我们要感谢他们的真知灼见丽莎Schneper和卡特琳Duevel发展这个实验。

<ol><li>将200ul合成介质板所需的饥饿条件（SC SC 2％和0.2％的葡萄糖与葡萄糖，例如）的兴趣不断增长的文化，如果文化的密度过彼此不同，调整细胞计数每200ul文化所以每下降大约相同数量的细胞。 </li><li>请一定要保持板块的下跌是文化的记录。 </li><li>保持板盖半掩，留在室温或在30 ° C，直到滴干。 </li><li>封口膜和保鲜膜（可选）的密封板和留在30℃为3-7天。 </li><li>文件（通过扫描，数字图片等）平板上的细胞的生长</li><li>用高压水（最好是去离子水）的琼脂表面清洗细胞分钟服用琼脂的照顾，不取消和改变方向。 </li><li>获取摆脱攻板纸巾，让他们干多余的水分。 </li><li>一旦板每块板的干笔的附着力（扫描或数码照片） </li><li>戴手套的手指，轻轻揉搓细胞形成的自来水琼脂表面下约一分钟。 </li><li>像以前那样干板。 </li><li>如果使用解剖范围，之前取出放在显微镜平台上板的针。 </li><li>与10倍或40倍放大倍率的文件入侵。确保把重点放在每个圆圈的中间部分，将过于拥挤的边缘，看到单个细胞形态。边缘可以表明丝状增长水平，并可以记录以供将来参考。扫描或采取整板的数码照片也可以是有益的。 </li></ol>

<ol>
	<li>Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R., Lappin-Scott, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+biofilms.">Microbial biofilms.</a> Annu Rev Microbiol. 49, 711-745 (1995).</li><li>Elortondo, F. J. P., Salmeron, J., Albisu, M., Casas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms+in+the+food+industry.">Biofilms in the food industry.</a> Food Science and Technology International. 5, 25-30 (1999).</li><li>Keinanen, M. M., Martikainen, P. J., Kontro, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+community+structure+and+biomass+in+developing+drinking+water+biofilms.">Microbial community structure and biomass in developing drinking water biofilms.</a> Can J Microbiol. 50, 183-191 (2004).</li><li>Biffinger, J. C., Pietron, J., Ray, R., Little, B., Ringeisen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biofilm+enhanced+miniature+microbial+fuel+cell+using+Shewanella+oneidensis+DSP10+and+oxygen+reduction+cathodes.">A biofilm enhanced miniature microbial fuel cell using Shewanella oneidensis DSP10 and oxygen reduction cathodes.</a> Biosens Bioelectron. 22, 1672-1679 (2007).</li><li>Kim, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+community+structure,+compartmentalization+and+activity+in+a+microbial+fuel+cell.">Bacterial community structure, compartmentalization and activity in a microbial fuel cell.</a> J Appl Microbiol. 101, 698-710 (2006).</li><li>Kim, J. R., Jung, S. H., Regan, J. M., Logan, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electricity+generation+and+microbial+community+analysis+of+alcohol+powered+microbial+fuel+cells.">Electricity generation and microbial community analysis of alcohol powered microbial fuel cells.</a> Bioresour Technol. 98, 2568-2577 (2007).</li><li>Picioreanu, C., Head, I. M., Katuri, K. P., Loosdrecht, M. C. v. a. n., Scott, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computational+model+for+biofilm-based+microbial+fuel+cells.">A computational model for biofilm-based microbial fuel cells.</a> Water Res. 41, 2921-2940 (2007).</li><li>Singh, R., Paul, D., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+implications+in+bioremediation.">Biofilms: implications in bioremediation.</a> Trends in Microbiology. 14, 389-397 (2006).</li><li>Cullen, P. J., Sprague, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+depletion+causes+haploid+invasive+growth+in+yeast.">Glucose depletion causes haploid invasive growth in yeast.</a> Proc Natl Acad Sci U S A. 97, 13619-13224 (2000).</li><li>Gimeno, C. J., Ljungdahl, P. O., Styles, C. A., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unipolar+cell+divisions+in+the+yeast+S.+cerevisiae+lead+to+filamentous+growth:+regulation+by+starvation+and+RAS.">Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS.</a> Cell. 68, 1077-1090 (1992).</li><li>Blankenship, J. R., Mitchell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+build+a+biofilm:+a+fungal+perspective.">How to build a biofilm: a fungal perspective.</a> Curr Opin Microbiol. 9, 588-594 (2006).</li><li>Reynolds, T. B., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bakers'+yeast,+a+model+for+fungal+biofilm+formation.">Bakers' yeast, a model for fungal biofilm formation.</a> Science. 291, 878-881 (2001).</li><li>Verstrepen, K. J., Klis, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flocculation,+adhesion+and+biofilm+formation+in+yeasts.">Flocculation, adhesion and biofilm formation in yeasts.</a> Mol Microbiol. 60, 5-15 (2006).</li><li>Liu, H., Styles, C. A., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elements+of+the+yeast+pheromone+response+pathway+required+for+filamentous+growth+of+diploids.">Elements of the yeast pheromone response pathway required for filamentous growth of diploids.</a> Science. 262, 1741-1744 (1993).</li><li>Madhani, H. D., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+filamentous+differentiation+and+virulence+in+fungi.">The control of filamentous differentiation and virulence in fungi.</a> Trends Cell Biol. 8, 348-353 (1998).</li><li>Mosch, H. U., Kubler, E., Krappmann, S., Fink, G. R., Braus, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosstalk+between+the+Ras2p-controlled+mitogen-activated+protein+kinase+and+cAMP+pathways+during+invasive+growth+of+Saccharomyces+cerevisiae.">Crosstalk between the Ras2p-controlled mitogen-activated protein kinase and cAMP pathways during invasive growth of Saccharomyces cerevisiae.</a> Mol Biol Cell. 10, 1325-1335 (1999).</li><li>Mosch, H. U., Roberts, R. L., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ras2+signals+via+the+Cdc42/Ste20/mitogen-activated+protein+kinase+module+to+induce+filamentous+growth+in+Saccharomyces+cerevisiae.">Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae.</a> Proc Natl Acad Sci U S A. 93, 5352-5356 (1996).</li><li>Pan, X., Heitman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP-dependent+protein+kinase+regulates+pseudohyphal+differentiation+in+Saccharomyces+cerevisiae.">Cyclic AMP-dependent protein kinase regulates pseudohyphal differentiation in Saccharomyces cerevisiae.</a> Mol Cell Biol. 19, 4874-4887 (1999).</li><li>Roberts, R. L., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elements+of+a+single+MAP+kinase+cascade+in+Saccharomyces+cerevisiae+mediate+two+developmental+programs+in+the+same+cell+type:+mating+and+invasive+growth.">Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth.</a> Genes Dev. 8, 2974-2985 (1994).</li><li>Robertson, L. S., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+three+yeast+A+kinases+have+specific+signaling+functions+in+pseudohyphal+growth.">The three yeast A kinases have specific signaling functions in pseudohyphal growth.</a> Proc Natl Acad Sci U S A. 95, 13783-13787 (1998).</li><li>Reynolds, T. B., Jansen, A., Peng, X., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mat+formation+in+Saccharomyces+cerevisiae+requires+nutrient+and+pH+gradients.">Mat formation in Saccharomyces cerevisiae requires nutrient and pH gradients.</a> Eukaryot Cell. , (2007).</li></ol>

酵母细胞显示各种分化的模式，根据营养可用性和环境条件，包括饥饿和压力的条件下，根据各种营养素强调的细丝，和絮凝孢子形成。各种酵母菌，包括酵母和白色念珠菌，也可以发现在不同的微生物形成的生物膜。虽然有一些丝和入侵行为的相关性，目前尚不清楚究竟如何丝可能会导致表面和组织的入侵和定植。酵母可以肯定会发现在营养和丝状的形式在自然界中生物膜，以及威胁人类健康的地方，如导管?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Moticam 350</td>
<td>Camera</td>
<td>Motic</td>
<td>discontinued (new model: Moticam 352)</td>
<td>A relatively cheap camera that attaches to eye pieces of microscopes and captures digital images for PC or Mac.</td>
</tr>		</tbody>
		</table>
		</div>

assay for adhesion and agar invasion in s. cerevisiae

酵母菌是在自然生物膜发现，许多微生物殖民表面。在人工环境，如人造物体表面，生物膜可以减少工业生产力，破坏结构，威胁着人类的生活。 1-3另一方面，利用生物膜的力量可以帮助清洁环境，并产生可持续的能源。 4-8酿酒酵母拓殖表面，并参与复杂的生物膜的能力主要是通过各种信号转导通路，并在这个有机体的环境因素引发的分化程序的重新发现被忽略，直到。 9，10使用酿酒酵母作为一种模式生物，理解的互动和衔接的信号转导通路的Ras - PKA，Kss1 MAPK和Hog1渗透压的途径，如迅速放置在酿酒酵母中生物膜的交界处，持续关注生物学和信号转导研究。 11-20为此，酵母细胞分化成长，胶粘剂，假菌丝细丝成为一个方便读数在各种环境变化的信号转导通路的激活。然而，丝是一个复杂的表型，这使得它的化验就好像它是一个简单的表型误导的集合。在过去的十年中，成功地采用了一些分析，从细菌生物膜研究酵母的研究，如垫形成实验测量软琼脂和结晶紫染色法，定量测量细胞表面坚持殖民地蔓延，。 12日，21然而，有一些发达国家的检测，定性评估的粘合剂和侵入表型酵母琼脂混乱。在这里，我们提出了一个简单和可靠的方法评估酵母菌株的粘合剂和侵入性的质量易于理解的步骤，以隔离入侵评估附着力评估。我们从以往的研究，10，16中采用的方法，包括生长的细胞在液体介质和电镀差大点，然后用清水洗净，以评估附着力和琼脂表面的细胞完全擦掉，以评估入侵到生长的营养条件琼脂。我们消除裸奔到琼脂细胞的需要，从而影响到琼脂细胞的侵袭。在一般情况下，我们观察到侵入单倍体菌株琼脂胶，但不是所有的粘合剂株可侵入琼脂培养基。我们的方法可以用来结合其他实验中，要认真剖析酵母的信号转导，分化，群体感应，和生物膜形成的分化步骤和要求。

Watch this Scientific Journal Video about Assay for Adhesion and Agar Invasion in S. cerevisiae at JoVE.com

Assay for Adhesion and Agar Invasion in S. cerevisiae

我们描述了酵母的附着力和琼脂作为衡量侵入和假菌丝分化入侵的定性分析。这个简单的实验，可用于评估各种突变体的侵入的表型，以及环境因素的影响，对酵母分化的信号通路。

化验粘附和酿酒酵母琼脂入侵

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

assay-for-adhesion-and-agar-invasion-in-s-cerevisiae

Research

JoVE Journal

Biology

14.8K 次观看.  Princeton University. 我们描述了酵母的附着力和琼脂作为衡量侵入和假菌丝分化入侵的定性分析。这个简单的实验，可用于评估各种突变体的侵入的表型，以及环境因素的影响，对酵母分化的信号通路。

视频: Assay for Adhesion and Agar Invasion in S. cerevisiae

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

科研

生物学

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

一个<em在体外 FluoroBlok肿瘤侵袭实验

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

为控制植物组织分解好氧菌的琼脂块缩影

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. The simplicity and power of the yeast aging model can also be explored to study DNA damage and genome maintenance as well as their contributions to diseases during aging. Here, we describe a system to study age-dependent DNA mutations, including base substitutions, frame-shift mutations, gross chromosomal rearrangements, and homologous/homeologous recombination, as well as nuclear DNA repair activity by combining the yeast chronological life span with simple DNA damage and mutation assays. The methods described here should facilitate the identification of genes/pathways that regulate genomic instability and the mechanisms that underlie age-dependent DNA mutations and cancer in mammals.

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Here we describe a set of DNA mutation assays that can be combined with the yeast chronological life span model to study the genes/pathways that regulate or contribute to genomic DNA instability during aging.

研究年龄相关的基因组不稳定性使用 S。酵母年代的寿命模型

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

流式细胞仪分离纯化<em&gt; S.酵母</em&gt;合子

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

软琼脂集落形成分析

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...