We would like to thank Gero Miesenb&#246;ck and Tim Ryan for sharing pHluorin cDNA used to generate reagents in this study, Nathan Wright, Joanna Poprawski and Lydia Nyasae for excellent technical assistance, members of the Wendland lab for helpful discussions, and Michael McCaffery and Erin Pryce at the Integrated Imaging Center (Johns Hopkins) for advice and assistance with microscopy and flow cytometry. This work was supported by grants from the National Institutes of Health (to B.W., GM60979) and the National Science Foundation (to B.W., MCB 1024818). K.W. was supported in part by a training grant from the National Institutes of Health (T32-GM007231). A.F.O. was supported by developmental funds from the Department of Biological Sciences at Duquesne University and National Science Foundation CAREER grant 553143.

The authors declare no competing interests.

יישומים של pHluorin כימות של אירועי endocytic בתאי שמרים עושים שימוש מטענים הטרנסממברני שבו תג הניאון הוא קבע את זנב cytoplasmic של החלבון (איור 1 א). עבור המבחנים המתוארים כאן, מטענים הם פלורסנט במאור פנים כאשר תג pHluorin חשוף בסביבות ניטראליות, אבל להיות רווה כאשר תג pHluorin מפ?...

סחר שלפוחי ממלא תפקיד חשוב בשמירה על הזהות אברון ותפקוד בתאים איקריוטיים, והוא מכשיר מרכזי להסדרת הרכב חלבונים בממברנה של תאים סלולריים הפרט. על הממברנה הפלזמה, שילוב של שלפוחית ​​exocytic מספק חלבונים וממברנות חדשים אל פני השטח של התא, ואילו שלפוחית ​​שנוצרה באמצעות אנדוציטוזה להסיר קרום וחלבונים מפני השטח למחזור עקב או מיקוד אל ליזוזום. לפיכך, אנדוציטוזה חשוב ספיגת חומרים מזינה ועל תגובות הסביבה התאית. Exocytosis ו אנדוציטוזה מאוזנים להסדיר שטח הממברנה, וכדי לאפשר מחזור של חלבונים פגומים. עיונים שמרים בתאי יונקים זיהו מספר רב של חלבונים המעורבים אנדוציטוזה, כמו גם מסלולים endocytic מרובים שמקדמים הפנמה של מטענים ספציפיים או מעשה שיש בהם בתגובה במגוון enתנאים סביבתיים. מסלול-למד הכי טוב הוא אנדוציטוזה מתווכת clathrin (CME), שבו clathrin אביזר cytosolic חלבונים להרכיב למבנה מעיל לייצב את שלפוחית ​​endocytic המתהווה. הניסויים cerevisiae Saccharomyces שמרים ניצני הניבו תובנות חשובות על הדינמיקה והסדר של גיוס חלבונים endocytic רבים 1-3. יש לציין, כי מכונה CME הוא שמור ביותר בתהליך האבולוציה כזה שרוב החלבונים הקשורים CME בשמרים יש orthologs אדם; וכך, נבגי שמרים כבר כלי חשוב להבנת המנגנונים של אנדוציטוזה כי הם משומר אאוקריוטים גבוה. לדוגמא, מחקרים באמצעות שמרי ניצנים נקבעו כי היווצרות והתבגרות של מבני CME (המכונה טלאים יקטינו קליפת מוח כמו שמרים) כרוך הגיוס הרציף של חלבונים רבים, החל clathrin וחלבוני מתאם מחייב למשא ואחריו גיוס של אבזר endocytic נוסף חלבונים,הפעלת Arp2 / פילמור אקטין 3 בתיווך, וגיוס של חלבונים המעורבים 1,2 scission שלפוחית. גיוס של חלבונים מכונים CME כדי קליפת מוח טלאים יקטינו הוא תהליך הורה מאוד וסטריאוטיפי, ואת הסדר המדויק של גיוס חלבונים רבים כבר נקבע ביחס לרכיבים אחרים של מכונות CME. חשוב לציין, מחקרים שנעשו לאחרונה אישרו צו דומה של גיוס חלבונים CME בבורות מצופה clathrin בתאי יונקים 4. בנוסף CME, סוגי תאים רבים להחזיק אחד או יותר מסלולי endocytic עצמאי-clathrin (CIE) המסתמכים על מנגנונים חלופיים לקדם היווצרות שלפוחית והפנמה מן קרום הפלזמה 5-7. בשמרים, אנו לאחרונה זיהינו מסלול CIE אשר מנצל את Rho1 GTPase הקטן גורם חליפי נוקלאוטיד גואנין ההפעלה שלה (GEF), Rom1 8,9. Rho1 מפעיל את formin Bni1 לקדם פילמור אקטין 10,11, אשר נדרש עבור סוג זה של אנדוציטוזה עצמאית-clathrin בשמרים 12. יתר על כן, המשפחה-arrestin α של חלבונים לגייס את האנזים היוביקוויטין Rsp5 לקדם ubiquitination מטען והפנמה עוקבות דרך CME 13-17, וגם לקדם הפנמה מטענים דרך מסלול CIE, ואולי באמצעות אינטראקציה ישירה עם חלבונים המעורבים CIE 18. מגוון מסלולי CIE קיים גם בתאי יונקים, כוללים מסלולי clathrin-עצמאי phagocytic המסתמכים על ortholog Rho1, RhoA 9,19. תפקידו של RhoA ב יונקים CIE הוא הבין היטב; לכן, מחקרים בשמרי ניצנים עשויים לספק תובנות מכניסטית נוספים החלימו על CIE היונק. כימות מדויק של אירועי endocytic יכול לספק מידע חשוב על התפקידים של חלבונים ספציפיים בויסות אנדוציטוזה, והוא יכול לחשוף את ההשפעות של מוטציות על הפנמת מטען או progression דרך מסלול endocytic. לשם כך, בשיטות ביוכימיות יכולות להיות מנוצלות כדי לפקח ספיג ליגנד או כדי למדוד את שיעורים של פירוק של חלבוני מטען endocytic. בתאים חיים, שילוב של חלבון פלואורסצנטי ירוק (GFP) על גרסותיה כדי מטעני עניין מאפשר הדמיה ישירה של כלי רכב כבדים. עם זאת, מטענים מתויג GFP הם של שימוש מוגבל כימות של אנדוציטוזה כי GFP הוא עמיד בפני פירוק בתוך ליזוזום (או vacuole בשמרים). יתר על כן, GFP הקרינה היא רק רגיש באופן חלקי לשינויים ב- pH, ונשאר לזיהוי בתוך vacuole לומן 20,21. כתוצאה מכך, הקרינה של תג ה- GFP נמשכת vacuole הרבה אחרי שאר המטען כבר מושפלת, ושלמי כימותים מבוססי תאים של עוצמת מטען עשויים להיות לא מדויקים בשל קרינת GFP לטווח ארוכה של vacuole. על מנת להתגבר על החסרונות של GFP הקרינה vacuolar, אנחנו קודם לכן עשו שימוש pH supereclipticluorin, וריאנט רגיש-pH של GFP כי מאיר במאור ב- pH נייטרלי, אבל מאבד הקרינה בסביבה חומצית כגון לומן של vacuole / ליזוזום 20,22,23. הצבת תג pHluorin על הזנב cytoplasmic של מטענים endocytic היתרי להדמיה של מטענים על קרום פלזמה על endosomes מוקדם, שבו תג pHluorin נשאר חשוף אל הציטופלסמה (איור 1 א). כפי endosomes מוקדם להתבגר, במתחם מיון endosomal הנדרש להובלה (ESCRT) מטענים משטח-מקומי חבילות מכונות לתוך שלפוחית כי ניצן לתוך לומן של אנדוזום, יצירת גופים multivesicular (MVBs) 24. עבור מטענים כי שולבו שלפוחית ​​MVB פנימית, תג pHluorin פונה כלפי לומן השלפוחית. שלפוחית ​​MVB הפנימית acidified; וכך, מטענים מתויגים pHluorin לאבד קרינה על endosomes מוקדם כשהם מתבגרים לתוך MVBs 20. ההיתוך הבא של MVB עם vacuole מספק את תוכן luminal MVBהשפלה, ותגיות pHluorin נשארים הרווה בסביבה החומצית של לומן vacuole. מאמר זה מספק תיאורים מפורטים של מבחני endocytic כמותית באמצעות מטענים עם תגי pHluorin ציטופלסמית בשמרים. זנים להביע מטענים מתויג pHluorin יכול לשמש מבחני קינטית ו / או נקודות קצה, בהתאם להתנהגות נכסים וסחר של מטענים ספציפיים. בנוסף, כמה מטענים מתויג pHluorin ניתנים שיטות ניתוח תפוקה גבוהה, כולל זרימת cytometry. חשוב לציין כי תג pHluorin הוא כלי תכליתי עבור חוקר ענייני endocytic בתאים חיים, המאפשר כימות של אנדוציטוזה והשוואה של פונקציה endocytic ב wild-type ו זנים מוטנטים.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adenine</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A8626-25G</td>
			<td style="width:167px;">Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto-agar</td>
			<td>Fisher</td>
			<td>BP1423-2</td>
			<td>Use for preparation of plate media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Difco Yeast Nitrogen Base (without amino acids)</td>
			<td>BD</td>
			<td>291920</td>
			<td>Use for preparation of liquid and plate media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Concanavalin A</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C5275-5MG</td>
			<td>Use for coating of chamber slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextrose</td>
			<td>Fisher</td>
			<td>BP350-1</td>
			<td>Use for preparation of liquid and plate media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Histidine</td>
			<td>Fisher</td>
			<td>BP382-100</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Leucine</td>
			<td>Acros</td>
			<td>125121000</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Lysine</td>
			<td>Fisher</td>
			<td>BP386-100</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Methionine</td>
			<td>Fisher</td>
			<td>BP388-100</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Tryptophan</td>
			<td>Fisher</td>
			<td>BP395-100</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Tyrosine</td>
			<td>Acros</td>
			<td>140641000</td>
			<td>Use for preparation of amino acid mixture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nunc Lab-Tek Chambered Coverglass (8-well)</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">155411</td>
			<td>Use for kinetic and endpoint assays of Mup1-pHluorin internalization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uracil</td>
			<td>Sigma</td>
			<td>U0750-100G</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Axiovert 200 inverted microscope</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;">Custom Build</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100X/1.4 Plan-Apochromat Oil Immersion Objective Lens</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>Objective should be 100X, 1.4NA or higher</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sensicam</td>
			<td style="width:71px;">Cooke Corporation</td>
			<td style="width:119px;"></td>
			<td>Camera should have 12-bit or higher dynamic range</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">X-Cite 120PC Q Illumination Source</td>
			<td style="width:71px;">Excelitas Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Slidebook 5 software</td>
			<td style="width:71px;">Intelligent Imaging Innovations</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">ImageJ software</td>
			<td style="width:71px;">National Institutes of Health</td>
			<td style="width:119px;"></td>
			<td>http://imagej.nih.gov/ij/</td>
		</tr>
	</tbody>
</table>

applications of phluorin for quantitative, kinetic and high-throughput analysis of endocytosis in budding yeast

חלבון פלואורסצנטי ירוק (GFP) על גירסאותיה משמשים כלים נרחב ללימוד לוקליזציה חלבון ודינמיקה של אירועים כגון שיפוץ cytoskeletal וסחר שלפוחי בתאים חיים. מתודולוגיות כמותיות באמצעות התכת GFP כימרי פותחו עבור יישומים רבים; עם זאת, GFP הוא מעט עמיד proteolysis, ובכך הקרינה שלה נמשכת ליזוזום / vacuole, אשר יכול לעכב כימות של סחר המטען במסלול endocytic. שיטה חלופית לכימות אנדוציטוזה ופוסט endocytic אירועים סחר עושה שימוש superecliptic pHluorin, וריאנט pH רגיש של GFP כי הוא הרווה בסביבה חומצית. היתוך Chimeric של pHluorin לזנב cytoplasmic של תוצאות חלבונים מטען הטרנסממברני בתוך ריסון של הקרינה על ההתאגדות של המטען לתוך הגוף multivesicular (MVBs) והסעה לבית לומן ליזוזום / vacuole. לכן, מרווה הקרינה vacuolar מקלה quantifiקטיון של אנדוציטוזה ואירועים מוקדם מסלול endocytic. מאמר זה מתאר שיטות באמצעות מטענים מתויגים pHluorin כימות של אנדוציטוזה באמצעות מיקרוסקופ פלואורסצנטי, כמו גם מבחנים מבוססי אוכלוסייה באמצעות cytometry זרימה.

לוקליזציה וכימות יציבים של חלבון מטען endocytic הפנים constitutively Ste3 על מנת להוכיח כי מטענים מתויג pHluorin יכול להיות מנוצל עבור כימות של אנדוציטוזה בתאים חיים, לוקליזציה של ה- GFP כימרי ו התכה pHluor...

Watch this Scientific Journal Video about Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast at JoVE.com

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

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.

יישומים של pHluorin עבור כמותי, קינטי וניתוח תפוקה גבוהה של אנדוציטוזה שמרים הנצה

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

applications-phluorin-for-quantitative-kinetic-high-throughput

Research

JoVE Journal

Biology

10.8K Views.  The Johns Hopkins University. 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.

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

Measuring Replicative Life Span in the Budding Yeast

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

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

מדידת תוחלת החיים בשמרים replicative הנצה

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

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

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

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

ניתוח ופיתוח של הפנוטיפ מורפולוגי כפונקציה של ריכוז חלבון שמרים הנצה

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

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

תפוקה גבוהה Overexpression פלסמיד שמרים מסך

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

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 ...

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.

הפקת ניצני שמרים וטיהור חלבון לחקר תפקוד, האינטראקציות, ולאחר translational השינויים

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

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Assay עקידת התפוקה גבוה MHC II לניתוח כמותי של פפטיד אפיטופים

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

שחזור של לבבות למבוגרים דג הזברה עבור יישומי תפוקה גבוהה

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

הקרנת תפוקה גבוהה עבור כימית מאפננים של גני Post-תעתיק מוסדר

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

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.

באמצעות מכשירים microfluidic למדוד פנוטיפים תוחלת החיים סלולר בתאי שמרים ניצני יחיד

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.

הדמיה ברזולוציה גבוהה וניתוח של Dynamics microtubule אסטרל הבודד שמרים ניצנים

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