Çeşitli FP-TUB1 plazmidler paylaşımı için Kerry Bloom (Kuzey Carolina Üniversitesi), Kyung Lee (NCI), Steven Markus (Colorado Eyalet Üniversitesi), ve Elmar Schiebel (Universität Heidelberg) teşekkür ederim. Biz slayt odası hazırlama yönteminde bizi eğitimi için Melissa Gardner (Minnesota Üniversitesi) minnettarız. Bu çalışma Ulusal Sağlık Enstitüleri (NIH) (JKM kadar) R01GM112893-01A1 ve (CE) T32GM008730 hibe tarafından desteklenmiştir.

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A., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergy+between+XMAP215+and+EB1+increases+microtubule+growth+rates+to+physiological+levels.">Synergy between XMAP215 and EB1 increases microtubule growth rates to physiological levels.</a> Nat Cell Biol. 15 (6), 688-693 (2013).</li><li>Mimori-Kiyosue, Y., Shiina, N., Tsukita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+behavior+of+the+APC-binding+protein+EB1+on+the+distal+ends+of+microtubules.">The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules.</a> Curr Biol. 10 (14), 865-868 (2000).</li><li>Byers, B., Goetsch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+of+spindles+and+spindle+plaques+in+the+cell+cycle+and+conjugation+of+Saccharomyces+cerevisiae.">Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae.</a> J Bacteriol. 124 (1), 511-523 (1975).</li><li>Marschall, L. 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L. J., 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=beta-Tubulin+C354+mutations+that+severely+decrease+microtubule+dynamics+do+not+prevent+nuclear+migration+in+yeast.">beta-Tubulin C354 mutations that severely decrease microtubule dynamics do not prevent nuclear migration in yeast.</a> Mol Biol Cell. 13 (8), 2919-2932 (2002).</li><li>Moore, J. K., Cooper, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinating+mitosis+with+cell+polarity:+Molecular+motors+at+the+cell+cortex.">Coordinating mitosis with cell polarity: Molecular motors at the cell cortex.</a> Semin Cell Dev Biol. 21 (3), 283-289 (2010).</li><li>Gartz Hanson, 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=Novel+alpha-tubulin+mutation+disrupts+neural+development+and+tubulin+proteostasis.">Novel alpha-tubulin mutation disrupts neural development and tubulin proteostasis.</a> Dev Biol. 409 (2), 406-419 (2016).</li><li>Fees, C. P., Aiken, J., O'Toole, E. T., Giddings, T. H. J., Moore, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+negatively+charged+carboxy-terminal+tail+of+beta-tubulin+promotes+proper+chromosome+segregation.">The negatively charged carboxy-terminal tail of beta-tubulin promotes proper chromosome segregation.</a> Mol Biol Cell. 27 (11), 1786-1796 (2016).</li><li>Song, S., Lee, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+function+of+Saccharomyces+cerevisiae+CDC5+in+cytokinesis.">A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis.</a> J Cell Biol. 152 (3), 451-469 (2001).</li><li>Knop, 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=Epitope+tagging+of+yeast+genes+using+a+PCR-based+strategy:+more+tags+and+improved+practical+routines.">Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines.</a> Yeast. 15 (10B), 963-972 (1999).</li><li>Kosco, K. 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=Control+of+microtubule+dynamics+by+Stu2p+is+essential+for+spindle+orientation+and+metaphase+chromosome+alignment+in+yeast.">Control of microtubule dynamics by Stu2p is essential for spindle orientation and metaphase chromosome alignment in yeast.</a> Mol Biol Cell. 12 (9), 2870-2880 (2001).</li><li>Toso, R. J., Jordan, M. A., Farrell, K. W., Matsumoto, B., Wilson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+stabilization+of+microtubule+dynamic+instability+in+vitro+by+vinblastine.">Kinetic stabilization of microtubule dynamic instability in vitro by vinblastine.</a> Biochemistry. 32 (5), 1285-1293 (1993).</li><li>Aiken, J., Sept, D., Costanzo, M., Boone, C., Cooper, J. A., Moore, J. K. <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+reveals+novel+and+discrete+functions+for+tubulin+carboxy-terminal+tails.">Genome-wide analysis reveals novel and discrete functions for tubulin carboxy-terminal tails.</a> Curr Biol. 24 (12), 1295-1303 (2014).</li><li>Straight, A. F., Marshall, W. F., Sedat, J. W., Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis+in+living+budding+yeast:+anaphase+A+but+no+metaphase+plate.">Mitosis in living budding yeast: anaphase A but no metaphase plate.</a> Science. 277 (5325), 574-578 (1997).</li><li>Khmelinskii, A., Lawrence, C., Roostalu, J., Schiebel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc14-regulated+midzone+assembly+controls+anaphase+B.">Cdc14-regulated midzone assembly controls anaphase B.</a> J Cell Biol. 177 (6), 981-993 (2007).</li><li>Markus, S. M., Omer, S., Baranowski, K., Lee, W. 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Yazarlar hiçbir rakip mali çıkarları olduğunu beyan ederim.

Tomurcuklanan maya modeli zamanla görüntü tek mikrotüpcüklere yetenek ve maya genetik araçlarını kullanarak tübülinler ve mikrotübül düzenleyicileri manipüle etme yeteneği dahil olmak üzere, bir in vivo ortamda mikrotübül dinamiklerinin yüksek çözünürlüklü ölçümler toplamak için önemli avantajlar sunuyor. Konkanavalin A ile kaplı bölmeler erimiş agar pedleri de dahil olmak üzere daha önce tarif edilen tertibatlarda, üzerinde bir çok avantaj sağl...

Mikrotübüller αβ-tübülin proteini alt ünitelerinin yapılmış iskelet polimerlerdir ve hücre içi ulaşım, hücre bölünmesi, morfojenez ve motilite içeren hücresel bağlamlarda çeşitli kullanılmaktadır. nerede ve ne zaman mikrotubuller formu bu çeşitli roller için mikrotübül ağları oluşturmak için, hücreler dikkatle düzenleyen gerekir. Bu düzenleme, serbest alt-birimler halinde mikrotübül polimerler veya demonte mikrotübüllere αβ-tübülin alt-birimleri monte ya da bu reaksiyonların kontrol edilmesi ile elde edilir; Bu mikrotübül dinamiklerinin olarak bilinir. mikrotübül alanının önemli bir amacı, tübülin ve / ya da mikro tüpler bağlanan αβ-tübülin alt-birimleri ve dışsal düzenleyiciler çalışmalar dahil olmak üzere mikrotübül dinamiklerine, düzenleyen moleküler mekanizmalarını etmektir. Ve iyi bilinen bir deneysel yaklaşım com genellikle, saflaştırılmış αβ-tübülin proteini kullanılarak, in vitro olarak, bu sistem tekrar oluşturmak için olanSaflaştırılmış dış düzenleyicileri ile bileflimini. Bu yararlı bir yaklaşım olsa da, yeniden oluşturulmuş bir sistemde mikrotübül dinamikleri canlı hücrelerde gözlenen olandan kuvvetle farklılık olduğu açıktır. Örneğin, mikrotübüller daha hızlı büyür ve in vitro in vivo daha yavaş küçültmek. Bu farklılıklar, hücreler bilinen dış düzenleyiciler 1 mevcudiyeti, aynı zamanda, henüz tanımlanmamış-faktörlere bağlı olabilir. Nedenle, mikrotübül düzenleyiciler ve bir yerli hücresel bağlamda dinamiklerini bozacak mutantların faaliyetlerini belirlemek için çok önemlidir. metazoan modelleri mikrotübül fonksiyonunu ve üst düzey örgüt araştırmak için geçerli olan sistemler olduğu kanıtlanmış olmasına rağmen, birkaç pratik kaygılar ciddi mikrotübül dinamiklerinin hassas ölçümü için bu modellerin yarar sınırlar. İlk olarak, hücre başına binlerce onlarca arasında değişen mikrotübül yüksek sayıda, güvenle etmek zorlaştırırzamanla bireysel Mikrotübülleri izlemek. Birçok çalışmada seçici böyle uç bağlayıcı (EB) ailede proteinler gibi mikrotübül uçlarına lokalize proteinler görüntüleme yoluyla bu sorunu aşmak. Bununla birlikte, bu proteinler, sadece Metazoan&#39;da 2 mikrotübülleri büyüyen uçları lokalize bilinmektedir. Bu nedenle, bu proteinlerin programı yalnızca dolaylı olarak felaket sıklığı dinamikleri diğer yönleri, ölçüm yaparken doğrudan büyüme oranları ölçümü ile sınırlıdır. İkincisi, genom düzenleme teknolojisindeki gelişmeler rağmen kararlı bir şekilde floresan etiketli tubulin veya selektif tübülin veya mikrotübül düzenleyiciler işlemek için mutasyonlar tanıtarak ifade eden hücreleri oluşturarak önemli bir sorun olmaya devam etmektedir. Üstelik Metazoan&#39;da birçok tübülin izotiplerinin varlığı mutasyonları bireysel tübülin genleri nasıl etkilediğini çalışma boşa çıkarıcıdır. Çiçek mayası sistemi in vivo microtubu ölçüm için çeşitli önemli avantajlar sağlarle dinamiği. Maya bireysel mikrotübül görselleştirme izin basitleştirilmiş mikrotübül ağı bulunmaktadır. Mayada, mikrotübüller çekirdek zarfına 3 gömülü mil kutup gövdelerinin (SPBs) olarak bilinen merkezi organize kaynaklanmaktadır. SPBs mikrotübül 4, 5 çekirdeklenmesi γ-tübülin küçük kompleksleri için iskelet olarak hizmet vermektedir. SPBs mikrotübül, mil mikrotübül ve astral mikrotübül iki sınıfları çekirdekleştirmek. Nükleolazma içine Mil mikrotubuller projesi ve kinetochore mikrotübül aracılığıyla kromozomların bağlayabilmek için ve interpolar Mikrotübülleri 6 örtüşen yoluyla mil stabilize için önemlidir. Buna karşılık, astral mikrotübül dışa sitozol içine proje ve iğ mikrotübüller yoğun ağına nispeten nadir karşılaştırılır. Mitoz sırasında önceden anafaz hücreler SPB ya çıkan sadece 1-2 astral mikrotübülleri sahiptir; Bu i olarak varyerine 7 demetleri olarak daha ndividual mikrotübüller. mitoz sırasında mikrotübüllerin Astral rolü tomurcuk boyun olarak bilinen anne ve tomurcuk bölmeleri arasındaki birleşme içine çekirdeği ve mil taşımaktır. Bu hareket, doğru ve en sonunda tomurcuk boyun 8 içine çekirdeği ve mil çekme astral mikrotübüller üzerindeki kuvvet üretir iyi tanımlanmış yolları kapsar. maya sisteminin bir diğer avantajı, eşsiz bir hassasiyetle mikrotübül düzenleyiciler ve tübülin alt-birimleri araştırmak için kullanılabilir olan genetik bir programı. Tek bir β-tübülin geni (TUB2) ve iki α-tubulin genleri (TUB1 ve TUB3): Maya da bir tübülin izotiplerinin basitleştirilmiş repertuar sahiptirler. Mutasyonlar hali hazırda bu genlerin sokulur ve böylece homojen bir tübülin nüfus 9, 10 olarak incelenebilir. yaygın bir dizi vardırMevcut etiketleme mikrotübül için yapılar ve bu kararlı ifadesi için kromozom lokuslarındaki entegrasyon için hedeflenebilir (Tartışma). Bu yöntemin genel amacı, mikrotübül dinamiklerinin yüksek çözünürlüklü ölçümler için dört boyutta (X, Y, Z ve T), maya hücreleri, canlı görüntü tek mikrotübüllere etmektir. maya hücrelerinde floresan etiketli tubulin konstitütif, düşük seviyeli bir ekspresyon için yapıları birleştirmek için yöntemler tarif edilmiştir. görüntüleme önce, canlı hücreler uzun süreli görüntüleme için hücrelerini kararlı hale lektin Concanavalin A ile kaplanmış kayar odalarına monte edilmiştir. görüntü elde etmek için en uygun parametreler, hem de veri analizi için bir iş akışı, ayrıca açıklanmıştır.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DOB (dropout bases)</td>
			<td>Sunrise science&#160;</td>
			<td>1650</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Leu</td>
			<td>Sunrise science&#160;</td>
			<td>1005</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Ameresco</td>
			<td>N833</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm polystyrene plates</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>ssDNA (Samon Sperm)&#160; in sterile DiH2O</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>&#160;D7656</td>
			<td>resuspend at 10 mg/mL in DiH2O. Store aliquots at -20 &#186;C</td>
		</tr>
		<tr>
			<td>Synthetic Complete Media&#160;</td>
			<td>Sunrise science&#160;</td>
			<td>1459-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Concanavalin A</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>L7647</td>
			<td>resuspend at 2 mg/mL in DiH2O. Store aliquots at -20 &#186;C</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-541-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>13-374-12</td>
			<td>paraffin film</td>
		</tr>
		<tr>
			<td>VALAP (Equal parts of Vaseline, lanolin and paraffin)</td>
			<td></td>
			<td></td>
			<td>melt at 75 &#186;C before use</td>
		</tr>
		<tr>
			<td>forceps&#160;</td>
			<td>Fisher Scientific</td>
			<td>16-100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Poyethylene glycol (PEG) 3350</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>202444</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ti E inverted Perfect Focus microscope</td>
			<td>Nikon Instruments</td>
			<td>MEA53100</td>
			<td></td>
		</tr>
		<tr>
			<td>1.45 NA 100x CFI Plan Apo objective</td>
			<td>Nikon Instruments</td>
			<td>MRD01905</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezo electric stage&#160;</td>
			<td>Physik Instrumente</td>
			<td>P-736</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disk scanner</td>
			<td>Yokogawa</td>
			<td>CSU10</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser combiner module</td>
			<td>Agilent Technologies</td>
			<td>MCL400B</td>
			<td></td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor Technology</td>
			<td>iXon Ultra 897&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements software</td>
			<td>Nikon Instruments</td>
			<td>MQS31100</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel software</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB software</td>
			<td>MathWorks, Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ64</td>
			<td>NIH</td>
			<td></td>
			<td>Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2016.</td>
		</tr>
		<tr>
			<td>Bio-Formats Importer plug-in</td>
			<td>Open Microscopy Environment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pUC19-LEU2::GFP-TUB1</td>
			<td></td>
			<td>pSK1050</td>
			<td>reference: Song, S. and Lee, K. S. A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis. J Cell Biol. 152 (3), 451-469 (2001)</td>
		</tr>
	</tbody>
</table>

high-resolution imaging and analysis of individual astral microtubule dynamics in budding yeast

Dinamik mikrotübülüsler birçok hücresel süreçlere temeldir ve mikrotübül dinamiklerinin doğru ölçümler hücreleri bu süreçleri düzenleyen nasıl içine ve nasıl genetik mutasyonlar darbe düzenleme öngörü sağlayabilir. metazoan modellerinde mikrotübül dinamiklerinin miktar genetik manipülasyonlara yüksek mikrotübül yoğunluğu ve ilişkin kısıtlamalar da bulunmaktadır ilişkili zorluklar, bir dizi vardır. Buna karşılık, tomurcuklanan maya modeli bu zorlukların üstesinden avantajlar sunuyor. Bu protokol, maya hücreleri, canlı bir mikrotübül dinamiklerinin ölçmek için bir yöntemi tarif etmektedir. floresan etiketli tubulin ifade eden hücreler, sabit zaman atlamalı görüntü elde etmek için izin monte kayar odalarına yapıştırılır. Yüksek hız için ayrıntılı bir rehber, dört boyutlu görüntü elde etme de eş odaklı görüntü yığınlarının dinamik mikrotübül özelliklerinin ölçülmesi için bir protokol, hem de temin edilmektedir. Bu yöntem, geleneksel maya genetik ile birlikte, atakantitatif tübülin alt-birimleri aktivitesini değiştiren mikrotübül regülatörleri veya mutasyonların etkilerini değerlendirmek için çok uygundur bir yaklaşım IdeS.

maya hücreleri canlı mikrotübül dinamiklerinin Ölçüm mikrotübül regülatörleri veya tubulin kodlayan genlerdeki mutasyonların etki polimerizasyonu ve depolimerizasyon oranları, aynı zamanda bu durumları arasındaki geçişin frekans alt-birimlerini ne kadar değerlendirmek için ilgi çekici bir araç sağlar. Şekil 1&#39;de, bir vahşi tip hücre astral mikrotübül dinamiklerinin bir zaman serisi ve (430Δ tub2-) β-tubulin bir mutasyona sahip b...

Watch this Scientific Journal Video about High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast at JoVE.com

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Maya tomurcuklanan nedeniyle güçlü genetik ve mikrotübül hücre iskeletinin basitliği için in vivo olarak mikrotübül dinamiklerinin çalışmak için avantajlı bir modeldir. Aşağıdaki protokol dönüşümü ve kültür maya hücreleri eş odaklı mikroskopi görüntü elde ve kantitatif maya hücrelerini canlı mikrotübül dinamiklerini analiz etmek açıklamaktadır.

Yüksek çözünürlüklü görüntüleme ve Bireysel Astral Mikrotubul Dynamics Analizi Budding Maya

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

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Research

JoVE Journal

Biology

9.1K Görüntüleme.  University of Colorado School of Medicine.  Maya tomurcuklanan nedeniyle güçlü genetik ve mikrotübül hücre iskeletinin basitliği için in vivo olarak mikrotübül dinamiklerinin çalışmak için avantajlı bir modeldir. Aşağıdaki protokol dönüşümü ve kültür maya hücreleri eş odaklı mikroskopi görüntü elde ve kantitatif maya hücrelerini canlı mikrotübül dinamiklerini analiz etmek açıklamaktadır. 

Video: High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

Geliştirici Maya replikatif Ömrü Ölçüm

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

Biyoloji

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Geliştirici Maya Protein Konsantrasyonu bir işlev olarak Morfolojik Fenotip Kalkınma Analizi

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

Tomurcuklanma Maya Floresan Time-lapse Filmler Kazanılması ve GREFTLERİ kullanarak Tek hücreli Dynamics analiz

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

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 ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Yüksek çözünürlüklü Melt Analizi ile PCR kullanarak hızlı ve verimli Zebra Genotiplemesi

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Yüksek çözünürlüklü Time-lapse Görüntüleme ve Yaşayan İnsan Umbilikal Ven Endotelyal Hücrelerinde Mikrotubul Dinamiği Otomatik Analizi

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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 ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

Fisyon Maya Cytokinetic Olayların zamanmekansal Analizi

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Mikroakışkan Aygıtlar Kullanma Tek Budding Maya Hücreleri ömrü ve Hücresel fenotipleri Ölçmek için

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Floresans canlı hücre mikroskobu ile mitotik ve meiotic Fission Maya nükleer dinamiklerinin incelenmesi

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...