozitif yüklü parçacıkların nanogold 4 nmol ile sferoplast karıştırın. Karışımın nihai hacmi 1 ml olmak zorundadır. </li><li> 15 dakika boyunca buz üzerinde, elde edilen hücre süspansiyonu yerleştirin. </li><li> Oda sıcaklığında süspansiyonu aktarın ve e nanogold içselleştirilmesi izin gerekli süre boyunca inkübendocytosis. NOT: 15 dakika alımı (erken ve geç endozomları) tüm endozomal sistemi işaretlemek için izin verir ise bir 5 dk alımı başta, endositik kesecikler ve erken endozomal bölmeleri etiketlenmesine yol açacaktır. Daha uzun inkübasyon zamanları (en fazla 30 dakika) de vakuol etiket için izin verir. NOT: Darbe kovalayıcı etiketleme deneyleri, bu yöntem ile gerçekleştirilemez. LE analizinde etiketleme, bu nedenle, aynı zamanda nanogold AM ve INEE de tespit edilecektir. </li></ol> 3.. Fiksasyon ve Bölünmesi <ol><li> Çift gücü fiksatif 1 ml ilave etmek suretiyle nanogold alımını durdurma [% 4 paraformaldehit (PFA), 0.1 M PHEM tampon maddesi içinde% 0.4 glutaraldehid (GA) (20 mM PIPES, 50 mM HEPES, pH 6.9, 20 mM EGTA, 4 mM MgC! 2)] hücre süspansiyonuna 1 M sorbitol ihtiva etmektedir. Oda sıcaklığında tüpü devam etti. </li><li> Yavaşça (bu spheroplastlar tutmaya izin verir 30 dakika boyunca micocentrifuge Tüpleri el ile birkaç kez ters çevirinsüspansiyon) ve daha sonra 25 saniye boyunca 1700 xg iki kez santrifüj. </li><li> Süpernatantı atarak ve taze standart gücü fiksatif 1 ml (% 2 PFA, 0.1 M PHEM tampon maddesi içinde% 0.2 GA) 1 M sorbitol ihtiva eden, yavaşça dönen bir tekerlek üzerinde, oda sıcaklığında 2 saat boyunca inkübe ekleyerek fiksatif değiştirin. </li><li> Daha önce tarif edildiği gibi, 6 cryo-kesit için hücreleri işleyin. Not: nanogold alımı ve gümüş geliştirme prosedürleri için, belirtilen protokol açıklanan periyodik asit muamele yapılması için değil. Periyodik asit muamelesi daha hücre duvarı permeabilize 26, ancak bu yapı spheroplastlar üretimi sırasında elimine edilmiştir. </li><li> Önce 8 açıklandığı gibi bir UCT&#39;de ultramikrotom kullanılarak kuru elmas bıçak ile -120 ° C de 50 nm ince cryosections kesilmiş ve 50 nikel ızgaralarını hasır kaplı Formvar karbon üzerine koyun. </li></ol> Nanogold Partikül Visua boyunca 4. Silver Cinsellization Not: Reaksiyon karışımları hazırlamak için prosedür temelde, üretici tarafından belirtilen biridir. Bu protokolü kullanarak pratik uyarlamaları, cryosections üzerinde gümüş geliştirme prosedürü etkin ve güvenilir kılar. <ol><li> Dondurucu gümüş güçlendirme seti çıkarın ve bir 37 ° C kuluçka makinesi ya da su banyosu içinde eritin. Çözülmüş zaman, (4.7 bakınız) kullanıma kadar karanlık bir odaya yerleştirilen bir 24 ° C inkübatör kiti yerleştirin. </li><li> Karanlık bir odada, ısıtma plakasını ve 24 ° C nihai sıcaklığa ısınmasına </li><li> Yüzey koruyucu, parlak tarafı üste, ve bir bantla sabitleyin ısıtma plakasının üst kapağı. </li><li> Benchkote üzerinde Parafilm koyun ve karanlıkta Parafilm kenarları görmek mümkün siyah işaretleyici ile kenarları işaretleyin. </li><li> Isıtma plakasının sıcaklığını izlemek için Benchkote üstündeki bir termometre bantlayın. Yavaş yavaş sıcaklığını ayarlamak değilse 24 ° C. </li><li> Plaiki kez damıtılmış su ve içinde gümüş güçlendirme seti ile 50 ml tüp ce. </li><li> Iki kez damıtılmış su ile küçük bir Petri kapları doldurmak, 37 ° C&#39;de önceden ısıtılmış Suda ızgaraları yerleştirin, 30 dakika boyunca, aşağı tarafı numune. </li><li> Bu 4.7 adımı bir kez daha tekrarlayın. </li><li> Saklama kutusunda ızgaraları aktarın ve karanlık odaya getirmek. </li><li> Isıtma plakası üzerinde sabitlenmiş olan Parafilm yerleştirilen 24 ° C &#39;de iki kere damıtılmış su içinde birkaç damla on (aşağı numune yüzü) geçirilerek yeniden ızgaraları durulayın. </li><li> 9 ek olarak çift distile su damlaları gümüş güçlendirme reaksiyondan sonra durulama için, Parafilm hazır olduğundan emin olun. </li><li> Işığı kapatın ve tüm ışıklar kapalı olduğundan emin olun. Kırmızı ışık açın. </li><li> 24 ° C inkübatör gümüş geliştirme kiti A ve B çözümler dışarı atın. </li><li> Ilk olarak bir çözelti, 6 damla ve 1,5 m olarak B çözeltisi daha sonra 6 damla koymakl mikrosantrifüj tüp ve baloncuklar yapmak için kaçınarak bir cam Pasteur pipeti ile iyice karıştırın. Çözelti, oldukça kalın olup, son olarak el ile kısaca girdaplama önce bir pipet ile karıştırılmalıdır. </li><li> Geri 24 ° C inkübatör A ve B çözümler koyun ve C çözüm çıkar. </li><li> A / B karışımına C çözeltisi 6 damla ekleyin. Sonra bir Pasteur pipeti ile birinci tekrar karıştırın ve sonra vorteks tarafından, kabarcıkları yapmamak. </li><li> Parafilm (~ 20 ul / damla) damla koyarak hemen nihai karışımı kullanın. </li><li> Temiz, anti-manyetik cımbız ile, elde edilecek olan arzu donanıma (altın parçacıkları, yani. Boyutu) bağlı olarak 6 to15 dakika için karışımın (örneğin,., Gümüş artırılması reaksiyonu) ile ilgili en ızgaraları yerleştirin. </li><li> Karışımdan ızgaraları çıkarın ve yıkama için 24 ° C &#39;de damla iki kez damıtılmış suyun üstüne onları geçmektedir. Birincisi, hızla arkaya 6 damla kullanmak ve daha sonra damla başına 7 dakikalık inkübasyon süresi ile 3 damla, Işıkları açmadan önce. </li><li> Bir sonraki adım, bir EM soruşturma sonucu görselleştirmek için bağışıklık altın etiketleme veya zar boyama 27 ya geçin. </li></ol>

<ol>
	<li>Gruenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endocytic+pathway:+a+mosaic+of+domains.">The endocytic pathway: a mosaic of domains.</a> Nat Rev Mol Cell Biol. 2, 721-730 (2001).</li><li>Sachse, M., Ramm, G., Strous, G., Klumperman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosomes:+multipurpose+designs+for+integrating+housekeeping+and+specialized+tasks.">Endosomes: multipurpose designs for integrating housekeeping and specialized tasks.</a> Histochem Cell Biol. 117, 91-104 (2002).</li><li>Luzio, J. P., Pryor, P. R., Bright, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomes:+fusion+and+function.">Lysosomes: fusion and function.</a> Nature Rev Mol Cell Biol. 8, 622-632 (2007).</li><li>Meel, E., Klumperman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+imagination:+understanding+the+endo-lysosomal+system.">Imaging and imagination: understanding the endo-lysosomal system.</a> Histochem Cell Biol. 129, 253-266 (2008).</li><li>Bonangelino, C. J., Chavez, E. M., Bonifacino, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+screen+for+vacuolar+protein+sorting+genes+in+Saccharomyces+cerevisiae.">Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae.</a> Mol Biol Cell. 13, 2486-2501 (2002).</li><li>Griffith, J., Reggiori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+nanogold-labeled+endocytic+compartments+of+yeast+Saccharomyces+cerevisiae+using+a+cryosectioning+procedure.">Ultrastructural analysis of nanogold-labeled endocytic compartments of yeast Saccharomyces cerevisiae using a cryosectioning procedure.</a> J Histochem Cytochem. 57, 801-809 (2009).</li><li>Babst, M., Katzmann, D. J., Estepa-Sabal, E. J., Meerloo, T., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESCRT-III:+an+endosome-associated+heterooligomeric+protein+complex+required+for+MVB+sorting.">ESCRT-III: an endosome-associated heterooligomeric protein complex required for MVB sorting.</a> Dev Cell. 3, 271-282 (2002).</li><li>Griffith, J., Mari, M., De Maziere, A., Reggiori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cryosectioning+procedure+for+the+ultrastructural+analysis+and+the+immunogold+labelling+of+yeast+Saccharomyces+cerevisiae.">A cryosectioning procedure for the ultrastructural analysis and the immunogold labelling of yeast Saccharomyces cerevisiae.</a> Traffic. 9, 1060-1072 (2008).</li><li>Lewis, M. J., Nichols, B. J., Prescianotto-Baschong, C., Riezman, H., Pelham, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+retrieval+of+the+exocytic+SNARE+Snc1p+from+early+yeast+endosomes.">Specific retrieval of the exocytic SNARE Snc1p from early yeast endosomes.</a> Mol Biol Cell. 11, 23-38 (2000).</li><li>Prescianotto-Baschong, C., Riezman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ordering+of+compartments+in+the+yeast+endocytic+pathway.">Ordering of compartments in the yeast endocytic pathway.</a> Traffic. 3, 37-49 (2002).</li><li>Odorizzi, G., Babst, M., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fab1p+PtdIns(3)P+5-kinase+function+essential+for+protein+sorting+in+the+multivesicular+body.">Fab1p PtdIns(3)P 5-kinase function essential for protein sorting in the multivesicular body.</a> Cell. 95 (3), 847-858 (1998).</li><li>Donselaar, E., Posthuma, G., Zeuschner, D., Humbel, B. M., Slot, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogold+labeling+of+cryosections+from+high-pressure+frozen+cells.">Immunogold labeling of cryosections from high-pressure frozen cells.</a> Traffic. 8, 471-485 (2007).</li><li>Lah, J. J., Hayes, D. M., Burry, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neutral+pH+silver+development+method+for+the+visualization+of+1-nanometer+gold+particles+in+pre-embedding+electron+microscopic+immunocytochemistry.">A neutral pH silver development method for the visualization of 1-nanometer gold particles in pre-embedding electron microscopic immunocytochemistry.</a> J Histochem Cytochem. 38, 503-508 (1990).</li><li>Shah, N., Zhang, S., Harada, S., Smith, R. M., Jarett, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopic+visualization+of+insulin+translocation+into+the+cytoplasm+and+nuclei+of+intact+H35+hepatoma+cells+using+covalently+linked+Nanogold-insulin.">Electron microscopic visualization of insulin translocation into the cytoplasm and nuclei of intact H35 hepatoma cells using covalently linked Nanogold-insulin.</a> Endocrinol. 136, 2825-2835 (1995).</li><li>Weipoltshammer, K., Schofer, C., Almeder, M., Wachtler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+enhancement+at+the+electron+microscopic+level+using+Nanogold+and+gold-based+autometallography.">Signal enhancement at the electron microscopic level using Nanogold and gold-based autometallography.</a> Histochem Cell Biol. 114, 489-495 (2000).</li><li>Tokuyasu, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+ultracryotomy+of+cell+suspensions+and+tissues.">A technique for ultracryotomy of cell suspensions and tissues.</a> J Cell Biol. 57, 551-565 (1973).</li><li>Cabrera, 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=Phosphorylation+of+a+membrane+curvature-sensing+motif+switches+function+of+the+HOPS+subunit+Vps41+in+membrane+tethering.">Phosphorylation of a membrane curvature-sensing motif switches function of the HOPS subunit Vps41 in membrane tethering.</a> J Cell Biol. 191, 845-859 (2010).</li><li>Harner, 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=The+mitochondrial+contact+site+complex,+a+determinant+of+mitochondrial+architecture.">The mitochondrial contact site complex, a determinant of mitochondrial architecture.</a> EMBO J. 30, 4356-4370 (2011).</li><li>Nair, U., 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=SNARE+proteins+are+required+for+macroautophagy.">SNARE proteins are required for macroautophagy.</a> Cell. 146, 290-302 (2011).</li><li>Jacquier, N., 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=Lipid+droplets+are+functionally+connected+to+the+endoplasmic+reticulum+in+Saccharomyces+cerevisiae.">Lipid droplets are functionally connected to the endoplasmic reticulum in Saccharomyces cerevisiae.</a> J Cell Sci. 124, 2424-2437 (2011).</li><li>Karanasios, 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=Regulation+of+lipid+droplet+and+membrane+biogenesis+by+the+acidic+tail+of+the+phosphatidate+phosphatase+Pah1p.">Regulation of lipid droplet and membrane biogenesis by the acidic tail of the phosphatidate phosphatase Pah1p.</a> Mol Biol Cell. 24, 2124-2133 (2013).</li><li>Mari, 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=An+Atg9-containing+compartment+that+functions+in+the+early+steps+of+autophagosome+biogenesis.">An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis.</a> J Cell Biol. 190, 1005-1022 (2010).</li><li>Nair, U., 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+role+for+Atg8-PE+deconjugation+in+autophagosome+biogenesis.">A role for Atg8-PE deconjugation in autophagosome biogenesis.</a> Autophagy. 8, 780-793 (2012).</li><li>Vaart, A., Griffith, J., Reggiori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exit+from+the+Golgi+is+required+for+the+expansion+of+the+autophagosomal+phagophore+in+yeast+Saccharomyces+cerevisiae.">Exit from the Golgi is required for the expansion of the autophagosomal phagophore in yeast Saccharomyces cerevisiae.</a> Mol Biol Cell. 21, 2270-2284 (2010).</li><li>Erpapazoglou, Z., 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+dual+role+for+K63-linked+ubiquitin+chains+in+multivesicular+body+biogenesis+and+cargo+sorting.">A dual role for K63-linked ubiquitin chains in multivesicular body biogenesis and cargo sorting.</a> Mol Biol Cell. 23, 2170-2183 (2012).</li><li>Tuinen, E., Riezman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolocalization+of+glyceraldehyde-3-phosphate+dehydrogenase,+hexokinase,+and+carboxypeptidase+Y+in+yeast+cells+at+the+ultrastructural+level.">Immunolocalization of glyceraldehyde-3-phosphate dehydrogenase, hexokinase, and carboxypeptidase Y in yeast cells at the ultrastructural level.</a> J Histochem Cytochem. 35, 327-333 (1987).</li><li>Slot, J. W., Geuze, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryosectioning+and+immunolabeling.">Cryosectioning and immunolabeling.</a> Nat Protoc. 2, 2480-2491 (2007).</li><li>Prescianotto-Baschong, C., Riezman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+of+the+yeast+endocytic+pathway.">Morphology of the yeast endocytic pathway.</a> Mol Biol Cell. 9, 173-189 (1998).</li><li>Vida, T. A., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+vital+stain+for+visualizing+vacuolar+membrane+dynamics+and+endocytosis+in+yeast.">A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast.</a> J Cell Biol. 128, 779-792 (1995).</li><li>Hainfeld, J. F., Powell, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+frontiers+in+gold+labeling.">New frontiers in gold labeling.</a> J Histochem Cytochem. 48, 471-480 (2000).</li></ol>

The authors have nothing to disclose.

Immuno-elektron-mikroskopisi, bu proteinlerin bulunan taşıyıcıları ve organellerin ultrastrüktürel çözünürlüğe sahip proteinlerin birleştirilmesi lokalizasyonu sağlayan bir tekniktir. Onun bölmeleri floresan mikroskobu olarak noktasal yapıları görünür, çünkü maya endozomal sistemini inceleyerek bu özellikle önemlidir. Bu, onları ayırmak için zordur. Bu nedenle EM tarafından algılanabilir ve bir zaman bağlı bir şekilde endositik yol giren için çok önemlidir bir probun kullanılması, ...

Endozomal Sistem 1,2 reseptörleri lizozomal enzim ayırma reseptörlerinin ticareti ve plazma membranı (PM) geri dönüşüm de dahil olmak üzere çok sayıda çok önemli hücresel rol oynayan önemli bir zar sıralama aletidir. Endosomlar üç farklı bölümlerinde, yani ayrılır. Erken endozomlar (EE), geç endozomlar (LE) ve geri dönüşüm endozomlar. Bu sınıflandırma da spesifik bir işaretleyici proteinler ve onların morfolojisi üzerine, onlara ulaşmak için endositeze malzeme için gereken zaman dayanmaktadır. PM den içselleştirilmiş membranlar, yani protein ve lipid çift, iki bozulması için Endozomlar yoluyla lızozomlara teslim edilebilir ya da geri dönüştürülebilir. Membranlar da lızozomlara devam veya geri Golgi&#39;ye alınmak ya, benzer Golgi&#39;ye gelen Endozomlar taşınan ve edilmektedir. Ayrıca, protein endozomal sınırlayıcı zarın oluşumuna yol açan bir işlem içeri doğru tomurcuklanma lümen vezikülleri içine sıralanabilirLE bir alt kategori, multiveziküler organları. Maya endozomal sistemi göreceli olarak daha az karmaşık, yüksek ökaryotik hücrelerin olandan. Maya endozomlar EE ve LE ayrılır. Bu geri dönüşüm endozomları değil, aynı zamanda dokuya özel lizozom ilgili organellere içermeyen memeli hücrelerinin tersine. Dolayısıyla onlar endozomal kaçakçılık yollarının 3,4 daha az karmaşık bir ağ var. Bu nedenle, maya temsil hala endozomal sistemindeki temel ilkeler zar trafiğin bir çalışma için avantajlı bir deneysel sistem temsil vardır. Bu avantaj, endozomal yollarıyla ilgili çok sayıda gen başlangıçta 5 maya genetik ekranlar ile izole edilmiş gerçeği ile vurgulanmıştır. Vahşi tip ve mutant maya hücrelerinde endozomal sisteminin kapsamlı biyokimyasal ve floresan mikroskobu yaklaşımlar kullanılarak incelenmiştir da, ultra-yapı seviyesinde bu araştırma tek olmuşturminimal. Endozomal organellerin çoğunun kesin tanımlama 6 zorlaştırır floresan mikroskobu ile noktasal yapılar olarak tespit edilir, çünkü Şekil incelemesi maya içinde özellikle önemlidir. Ne yazık ki maya endozomal protein belirteçlerinin tanıma antiserumların sadece sınırlı sayıda (IEM) preparatları 7-10 immün elektron-mikroskopi çalışıyor. Bazı proteinleri için, bu sorun, ilgi konusu genin endojen etiketleme ve 7,11,12 tespit etmek için etiketi tanıyan bir antikorun kullanılması engellenebilecektir edilmiştir. Ancak genellikle, proteinler, bunların düşük sentezleme seviyeleri IEM ile tespit edilemez. Bu yaklaşım, morfoloji / fonksiyonları yanlış lokalizasyonu ve / veya değişiklik oluşturabileceği nedeniyle aşırı ifadesi bir çözüm değildir. Böylece elektron mikroskobu EM tarafından saptanabilen bir prob ile endositik bölmelerin etiketleme etkili bir seçenektir. Bu, özellikle eğer iyi bir çözüm değildir prObe belirli bir organelini 6 işaretlemek ne zaman bilmek sağlayan bir zamana bağlı bir şekilde endositik güzergahı giriyor. Maya spheroplastlar (hücre duvarı enzimatik kaldırılmıştır, yani. Maya) ile pozitif yüklü nanogold alımı, başarılı bir şekilde, maya endozomal 10 bölmeleri tanımlamak için kullanılmıştır. Bu partiküller biyolojik zarların oluşturan negatif yüklü lipidler bağlanır. Böylece AM ile pozitif yüklü nanogold ortakları, endositoz ile hücre nüfuz eder ve vakuol ulaşmadan önce EE ve LE geçer. Bu küçük altın partikülleri, ancak, EM tarafından görülecek uygun bir büyüklüğe sahip değildir. Onları görünür kılmak için, boyutları altın prob yaklaşık 13-15 gümüş ya da altın çökelmesi yol açan kimyasal reaksiyonlar ile büyütülebilir. Biz geliştirdik ve başarıyla subsellüler gerçekleştirmek için Tokuyasu yöntemine dayalı bir IEM yaklaşım uyguladıkyerelleştirme 8,16 çalışmalar. Bu yöntem, morfolojisi 8,17-24 mükemmel bir çözünürlüğe sahip maya hazırlıkları immunogold etiketleme performans sağlar. Biz de 6 bölmeleri maya endozomal sisteminin nanogold etiketleme ile bu IEM protokolü birleştirerek bir prosedür kurduk. Biz morfolojik kusur 6,25 bir endozomal kaçakçılığı ile mutantlar endozomların farklı alt sınıflarını karakterize ve ultrastruktural inceleyen bu yaklaşımı kullanarak. Ayrıca, bu nanogold etiketleme immünolojik etiketleri en farklı endozom alt grupları üzerinde ilgi konusu bir proteinin dağılımını keşfetmek için imkanı sağlayan kombine edilebilir olduğunu göstermiştir. Burada, pozitif yüklü nanogold ile maya endozomal sisteminin etiketleme pratikte nasıl gerçekleştirildiğini mevcut.

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nanogold labeling of the yeast endosomal system for ultrastructural analyses

Endosomlar ökaryotik hücrelerde kontrol noktaları sıralama önemli membran biri olan ve daha çok plazma zarı ve Golgi proteinlerin geri dönüştürülmesi ya da imha düzenler. Bunun bir sonucu olarak endozomal sistemi, hücre homeostazın sürdürülmesinde bir rol oynar ve veziküler taşıma araçları ile birbirine organel bu ağa ait olan genlerdeki mutasyonlar, kanser ve nörobiyolojik bozukluklar da dahil olmak üzere ciddi bir probleme yol açar. Bu endozomal sisteminin biogenezi ve organizasyon altında yatan mekanizmaları anlamak için bu nedenle asal öneme sahiptir. Maya Saccharomyces cerevisiae bu görevde önemli olmuştur. Özellikle etiket ve ultrastrüktürel düzeyde bu model organizmanın endozomal sistemi analiz etmek için, biz burada bir gümüş güçlendirme reaksiyonu ile bu parçacıkların görselleştirme ardından spheroplastlar tarafından pozitif yüklü nanogold alımı için ayrıntılı bir protokol mevcut. Bu yöntem, aynı zamanda, bir çok değerli olduğuendozomal kaçakçılığı kusurları ile mutantların morfolojik inceleme için l. Ayrıca, sadece elektron mikroskobik inceleme için geçerlidir, ama aynı zamanda protein lokalizasyonu araştırmalar için immünolojik labelings ile kombine edilebilir.

Sunulan protokole göre, maya endozom sisteminin morfolojisi iletim EM erişim olabilir. Şekil 1 ulaştı ve bu nedenle nanogold ile etiketlenmiş endozomal bölmelere farklı gösterir. Gümüş geliştirilmiş nanogold net elektron yoğun parçacıklar olarak görülebilir. Gümüş geliştirme reaksiyonu için kurulmuş uygun ayarlar maya hücresinin ultrastrüktürdeki müdahale etmez 5 ve 15 nm arasında, homojen bir boyut aralığı içinde parçacıklara sahip olan altın sağlar. Plazma membran...

Watch this Scientific Journal Video about Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses at JoVE.com

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Maya, Saccharomyces cerevisiae, endozomal sistemin biogenezi ve fonksiyonlarını düzenleyen genleri tanımlamak ve incelemek için bir anahtar bir model organizma olmuştur. Burada ince yapı çalışmaları için endozomal bölmelere spesifik etiketlenmesi için detaylı bir protokol mevcut.

Ultrastrüktürel Analizler için Maya endozomal Sisteminin Nanogold Etiketleme

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

nanogold-labeling-yeast-endosomal-system-for-ultrastructural

Research

JoVE Journal

Biology

10.2K Görüntüleme.  University Medical Center Utrecht.  Maya, Saccharomyces cerevisiae, endozomal sistemin biogenezi ve fonksiyonlarını düzenleyen genleri tanımlamak ve incelemek için bir anahtar bir model organizma olmuştur. Burada ince yapı çalışmaları için endozomal bölmelere spesifik etiketlenmesi için detaylı bir protokol mevcut. 

Video: Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

Canlı İmmobilize Maya Hücreleri Görselleştirme Hızlı Tekniği

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

Biyoloji

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

Ampüteler Osseointegrasyon Akıllı İmplant Tasarım Sistemi Biyoelektrik Analizleri

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

VE Floresan Etiketleme Drosophila Kalp Yapıları

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

İnsan DNA Onarım Proteinler Genetik Araştırmalar Maya Model Sistemi olarak kullanma

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-Ubiquitin Dayalı Membran Maya İki Hybrid (MİT) Sistemi: protein-protein etkileşimleri belirlenmesi için güçlü bir araç

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

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

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

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

Büyüme Faktörü Progranulin ile etkileşimde Proteinler tanımlamak Modifiye Maya-İki Hybrid Sistemi

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

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

Çoklu Gen Delesyonlar Taşıma Maya Suşlarında Nesil için Yeşil Canavar Süreci

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Fonksiyon Çalışma, Etkileşimler, ve Post-translasyonel değişiklikler için tomurcuklanan Maya Protein Ekstraksiyonu ve Saflaştırılması

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

Memeli Nükleer Reseptör kalıntıları tespit Maya İki melez Sistemi ters olduğunu Ligandlannın ve / veya antagonistleri ile Etkileşim

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

Budding Maya endositozun kantitatif, kinetik ve Yüksek verim Analizi pHluorin Uygulamaları

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...