CQS receives a fellowship from the LOEWE Excellence Cluster for Integrative Fungal Research (IPF). The author would like to thank Ida J. van der Klei for the P. chrysogenum strains used in this work and Andreas S. Reichert for the gift of rapamycin.

1. הכנת P. chrysogenum לניסויי הרעב <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אם P. זן chrysogenum עניין נשמר על אורז (&quot;אורז ירוק&quot;), מקום 2-3 גרגרי אורז מכוסים המתנבג תפטיר לתוך צינור 1.5 מיליליטר microcentrifuge. מלא אותו בYGG 500 μl (10 גר &#39;/ ליטר KCl, 20 גר&#39; / גלוקוז l, 10 גר &#39;/ בסיס חנקן שמרי l, 5 גר&#39; / ליטר K 2 HPO תמצית שמרי 4, 20 גר &#39;/ ליטר). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מערבולת הצינור למשך 30 שניות כדי שנבגים יכולים להתנתק מהאורז ביעילות. </li><li style=";text-align:right;direction:rtl"> דגירה הצינור ליום 1 בטמפרטורת חדר (למשל, בין 20 ל 25 מעלות צלזיוס). </li><li style=";text-align:right;direction:rtl"> הכן דילול 1/50 של השעיה נבג זה במים ברז סטרילי, ומערבב היטב. </li></ol></li><li style=";text-align:right;direction:rtl"> אם P. זן chrysogenum עניין נשמר על צלחת אגר (למשל, צלחת אגר YGG), להעביר חתיכות קטנות של התפטיר לתוך צינור 1.5 מיליליטר microcentrifuge המכיל 400 μl מים ברז סטרילי וכ GL 100 מ&quot;גחרוזים התחת באמצעות איסוף שן סטרילי או פיפטה קצה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מערבולת הצינור למשך 2 דקות, כך שהתפטיר הופך מקוטעת. השתמש בתוכן של הצינור באופן מיידי. </li></ol></li></ol> 2. שלבי הכנה לטיפוח P. chrysogenum על רפידות רעב <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לעבור על צלחת חימום ולהגדיר אותו כ 60 ° C. </li><li style=";text-align:right;direction:rtl"> לפזר 2 גרם של agarose ב100 מ&quot;ל מי ברז על ידי בישול הפתרון במיקרוגל. תשמור על עצמך כי פתרון זה הוא ברור לחלוטין לאחר חימום. אם זה לא, לבשל אותו שוב עד agarose הוא נמס לגמרי. תן פתרון agarose להתקרר כ 60 ° C לפני השימוש. </li><li style=";text-align:right;direction:rtl"> מלא 1.5 מיליליטר צינורות microcentrifuge (2-4, תלוי במספר דגימות) במים ברז 400 μl סטרילי כל אחת (לא תרכובות נוספות הוסיפו) או 400 x μl (תוספת של x μl של פתרון מתחם בשלב 2.5) ולמקם אותם לצלחת חימום דקות לפחות 1, כך שהוואטאה בתוך מתחמם. </li><li style=";text-align:right;direction:rtl"> הנח שקופיות מיקרוסקופ המכילות חלל מרכזי לצלחת חימום דקות לפחות 1. </li><li style=";text-align:right;direction:rtl"> להוסיף 400 μl 2% agarose לפתרון בצינורות 1.5 מיליליטר microcentrifuge ובקצרה מערבולת. לאחר שלב זה אם תרצה בכך, להוסיף תרכובות נוספות (כלומר, rapamycin 1 מיקרומטר). אם זה נעשה, מערבולת בקצרה לאחר כל חומר נוסף הוא pipetted לצינור. שים את הצינורות בחזרה על שולחן החימום כדי למנוע התמצקות מוקדמת. הערה: הנפח הכולל של צינור microcentrifuge צריך להיות 800 μl. </li></ol> 3. הכנת שקופיות מיקרוסקופ המכילה רפידות רעב <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 140 μl פיפטה של ​​פתרון agarose לתוך החלל של שקופיות מיקרוסקופ (שעדיין נמצא על צלחת החימום). נסה להימנע מבועות במידת האפשר. </li><li style=";text-align:right;direction:rtl"> מייד במקום להחליק כיסוי על פתרון agarose. הערה: זה יגרום משטח שטוח. </li><li style=";text-align:right;direction:rtl"> שים מיקרושקופית היקף על ספסל המעבדה לכ -4 דקות, כדי לאפשר את כרית agarose כדי לחזק. </li><li style=";text-align:right;direction:rtl"> מוציאים בזהירות את coverslip מפנקס agarose ידי הזזה אותו בעזרת אצבע אגודל או מדד (ללבוש כפפות, כדי למנוע זיהום!). </li><li style=";text-align:right;direction:rtl"> מניחים את שקופית מיקרוסקופ עם הכרית לחדר רטוב כדי למנוע התייבשות. המלצה: השתמש בתיבת קצה ריקה שעדיין יש להכניס להחזקת הטיפים פיפטה. מוסיף מים סטריליים לתיבה, כך שהחלק התחתון מכוסה. מניחים את שקופיות מיקרוסקופ על ההוספה ולסגור את התיבה. </li></ol> 4. ייחס רפידות הרעב עם פ chrysogenum ודגירה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 5 μl פיפטה של ​​דילול 1/50 הנבג (אם התרבויות גדלו על אורז) או 5 μl של הפתרון חי מכיל שברי תפטיר (אם התרבויות גדלו על צלחת אגר) על מרכז כרית הרעב. </li><li style=";text-align:right;direction:rtl"> סגור את התא הרטוב ולעטוף אותו בשקית ניילון (זה משמש כהגנה נוספת מפני התייבשות של רפידות הרעב). היזהר שלא להזיז את הקופסה יותר מדי כי אחר טיפות החיסון יהיו מוטרדות. </li><li style=";text-align:right;direction:rtl"> אחסן את התיבה העטופה בRT לפחות 20 שעות לפני ניתוח הדגימות. </li></ol> 5. ניתוח מיקרוסקופי של הדגימות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאחר 20 שעות של דגירה הדגימות מוכנות לניתוח מיקרוסקופי; לשים שקופית מיקרוסקופ על נייר טישו יבש (התחתון נוטה להיות רטוב). </li><li style=";text-align:right;direction:rtl"> פיפטה נפח קטן של מים (בערך 50 μl) על כרית הרעב. שים coverslip על הכרית ולסחוט את מים עודפים. הסר את עודפי מים עם נייר טישו. </li><li style=";text-align:right;direction:rtl"> שים את שקופיות מיקרוסקופ על שולחן ההתבוננות במיקרוסקופ פלואורסצנטי. כדי לקבל סקירה כללית של צמיחת תפטיר, להשתמש אובייקטיבי 20X. מטרות השימוש 63X או 100X ללימוד הלוקליזציה של ה- GFP בעובש. על מנת למנוע התייבשות הדרגתית של המדגם לא לנתח דגימות ליותר מ -15 דקות לפני שמכניס אותם בחזרה לתוך החדר הרטוב. </li><li style=";text-align:right;direction:rtl"> חזור על 5.3 במרווחי זמן קבועים (לדוגמא, לאחר 40 שעות ו -60 שעות של צמיחה). הערה: המדגם ניתן להחזיר לתא הרטוב. אין צורך להסיר את coverslip כפי שהוא אינו משפיע על צמיחת תפטיר. </li></ol>

<ol>
	<li>Fischer, F., Hamann, A., Osiewacz, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+quality+control:+an+integrated+network+of+pathways.">Mitochondrial quality control: an integrated network of pathways.</a> Trends Biochem. Sci. 37 (7), 284-292 (2012).</li><li>Pollack, J. K., Harris, S. D., Marten, M. R. <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+filamentous+fungi.">Autophagy in filamentous fungi.</a> Fungal Genet. Biol. 46 (1), 1-8 (2009).</li><li>Scheckhuber, C. Q., Osiewacz, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Podospora+anserina:+a+model+organism+to+study+mechanisms+of+healthy+ageing.">Podospora anserina: a model organism to study mechanisms of healthy ageing.</a> Mol. Genet. Genomics. 280 (5), 365-374 (2008).</li><li>Pinan-Lucarr&#233;, B., Iraqui, I., Clav&#233;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Podospora+anserina+target+of+rapamycin.">Podospora anserina target of rapamycin.</a> Curr. Genet. 50 (1), 23-31 (2006).</li><li>Kikuma, T., Ohneda, M., Arioka, M., Kitamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+the+ATG8+homologue+Aoatg8+and+role+of+autophagy+in+differentiation+and+germination+in+Aspergillus+oryzae.">Functional analysis of the ATG8 homologue Aoatg8 and role of autophagy in differentiation and germination in Aspergillus oryzae.</a> Eukaryot. Cell. 5 (8), 1328-1336 (2006).</li><li>Pinan-Lucarr&#233;, B., Balguerie, A., Clav&#233;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+cell+death+in+Podospora+autophagy+mutants.">Accelerated cell death in Podospora autophagy mutants.</a> Eukaryot. Cell. 4 (11), 1765-1774 (2005).</li><li>Veneault-Fourrey, C., Barooah, M., Egan, M., Wakley, G., Talbot, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagic+fungal+cell+death+is+necessary+for+infection+by+the+rice+blast+fungus.">Autophagic fungal cell death is necessary for infection by the rice blast fungus.</a> Science. 312 (5773), 580-583 (2006).</li><li>Bartoszewska, M., Kiel, J. A., Bovenberg, R. A., Veenhuis, M., van der Klei, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+deficiency+promotes+beta-lactam+production+in+Penicillium+chrysogenum.">Autophagy deficiency promotes beta-lactam production in Penicillium chrysogenum.</a> Appl. Environ. Microbiol. 77 (4), 1413-1422 (2011).</li><li>Meijer, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisomes+are+required+for+efficient+penicillin+biosynthesis+in+Penicillium+chrysogenum.">Peroxisomes are required for efficient penicillin biosynthesis in Penicillium chrysogenum.</a> Appl. Environ. Microbiol. 76 (17), 5702-5709 (2010).</li><li>Cubitt, A. B., Heim, R., Adams, S. R., Boyd, A. E., Gross, L. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding,+improving+and+using+green+fluorescent+proteins.">Understanding, improving and using green fluorescent proteins.</a> Trends Biochem. Sci. 20 (11), 448-455 (1995).</li><li>Jung, C. H., Ro, S. H., Cao, J., Otto, N. M., Kim, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTOR+regulation+of+autophagy.">mTOR regulation of autophagy.</a> FEBS Lett. 584 (7), 1287-1295 (2010).</li><li>Hickey, P. C., Read, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+living+cells+of+Aspergillus+in+vitro.">Imaging living cells of Aspergillus in vitro.</a> Med. Mycol. 47, S110-S119 (2009).</li></ol>

The author has nothing to disclose.

השיטה המוצגת כאן מאפשרת מחקר והנוח לשחזור של autophagy בפ chrysogenum. לדוגמא, ניתן להשתמש בו לסינון היעילות של תרכובות שונות אם הם מסוגלים ויסות תגובת autophagy של פטרייה זו או לא. התוצאות עם rapamycin להוכיח כי עיכוב של איתות TOR מוביל לאינדוקציה בולטת של autophagy בפ chrysogenum שגם הו?...

פטריות פילמנטיות הן מערכות מודל מצוינות לחקר תהליכים התפתחותיים. הם מציעים מספר יתרונות ניסיוניים כמו טיפוח זול, מספרים גבוהים של צאצאים ונגישות גנטית. הנקודה האחרונה היא רלוונטית במיוחד לבניית transformants המאפשרים לחקור את החשיבות של גנים, עד כה, uncharacterized למנגנונים תאיים שונים. פטריות פילמנטיות כבר סייעו להבהרה של מספר גורמים ומנגנונים של מסלולי בקרת איכות סלולריות כמו פעילות פרוטאז לפירוק של חלבונים חריגים, דינמיקה של המיטוכונדריה לשמירה על השלמות וautophagy המיטוכונדריה להסרת עודפים ו / או רכיבי תא מתפקדים ולשמירה כדאיות תא בזמנים של 1,2,3 רעב. ישנן מספר שיטות ניסיוניות זמינות ללימוד autophagy בפילמנטיות פטריות 2: חקירה (i) של vacuoles if הם מכילים גופי autophagic צפופים כאשר פרוטאזות מעוכבת על ידי מיקרוסקופי אלקטרונים הילוכים 4, (ii) להדמיה של autophagosomes על ידי ניטור מוקדי GFP-Atg8 באמצעות מיקרוסקופ פלואורסצנטי 5,6 וכן (iii) זיהוי של מבני autophagosomal acidified באמצעות קדברין monodansyl צבע פלואורסצנטי 7. כאן, שיטה חדשה של גובר chrysogenum Penicillium ללימודי autophagy מוצגת. האלמנט העיקרי הוא &#39;פנקס הרעב&#39;, שפשוט מורכב של 1% מומסים agarose במים ברז מעוקר. תרכובות נוספות (למשל, לחצים, אוכלי נבלות, מאפנני autophagy) ניתן להוסיף לפנקס כל עוד הם לא להציג אוטומטי הקרינה. הכרית ממוקמת בשקופיות מיקרוסקופ המכילות חלל מרכזי רדוד. פנקס זה במחוסן או עם השעיה נבג או עם שברי תפטיר קטנים. האחרון הוא מומלץ אם המתח של עניין אינו sporulatדואר יעילות (למשל, זני atg1 Δ 8). השקופיות ממוקמות בתאים רטובים (ניתן לבנות האלה בקלות על ידי שימוש בתיבות קצה פיפטה ריקות) כדי למנוע התייבשות של המדגם וטופחו בטמפרטורת חדר. P. chrysogenum הוא מסוגל לגדול לכמה ימים בתנאים אלה. Autophagy ניתן לצפות מיקרוסקופי על ידי הגדלת vacuolar אשר מהווה סמן חיובי לפטריית autophagy. בתרומה זו, P. זן chrysogenum (Wisconsin 54-1255) משמש המהווה חלבון פלואורסצנטי ירוק כי הוא ממוקד לperoxisomes על ידי הרצף &#39;SKL&#39; C-המסוף שלה 9. לכן ניתן לעקוב אחר השפלה של peroxisomes. זה אפשרי לתייג גם תאים אחרים של התא (לדוגמא, מיטוכונדריה) באמצעות אותות לוקליזציה מתאימים ולנתח שפלתם. למרות נתונים מפ chrysogenum Ws54-1255 (GFP-SKL) מוצג כאן, זה בהחלט אפשרילהשתמש בשיטת &#39;פנקס רעב &quot;גם לפטריות פילמנטיות אחרות (למשל, crassa Neurospora, macrospora Sordaria, מיני Aspergillus, וכו&#39;).

<table><tbody><tr><td>Microscope slides with central cavity</td><td>Carl Roth GmbH, Karlsruhe, Germany</td><td>H884.1</td><td>These can be used multiple times after cleaning.</td></tr><tr><td>Glass beads</td><td>Carl Roth GmbH, Karlsruhe, Germany</td><td>A553.1</td><td>Diameter: 0.25 - 0.50 mm</td></tr></tbody></table>

analysis of autophagy in penicillium chrysogenum by using starvation pads in combination with fluorescence microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

כדי להדגים את התועלת של הפרוטוקול המפורט לעיל השפלה peroxisome בפ זן chrysogenum Ws54-1255 (GFP-SKL) נותח. בזן זה GFP-SKL בדרך כלל מיובא לperoxisomes 9. התוצאה היא ההופעה של צורות כדוריות מרובות כאשר המדגם מנותח באמצעות מיקרוסקופ פלואורסצנטי. אם autophagy מתרחש, vacuoles להגדיל. GFP-SKL הופך ?...

Watch this Scientific Journal Video about Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy at JoVE.com

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

ניתוח של Autophagy ב<em&gt; Chrysogenum Penicillium</em&gt; על ידי שימוש ברפידות רעב בשילוב עם מיקרוסקופ פלואורסצנטי

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

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Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

8.5K Views.  Senckenberg Research Institute. A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

Video: Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Cecal קשירת ונקר-Induced אלח דם כמודל ללמוד Autophagy בעכברים

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

Immunology and Infection

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.

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 ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

ביולוגיה של התא כמותית של הקשורים ניוון מוחיים ב דרוזופילה דרך ניתוח לא משוחדת של חלבונים מתויגות Fluorescently באמצעות ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Neuroscience

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

ניתוח כמותי של Autophagy באמצעות מיקרוסקופ פלואורסצנטי 3D מתקדם

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

לחיות תא הדמיה של אירועי autophagy מוקדמים: Omegasomes ומעבר

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

הערכת השטף autofagic ידי מדידת LC3, p62, ו LAMP1 Co-localization באמצעות multispectral הדמיה cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

חקר אינטראקציות חלבון במחקר Autophagy

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

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

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

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

בחינת Mitotic ומיוטיק ביקוע הדינמיקה הגרעינית על ידי הזריחה לחיות-מיקרוסקופ התא

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

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

יישום של ממברנה וקיר תא צבעי פלורסנט סלקטיבית עבור הדמיה בשידור חי התא של פטריות Filamentous

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans.&#160;The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis.

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

The present protocol describes the induction of autophagy in the Drosophila melanogaster larval fat body via nutrient depletion and analyzes changes in autophagy using transgenic fly strains.

ניתוח אוטופגיה הנגרמת על ידי רעב בגוף השומן של הזחל Drosophila melanogaster

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. ...

ניתוח של Autophagy ב

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