עבודה זו נתמכה על ידי המועצה למחקר האוסטרלי גרנט DP0986937.

<ol>
	<li>Levine, B., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+the+pathogenesis+of+disease.">Autophagy in the pathogenesis of disease.</a> Cell. 132, 27-42 (2008).</li><li>Mizushima, N., Levine, B., Cuervo, A. M., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+fights+disease+through+cellular+self-digestion.">Autophagy fights disease through cellular self-digestion.</a> Nature. 451, 1069-1075 (2008).</li><li>He, C., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+mechanisms+and+signaling+pathways+of+autophagy.">Regulation mechanisms and signaling pathways of autophagy.</a> Annu. Rev. Genet. 43, 67-93 (2009).</li><li>Kroemer, G., Mari&#241;o, G., Levine, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+the+integrated+stress+response.">Autophagy and the integrated stress response.</a> Mol Cell. 40, 280-293 (2010).</li><li>Camougrand, N., Kissov&#225;, I., Salin, B., Devenish, R. 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+mitophagy+in+yeast.">Monitoring mitophagy in yeast.</a> Methods Enzymol. 451, 89-107 (2008).</li><li>Yen, W. L., Klionsky, D. J. <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+live+long+and+prosper:+autophagy,+mitochondria,+and+aging.">How to live long and prosper: autophagy, mitochondria, and aging.</a> Physiology (Bethesda). 23, 248-262 (2008).</li><li>Tolkovsky, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy.">Mitophagy.</a> Biochim. Biophys. Acta. 1793, 1508-1515 (2009).</li><li>Bhatia-Kissov&#225;, I., Camougrand, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+in+yeast:+actors+and+physiological+roles.">Mitophagy in yeast: actors and physiological roles.</a> FEMS Yeast Res. 10, 1023-1034 (2010).</li><li>Kanki, T., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+mechanism+of+mitochondria+autophagy+in+yeast.">The molecular mechanism of mitochondria autophagy in yeast.</a> Mol. Microbiol. 75, 795-800 (2010).</li><li>Nowikovsky, K., Reipert, S., Devenish, R. J., Schweyen, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mdm38+protein+depletion+causes+loss+of+mitochondrial+K+/H++exchange+activity,+osmotic+swelling+and+mitophagy.">Mdm38 protein depletion causes loss of mitochondrial K+/H+ exchange activity, osmotic swelling and mitophagy.</a> Cell Death Differ. 14, 1647-1656 (2007).</li><li>Devenish, R. J., Prescott, M., Turcic, K., Mijaljica, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+organelle+turnover+in+yeast+using+fluorescent+protein+tags.">Monitoring organelle turnover in yeast using fluorescent protein tags.</a> Methods Enzymol. 451, 109-131 (2008).</li><li>Rosado, C. J., Mijaljica, D., Hatzinisiriou, I., Prescott, M., Devenish, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rosella:+a+fluorescent+pH-biosensor+for+reporting+vacuolar+turnover+of+cytosol+and+organelles+in+yeast.">Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast.</a> Autophagy. 4, 205-213 (2008).</li><li>Gietz, R. D., Woods, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+yeast+by+lithium+acetate/single-stranded+carrier+DNA/polyethylene+glycol+method.">Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.</a> Methods Enzymol. 350, 87-96 (2002).</li></ol>

אין ניגודי אינטרסים הכריז.

בתמונות שהוצגו נציג איור. 2B ו - 2C ניתן לראות בבירור כי השימוש אברון ספציפי כתבים פלורסנט מיקרוסקופ פלואורסצנטי מספק ראיות של לוקליזציה והפצה המיטוכונדריה / בגידול והן חנקן מורעבים התאים, ומאפשר mitophagy להיות דמיינו. זה יש להדגיש כי שיטה זו אינה מוגבלת mitophagy הבאה. Autophagy של מרכיבים תאיים אחרים או אברונים (למשל, הריבוזומים, טיפות השומנים, peroxisomes, reticulum endoplasmic ואת הגרעין) עשוי להיות במעקב עם תיוג מתאים. פרטי לבנות את biosensor רוזלה, תרבות תנאים התא תוצאות אופייני מחזור של גרעין (nucleophagy) דווחו 11,12 הממחישות את היישום הרחב יותר של השיטה. השימוש אברון ספציפי כתבים ניאון עבור autophagy ניטור דורש כמה שיקולים של תכנון לבנות כדי לזכור. אלה כוללים: (1) סלקציה היתוך האות המיקוד המתאים כדי להשיג אברון ספציפי מיקוד, (2) בחירה של חלבון פלואורסצנטי מתאים (ים): (3) פליטת הקרינה חזקה; (4) לתקן לוקליזציה אברון ו / או חלוקה בתוך התא. לאחר הכתב אברון ספציפי באה לידי ביטוי בהצלחה מארח את השמרים נבחר זן קבוצה שנייה של שיקולים להיות: (1) צמיחה שמרים תנאים; (2) מדגם הכנה התא; (3) הדמיה פרמטרים כמו חשוב הפרמטרים שנבחרו עבור CLSM (למשל, עוצמת הלייזר, סריקה קצב ומצב, ערך חריר, ערך הזום, מטרות שימוש או חשיפה CCD-מצלמה) נשמרות לאורך ניסוי להבטיח כי ניתן לבצע השוואות בין שליטה (תאים גדל) דגימות רעב: ( 3) אינדוקציה autophagy ומוצלחת המסירה של כתב לתוך vacuole. בסך הכל, זו שיטה מיקרוסקופית פלורסנט קל, מהיר יחסית, ויש לו פוטנציאל היישום ההקרנה תפוקה גבוהה עבור תרופות חדישות המשפרות או לעכב autophagy, וכן גנים המווסתים או לווסת autophagy.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> שם מגיב / ציוד </th><th> חברה </th><th> מספר קטלוגי </th></tr><tr><td> Saccharomyces cerevisiae EasyComp טרנספורמציה Kit </td><td> Invitrogen </td><td> K5050-01 </td></tr><tr><td> חנקן שמרים ללא בסיס חומצות אמינו אמוניום סולפט </td><td> BD Difco </td><td> 200-0030 </td></tr><tr><td> התכה נמוכה agarose </td><td> Progen Biosciences </td><td> 200-0030 </td></tr><tr><td> מיקרוסקופ שקופיות (76 x 26 מ&quot;מ) </td><td> Menzel-גלזר </td><td></td></tr><tr><td> מיקרוסקופ coverslips (22 x 22 מ&quot;מ) </td><td> Menzel-גלזר </td><td> BB022022A1 </td></tr><tr><td> לייזר Confocal סריקת מיקרוסקופ Fluoview FV500 </td><td> אולימפוס </td><td></td></tr><tr><td> CMAC-Arg </td><td> Moleular בדיקות-Invitrogen </td><td> Y-7531 </td></tr></tbody></table> כל ריכוזי נתון w / v, אלא אם צוין אחרת. שמרים בינוני מינימלי (YMM): 0.67% חנקן שמרים ללא בסיס חומצות אמינו, גלוקוז 2%, אגר 1.5% (מוסף רק בעת ביצוע התקשורת מוצק) וכל תוספי auxotrophic הנדרש (0.01%)]. Saccharomyces מלחים (SS): (NH 4) 2 S0 4, 0.12%; KH 2 PO 4, 0.10%, 0.6% MgC1 2 H2O, 0.07; NaC1, 0.05%; CaCl2, 0.01%; FeC1 3, 0.0005%. בינוני צמיחה: אס ואתנול 2% (v / v) ותוספי auxotrophic המתאים. חנקן רעב בינוני: 0.17% חנקן שמרים ללא בסיס חומצות אמינו אמוניום סולפט (Difco), 2% אתנול (v / v).

a fluorescence microscopy assay for monitoring mitophagy in the yeast saccharomyces cerevisiae

Autophagy חשוב מחזור של רכיבים סלולריים תחת מגוון של תנאים שונים. זה ממלא תפקיד חיוני homeostatic וכן בקרת איכות מנגנון שיכול היעד סלקטיבי לבזות חומר הסלולר כולל organelles1-4. לדוגמה, פגום או מיותר המיטוכונדריה (איור 1), לא מסולק על ידי autophagy, יכול לייצג איום על הומאוסטזיס הסלולר הישרדות התא. ב השמרים, Saccharomyces cerevisiae, חסך תזונתי (למשל, רעב חנקן) או נזק יכול לקדם מחזור סלקטיבית של המיטוכונדריה על ידי autophagy בתהליך שמכונה mitophagy 5-9. אנו מתארים גישה פשוטה מיקרוסקופ פלואורסצנטי להעריך autophagy. לשם הבהירות אנו להגביל את התיאור שלנו כאן כדי להראות עד כמה הגישה ניתן להשתמש כדי לפקח mitophagy בתאי שמרים. Assay עושה שימוש כתב ניאון, רוזלה, המהווה biosensor כפול הפליטה הכוללת חלבון יחסית-pH יציב ניאון אדום קשור חלבון ה-pH רגיש פלואורסצנטי ירוק. המבצע של הכתב הזה מסתמך על ההבדלים בין ה-pH (5.0-5.5 pH ~) vacuole ו (pH 8.2 ~) המיטוכונדריה בתאים חיים. תחת תנאי גידול, סוג התאים בר התערוכה הן פלואורסצנטי אדום וירוק מופץ מאפיין בצורה של המיטוכונדריה. פליטת הקרינה לא קשורה vacuole. כאשר נתון רעב חנקן, מצב אשר גורם mitophagy, בנוסף פלואורסצנטי אדום וירוק תיוג במיטוכונדריה, תאים התערוכה הצטברות של האדומים, אבל לא פלואורסצנטי ירוק, את לומן vacuolar חומצי המייצג את המסירה של המיטוכונדריה כדי vacuole. ניקוד תאים עם אדום, אבל vacuoles ניאון ירוק לא יכול לשמש כמדד לפעילות mitophagic בתאים 5,10-12.

Watch this Scientific Journal Video about A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae at JoVE.com

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

גישה חזקים כדי לפקח על משלוח של האברונים את לומן חומצי של vacuole את השמרים במשך השפלה ומיחזור מתואר. השיטה מסתמכת על תיוג הספציפי של האברונים היעד עם פלואורסצנטי כפול פליטת מקודדים גנטית pH-biosensor, ויזואליזציה של תאים בודדים באמצעות מיקרוסקופ פלואורסצנטי.

Assay מיקרוסקופ פלואורסצנטי עבור Mitophagy ניטור השמרים 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 ...

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Nanyang Technological University

Research

JoVE Journal

Biology

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

25.4K Views.  Monash University. גישה חזקים כדי לפקח על משלוח של האברונים את לומן חומצי של vacuole את השמרים במשך השפלה ומיחזור מתואר. השיטה מסתמכת על תיוג הספציפי של האברונים היעד עם פלואורסצנטי כפול פליטת מקודדים גנטית pH-biosensor, ויזואליזציה של תאים בודדים באמצעות מיקרוסקופ פלואורסצנטי.

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

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Dissection של Saccharomyces cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

Phosphoproteomics כמותיים חומצת שומן מגורה Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

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.

זמן לשגות הדמיה של מיטוזה לאחר transfection siRNA

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

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

Microarray ניתוח עבור Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

כמותי תא חי פלואורסצנטי, מיקרוסקופ ניתוח של שמרים ביקוע

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

לטווח ארוך רעילות מבחן קטלני עם לסרטן הארטמיה franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

ביטוי וטיהור של חלבון פיברוזיס פיברוזיס הטרנסממברני הרגולטור מוליכות ב Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

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.

הדמיה וניתוח של מולקולות ה-mRNA באמצעות הקרינה<em&gt; באתרו</em&gt; הכלאה ב<em&gt; Cerevisiae Saccharomyces</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.

מבחני יחד לrefolding החלבון בניטור<em&gt; Cerevisiae Saccharomyces</em

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

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.