Biz yazılım yapılan yorumlar için Emily Jackson, Joshua Zeidman ve Nicholas Wren teşekkür ederim. Bu çalışma GM95733 (NM için), BBBE 103316 ve MIT başlangıç ​​fonları (NM için) tarafından finanse edildi.

<ol>
	<li>Locke, J. C. W., Elowitz, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+movies+to+analyse+gene+circuit+dynamics+in+single+cells.">Using movies to analyse gene circuit dynamics in single cells.</a> Nature Reviews. Microbiology. 7 (5), 383-392 (2009).</li><li>Rosenfeld, N., Young, J. W., Alon, U., Swain, P. S., Elowitz, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Regulation+at+the+Single-Cell+Level.">Gene Regulation at the Single-Cell Level.</a> Science. 307 (5717), 1962-1965 (2005).</li><li>Colman-Lerner, A., Gordon, 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=Regulated+cell-to-cell+variation+in+a+cell-fate+decision+system.">Regulated cell-to-cell variation in a cell-fate decision system.</a> Nature. 437 (7059), 699-706 (2005).</li><li>Vega, N. M., Allison, K. R., Khalil, A. S., Collins, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling-mediated+bacterial+persister+formation.">Signaling-mediated bacterial persister formation.</a> Nature Chemical Biology. 8 (5), 431-433 (2012).</li><li>Larson, D. R., Zenklusen, D., Wu, B., 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=Real-Time+Observation+of+Transcription+Initiation+and+Elongation+on+an+Endogenous+Yeast+Gene.">Real-Time Observation of Transcription Initiation and Elongation on an Endogenous Yeast Gene.</a> Science. 332 (6028), 475-478 (2011).</li><li>Golding, I., Paulsson, J., Zawilski, S., Cox, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+kinetics+of+gene+activity+in+individual+bacteria.">Real-time kinetics of gene activity in individual bacteria.</a> Cell. 123 (6), 1025-1061 (2005).</li><li>Suter, D. M., Molina, N., Gatfield, D., Schneider, K., Schibler, U., Naef, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+Genes+Are+Transcribed+with+Widely+Different+Bursting+Kinetics.">Mammalian Genes Are Transcribed with Widely Different Bursting Kinetics.</a> Science. 332 (6028), 472-474 (2011).</li><li>Cookson, N. A., Cookson, S. W., Tsimring, L. S., Hasty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle-dependent+variations+in+protein+concentration.">Cell cycle-dependent variations in protein concentration.</a> Nucleic Acids Research. 38 (8), 2676-2681 (2010).</li><li>Zopf, C. J., Quinn, K., Zeidman, J., Maheshri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cycle+dependence+of+transcription+dominates+noise+in+gene+expression.">Cell-cycle dependence of transcription dominates noise in gene expression.</a> , (2013).</li><li>Kaufmann, B. B., Yang, Q., Mettetal, J. T., Van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+stochastic+switching+revealed+by+single-cell+genealogy.">Heritable stochastic switching revealed by single-cell genealogy.</a> PLoS Biology. 5 (9), e239 (2007).</li><li>Cookson, S., Ostroff, N., Pang, W. L., Volfson, D., Hasty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+dynamics+of+single-cell+gene+expression+over+multiple+cell+cycles.">Monitoring dynamics of single-cell gene expression over multiple cell cycles.</a> Molecular Systems Biology. 1 (1), 2005.0024 (2005).</li><li>Paliwal, S., Iglesias, P. A., Campbell, K., Hilioti, Z., Groisman, A., Levchenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAPK-mediated+bimodal+gene+expression+and+adaptive+gradient+sensing+in+yeast.">MAPK-mediated bimodal gene expression and adaptive gradient sensing in yeast.</a> Nature. 446 (7131), 46-51 (2007).</li><li>Charvin, G., Cross, F. R., Siggia, E. 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+microfluidic+device+for+temporally+controlled+gene+expression+and+long-term+fluorescent+imaging+in+unperturbed+dividing+yeast+cells.">A microfluidic device for temporally controlled gene expression and long-term fluorescent imaging in unperturbed dividing yeast cells.</a> PloS One. 3 (1), e1468 (2008).</li><li>Gordon, A., Colman-Lerner, A., Chin, T. E., Benjamin, K. R., Yu, R. C., Brent, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+quantification+of+molecules+and+rates+using+open-source+microscope-based+cytometry.">Single-cell quantification of molecules and rates using open-source microscope-based cytometry.</a> Nat. Meth. 4 (2), 175-181 (2007).</li><li>Otsu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Threshold+Selection+Method+from+Gray-Level+Histograms.">A Threshold Selection Method from Gray-Level Histograms.</a> IEEE Trans. Sys. Man. Cyber. 9 (1), 62-66 (1979).</li><li>Goranov, A. I., Cook, 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+rate+of+cell+growth+is+governed+by+cell+cycle+stage.">The rate of cell growth is governed by cell cycle stage.</a> Genes &amp; Development. 23 (12), 1408-1422 (2009).</li><li>To, T. -. L., Maheshri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise+Can+Induce+Bimodality+in+Positive+Transcriptional+Feedback+Loops+Without+Bistability.">Noise Can Induce Bimodality in Positive Transcriptional Feedback Loops Without Bistability.</a> Science. 327 (5969), 1142-1145 (2010).</li><li>O'Neill, E. M., Kaffman, A., Jolly, E. R., O'Shea, E. K. <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+PHO4+nuclear+localization+by+the+PHO80-PHO85+cyclin-CDK+complex.">Regulation of PHO4 nuclear localization by the PHO80-PHO85 cyclin-CDK complex.</a> Science. 271 (5246), 209-212 (1996).</li><li>Raser, J. M., O'Shea, E. K. <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+stochasticity+in+eukaryotic+gene+expression.">Control of stochasticity in eukaryotic gene expression.</a> Science. 304 (5678), 1811-1814 (2004).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Yukarıdaki protokol Mikroakiskan veya yazılım geliştirme sınırlı deneyimi olan floresan zaman serisi verileri elde etmek ve analiz etmek için basit bir yöntem açıklanır. Bu tek bir maya hücrelerinin zaman sukut floresan film elde etmesini sağlar, papaz izleme ve soy atamaları,, ilgili hücre boyutu ve ifade ölçümleri ayıklamak ve bir ticari mikroakışkan kültür cihaz kullanarak zaman ve çok yönlü bir grafik kullanıcı üzerinde bütün hücrelerin davranışını analiz arayüzü (GUI). Deneyse...

Gen Tek hücre analizi gen regülasyonu birçok açıdan bizim anlaşılmasını kolaylaştırdı. Akım sitometri veya mikroskopi kullanılarak floresan muhabiri ifade statik anlık tek hücreli ifade dağılımı hakkında yararlı bilgiler sağlar, ancak tarih ve doğrudan gen dinamikleri bilgilendirmek için gerekli zaman serisi verileri evrimi eksikliği. Floresan zaman atlamalı mikroskopi tek hücreli ölçümleri ve tarih hem de elde etmek için bir araç sunar. Çeşitli deneysel ve analitik teknikler böylece, hücre-hücre varyasyon 2,3, bakteriyel persister oluşumu 4, transkripsiyon gibi gen regülasyonu özellikleri içine anlayışlar (bir yorum için 1) aktarmanın, floresan muhabiri ifade film elde etmek ve ölçmek için geliştirilmiştir başlatılması ve uzama 5, 6,7 patlama transkripsiyonel, hücre döngüsü bağımlılık 8,9, ve kalıtım 10. Bununla birlikte, OBT&#39;ninAining kaliteli tek hücreli floresan zaman serisi kültür önemli teknik sorunlar kontrol edilebilir bir ortam ve elde edilen floresan filmleri yüksek verimli miktarının bir hücre tek tabaka içerir. Burada, S. floresan film elde etmek ve analiz etmek için bir prosedür tarif hücre kültürü cihaz üretiminde veya yazılım geliştirme hiç gerekli deneyimi (Şekil 1) ile cerevisiae. İlk olarak, ayrıntılı bir örnek protokolü bir veya daha fazla floresan gazetecilere ifade tomurcuklanan maya için floresan zaman serisi film oluşturmak için. Özel mikroakışkan kültür odaları inşa edilmiş ve başarılı bir şekilde daha önce 11-13 istihdam edilmiştir rağmen, biz CellAsic (Hayward, CA) bir ticari mikroakışkan cihazı kullanın. Sistem, tek tabaka sınırları büyüme hücreleri ve perfüzyon ortamının sürekli olarak kontrol edilmesini sağlar. Biz mevcut mikroskopi protokolü fluoresc elde etmek için basit bir araçtırence tomurcuklanan maya film, ancak herhangi bir değişiklik deney protokolünde (özelleştirilmiş bir kültür cihaz, alternatif medya koşulları, vb.) tek maya hücrelerinin benzer floresan film veri verimli ikame edilebilir. Sonra, zaman serisi veri ayıklamak için bir grafik kullanıcı arayüzü (GUI) tabanlı MATLAB yazılım paketi (Mathworks, Natick, MA), Floresans Zaman Serisi Hızlı Analizi (GREFTLERİ) için GUI adlandırılan, kullanarak filmlerin analizi anahat tek hücreler için. GREFTLERİ hücreleri segmentlere ve izleme ve floresan ve geometrik bilgileri ayıklamak en çok yönlü, açık kaynak yazılım paketi Hücre-ID 14 benzer özelliklere sahiptir. Ancak, GREFTLERİ önemli ek özellikler sağlar. İlk olarak, segmentasyon ve veri doğruluğunu, yerine analizden sonra aykırı bölge izleri sadece istatistiksel yolluk doğrulamak için izleme sonuçlarının kolay interaktif düzenleme sunmaktadır. Ayrıca, automaticall için analizi uzanıry adayı soy ve tomurcuklanan maya ilgi hücre döngüsü noktaları. Anne ve kızı iki bağımsız hücre bölge oluşturmak için bölmek zamanını belirleme tüm hücre (herhangi bir bağlı tomurcuk gibi anne) hücre döngüsü 8 boyunca ölçümleri belirlemek için çok önemlidir. Suite bu görevleri yerine getirmek için üç modülden oluşmaktadır. Odaklı ve odaklanmamış parlak bir alan görüntüler arasında kontrast dayanan ve ilk bölümleri hücre bölgeleri kullanıcı segmentasyonu parametrelerini tanımlamak ve görsel olarak test etmenizi sağlar. İkinci (. Mevcut Blair ve Crocker ve ark IDL rutin Dufresne&#39;nin MATLAB uygulaması kullanarak,: parça <a href="http://physics.georgetown.edu/matlab/" target="_blank">http://physics.georgetown.edu/MATLAB/</a> ) ve zaman içinde hücre bölgelerde önlemler; otomatik soy atar ve görsel denetim sağlar ve hata düzeltme. Basit bir komplo GUI sorgu tek hücreli özelliklerine dahildir. Üçüncü modül tomurcuk ortaya çıkması ve bölme ti bağlıyormes, ve tüm hücre zaman serisi verilerin yanı sıra ilk ve ikinci kez türevleri (as 9 ele) verir. Analiz modülü tercih istatistiksel yazılım sonraki çalışma için bir boşluk ayrılmış metin dosyası olarak veri verir. Böylece paketi kullanıcı bir grafik arayüz üzerinden yüksek kaliteli zaman serisi veri ayıklamak sağlar. Biz hücre döngüsü 9 bir fonksiyonu olarak tek tomurcuklanan maya hücreleri gerçek zamanlı transkripsiyon oranları tahmin etmek için bu yöntemi kullandık. Modüllerin tomurcuklanma maya, parametreleri veya için optimize edilmiş olsa da, gerekirse, serbestçe kullanılabilir kod diğer organizmalar ve resim tipleri için adapte edilebilir. Segmentasyon, izleme, ve soy atama algoritmaları atanan görüntüleme ve söz konusu organizmanın türlerine özgü olabilir. Mevcut algoritmaları yerine, ama yine de kullanıcı dostu görsel denetim ve segmentasyon düzeltme ve her zaman istediğimiz an izleme hataları sağlar GUI arayüzü korumak olabilirherhangi bir algoritma ile r.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Y04C Yeast Perfusion Plate</td>
			<td>CellAsic</td>
			<td>Y04C-02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ONIX Microfluidic Perfusion Platform</td>
			<td>CellAsic</td>
			<td>EV-262</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Axio Observer.Z1 Microscope</td>
			<td>Zeiss</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plan-Apochromat 63X/1.40 Oil DIC objective</td>
			<td>Zeiss</td>
			<td>440762-9904-000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cascade II EMCCD camera</td>
			<td>Photometrics</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lumen 200 metal-halide arc lamp</td>
			<td>PRIOR Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triple-bandpass dichroic filter cube and excitation and emission filter set</td>
			<td>Chroma Technology Corp</td>
			<td>set #89006</td>
			<td>Used for YFP (Venus/Citrine), CFP (Cerulean), RFP (mCherry/tdTomato)</td>
		</tr>
		<tr>
			<td>MAC 5000 controller and filter wheels</td>
			<td>Ludl Electronic Products</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MATLAB R2011a</td>
			<td>Mathworks</td>
			<td>&nbsp;</td>
			<td>64-bit version handles large data files better than 32-bit</td>
		</tr>
	</tbody>
</table>

acquiring fluorescence time-lapse movies of budding yeast and analyzing single-cell dynamics using grafts

Floresan zaman atlamalı mikroskopi tek hücre düzeyinde birçok biyolojik süreçlerin çalışmada güçlü bir araç haline gelmiştir. Özellikle, gen ifadesinin zamansal bağımlılığı gösteren film kendi düzenleme dinamikleri ilişkin bilgi sağlar, ancak tek hücre floresan film elde ve analiz için birçok teknik zorluklar vardır. Biz burada bu tür veri oluşturmak için ticari olarak satılan mikroakışkan kültür cihaz kullanarak basit bir protokol ve bir MATLAB tabanlı, grafik kullanıcı arayüzü (GUI) tabanlı yazılım paketi floresan görüntüleri ölçmek için açıklar. Yazılım bölümleri ve parça hücreleri kullanıcı görsel veri hataları barındırmalarına sağlar, ve otomatik olarak soy ve bölme kez atar. GUI daha bütün bir hücre izleri yanı sıra birinci ve ikinci zaman türevleri üretmek için zaman serisi analiz eder. Yazılım S. tasarlanmış iken cerevisiae, modülerlik ve çok yönlü t izin vermelidirbirkaç değişiklik ile diğer hücre tipleri eğitim için bir platform olarak hizmet o.

Bir başarılı bir performans ve analiz deney gerçekçi atanan tomurcuk ortaya çıkması ve bölme kez tek bir bütün hücreler için daha çok sürekli zaman serisi verecektir. Örnek olarak, büyüme ve genel ifadesi tek bir hücre içinde süresi (Tablo 4, Y47 içinde değişebilir nasıl gözlemlemek için yapısal ADH1 promoteri tarafından tahrik Shibuya floresan protein bütünleşmiş bir kopyası (CFP) ifade eden bir maya suşu ile haploid Yukarıdaki protokol gerçekleşt...

Watch this Scientific Journal Video about Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS at JoVE.com

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

Biz floresan büyüyen maya hücreleri mikroskop film ve tek hücreli zaman serisi veri ayıklamak için bir GUI tabanlı yazılım paketi elde etmek için basit protokol mevcut. Analizi otomatik soy ve görsel denetim ve izlenen verileri manuel küratörlüğü ile entegre bölümü zaman atama içerir.

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 ...

acquiring-fluorescence-time-lapse-movies-budding-yeast-analyzing

Research

JoVE Journal

Biology

12.7K Görüntüleme.  Massachusetts Institute of Technology. Biz floresan büyüyen maya hücreleri mikroskop film ve tek hücreli zaman serisi veri ayıklamak için bir GUI tabanlı yazılım paketi elde etmek için basit protokol mevcut. Analizi otomatik soy ve görsel denetim ve izlenen verileri manuel küratörlüğü ile entegre bölümü zaman atama içerir.

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

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

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Zaman atlamalı Görüntüleme siRNA Transfeksiyon sonra Mitoz

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Time-lapse video Mikroskop ile Hücrelerinin Rastgele Göç Kantitatif Analiz

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

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 ...

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

Sumoylation ve geliştirici Maya Kinetokor Proteinler Ndc10 ve Ndc80 ve ubikuitinasyomınun Tespiti verir Protein Saflaştırma Tekniği

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

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 ...

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 ...

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

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

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 ...

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 ...