We thank the Dictyostelium Stock Center for strains and reagents. This study was financed by grants from the Swiss National Science Foundation and the Canton of Basel.

<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שים את התגובה הרעבה מוקדם של discoideum Dictyostelium ידי מיקרוסקופיה זמן לשגות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לגדל תאים DH1.10 או תאים -deficient קורה בתוך הבקבוק Erlenmayer המכיל בינוני HL-5 (1 L: 5 גרם proteose peptone, 5 גרם thiotone E peptone, 10 גלוקוז גרם, תמצית שמרים 5 גרם, 0.35 גרם Na 2 HPO 4 * 7H 2 O, 0.35 גרם KH 2 4 PO, 0.05 גרם dihydrostreptomycin-סולפט, pH 6.6) בשעה 22 ° C באינקובטור רועד עם סיבוב של 160 סל&quot;ד. שמור התאים בצפיפות בין 0.01 x 10 6 תאים / מ&quot;ל ו -2 x 10 6 תאים / מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> בדוק צבירת תא, על ידי תאי DH1.10 קצירת יומן פאזיים וגדל 21 או תאי -deficient קורה 10 שנוצרו ברקע DH1.10, גדלים רועדים תרבות עם מדיום HL-5 על 22 מעלות צלזיוס. כדי לעשות זאת, לקחת את הכמות המתאימה של תאים (בדרך כלל בין 10 ל 50 מ&quot;ל), צנטריפוגות במשך 3 דקות ב 400 XG לשטוף את התאים פעמיים BSS (10 mM NaCl, 10 מ&quot;מ KCl, 2.5 מ&quot;מ CaCl 2, pH 6.5). </li><li style=";text-align:right;direction:rtl"> ספירת תאים באמצעות hemocytometer. כתוצאה מכך, תאי צלחת בצפיפות של (5, 10, 20, או 40) x 10 4 תאים / 2 סנטימטר בצלחת 24 גם. לאפשר להם לדבוק עבור שעה 1 ב 22 מעלות צלזיוס BSS. </li><li style=";text-align:right;direction:rtl"> דמיינו צבירה ידי מיקרוסקופיה זמן לשגות כפי שתואר קודם לכן 10, לקחת תמונות כל 135 שניות. באמצעות הדמית תא חי להגדיר מצויד האובייקטיבי 5X ומצלמת מכשיר אלקטרונים-הכפלת תשלום מצמידים אוטומתית את התוכנה המתאימה (ראה לוח חומרים). </li></ol></li><li style=";text-align:right;direction:rtl"> פועם cAMP של תאים discoideum Dictyostelium במהלך רעב. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לבחון את השפעת פולסים cAMP לשימוש חיצוני על התפתחות תאים DH1.10 21 או תאים -deficient DH1.10 קורה 10, על ידי איסוף תאים. כדי לעשות זאת, לקחת את הכמות המתאימה של תאים (בדרך כלל בין 10 ל 50 מ&quot;ל), צנטריפוגות במשך 3 דקות ב 400 XG ולשטוף פעמיים BSS. </li><li style=";text-align:right;direction:rtl"> couNT תאים באמצעות hemocytometer ו resuspend התאים בצפיפות של 1 x 10 7 תאים / מ&quot;ל ב BSS. לנער את תרבויות (160 סל&quot;ד) בשעה 22 מעלות צלזיוס למשך 2 שעות לפני יישום קטניות. להוסיף קטניות cAMP באמצעות משאבת peristaltic נשלט טיימר. לתכנת את המשאבה כדי לספק דופק שניות 5 כל 6.5 דקות של 15 μl של 50 ננומטר cAMP (ריכוז סופי) על פני תקופה של 5 שעות. </li><li style=";text-align:right;direction:rtl"> ספירת תאים באמצעות hemocytometer. בהמשך לכך, צלחת התאים בצפיפות של (5, 10, 20, או 40) x 10 4 תאים / 2 ס&quot;מ בצלחת 24 גם. אפשר לדבוק עבור שעה 1 ב BSS. </li><li style=";text-align:right;direction:rtl"> דמיין צבירה לאחר 16 שעות ב 22 מעלות צלזיוס על ידי מיקרוסקופ שדה בהיר באמצעות מטרת 5X. </li></ol></li><li style=";text-align:right;direction:rtl"> אינדוקציה של פיתוח discoideum Dictyostelium באמצעות חשיפה בינונית מותנה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כן בינוני מותנה טרי כמתואר 22. איסוף תאי יומן פאזיים DH1.10 21 או תאי -deficient DH1.10 קורית 10, מן רעד כתures עם המדיום HL-5 ב 22 ° C בעזרת פיפטה, צנטריפוגות במשך 3 דקות ב 400 XG ולשטוף תאים שלוש פעמים PBM (0.02 M פוספט אשלגן, 10 מיקרומטר CaCl 2, ו 1 מ&quot;מ MgCl 2, pH 6.1). </li><li style=";text-align:right;direction:rtl"> ספירת תאים באמצעות hemocytometer, resuspend אלה PBM בצפיפות של 1 x 10 7 תאים / מ&quot;ל ולנער במשך 20 שעות ב 110 סל&quot;ד / 22 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> אסוף בינוני מותנה לאחר צנטריפוגה ב g 400 × 3 דקות ולהבהיר ידי צנטריפוגה ב 8000 גרם × במשך 15 דקות ב 4 ° C.. </li><li style=";text-align:right;direction:rtl"> מותנה מסנן בינוני דרך פילטר 0.45 מיקרומטר (ראה לוח חומרים) לדלל משולש ב PBM. </li><li style=";text-align:right;direction:rtl"> ספירת תאים באמצעות hemocytometer צלחת בצפיפות של (5, 10, 20, או 40) x 10 4 תאים / 2 ס&quot;מ בצלחת 24 גם. לאפשר להם לדבוק עבור שעה 1 ב 22 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> supernatant Exchange של תאים עם המדיום המותנה שהוכן קודם לכן. </li><li style=";text-align:right;direction:rtl"> דמיינו צבירהחרה 16 ​​שעות על ידי מיקרוסקופ שדה בהיר באמצעות מטרת 5X. </li></ol></li></ol>

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No conflicts of interest declared.

החלבונים coronin נמצאים מינים רוב clade איקריוטיים. Coronin Dictyostelium discoideum, את homologue של יונקים coronin 1, הוא מעורב בתגובה רעב מוקדם, מאז coronin A-לקוי התאים אינם מסוגלים ליצור מרכזי צבירה במהלך המוקדמות התפתחותי מחזור 10. כדי להיות מסוגל כמותית להעריך נכונת העיכוב בהתפתחות ב?...

משפחת coronin של חלבונים הוא שמור ביותר ברחבי אאוקריוטים. חלבונים אלה מאופיינים על ידי הנוכחות של אזור אמינו מסוף טריפטופן-aspartate (WD) לחזור המכיל ואחריו אזור ייחודי מחובר לתחום מפותל-סליל carboxy מסוף 13,14 (איור 1). Coronins היו מעורבים במגוון של תפקודים תאיים, כולל רגולציה cytoskeletal האות התמרה 12. ביונקים, עד שש מולקולות coronin קצרות (coronin 1-6) וכן &#39;טנדם&#39; coronin 7, יכולה להיות שותף הביע 12,15. Coronin 1 הוא בן המשפחה למד בצורה המקיפה ביותר, ו הוצג להיות מעורב הרס הפתוגן, הישרדות תא T ואיתות עצבית. איך, בדיוק, coronin 1 מבצע פעילויות אלה עדיין לא ברורים. בעוד coronin 1 הוצג להסדיר Ca 2 + ו cAMP תלוי איתות וכן F- תקטין cytoskeleton אפנון 16-18, שיתוף הפוטנציאל-expression של עד 7 בני משפחה ביונקים עשתה זה מאתגר ללמוד את הפונקציה מולקולרית של coronins במערכות אלה, בשל כפילויות פוטנציאליים. בניגוד אורגניזמים יונקים, discoideum Dictyostelium eukaryote נמוך מבטא רק שני בני משפחה coronin (א coronin, את ortholog של B יונקים coronin 1 ו coronin, את ortholog של יונקים coronin 7) עם כנראה לא יתירים פונקציות 15,19,20. עובדה זו הופכת discoideum Dictyostelium מודל חזק כדי ללמוד את הפונקציה של coronins. כדי ללמוד את התפקיד של coronin ב discoideum Dictyostelium, אנו מושרה מעגל ההתפתחות ברעב צלחות רקמות תרבות המכילות תמיסת מלח מאוזן (BSS) חיץ או באמצעות תאי סוג ברים או תאים חסרי coronin A 10. מצאנו כי התאים חסרי coronin לא הצליחו ליצור אגרגטים רבים-תאי על רעב. לקבלת הערכה כמותית מדויקת של פנוטיפ זההדמית תא חייה אוטומטית המתואר בפרוטוקול זה היא כלי חיוני. הפגם בייזום של התגובה רעב מוקדם בתאים חסר coronin A יכול להינצל על ידי אספקת פולסים של cAMP, דבר המצביע על כך coronin מתפקדת במעלה הזרם של מפל cAMP. היישום אקסוגני של פולסים cAMP לדמות לתחילת עבודות הפיתוח נוצל על ידי מספר מעבדות ב 8,9 בעבר. עם זאת, הליך זה ידוע גם להיות תלוי מאוד צפיפות תא ועיתוי. לכן, הפרוטוקול המתואר כאן שואף להפחית variabilities הללו על מנת להבטיח רמה גבוהה של שחזור. יחדיו, טכניקות נוצלו במחקרים אלה מספקים כלים חזקים לחקור פונקציות של חלבונים במהלך השלבים המוקדמים של מעגל ההתפתחות של discoideum Dictyostelium ויש להם את הפוטנציאל לזהות החצים למעלה וכן effectors במורד הזרם של coronin פונקציה.

<table><tbody><tr><td>HL-5 media (for 1 L: 5 g proteose peptone, 5 g thiotone E peptone, 10 g glucose, 5 g yeast extract, 0.35 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O, 0.35 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 0.05 g dihydrostreptomycin-sulfate, pH 6.6)</td><td></td><td></td><td></td></tr><tr><td>Proteose peptone</td><td>BD Bioscience</td><td>211693</td><td></td></tr><tr><td>Thiotone E peptone</td><td>BD Bioscience</td><td>211684</td><td></td></tr><tr><td>Yeast extract</td><td>BD Bioscience</td><td>212750</td><td></td></tr><tr><td>Glucose</td><td>AppliChem</td><td>A3666</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fluka</td><td>71643</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>AppliChem</td><td>A1043</td><td></td></tr><tr><td>dihydrostreptomycin-sulfate</td><td>Sigma-Aldrich</td><td>D1954000</td><td></td></tr><tr><td>PBM (0.02 M potassium phosphate, 10 &amp;mu;M CaCl&lt;sub&gt;2&lt;/sub&gt;, and l mM MgCl&lt;sub&gt;2&lt;/sub&gt;, pH 6.1)</td><td></td><td></td><td>self made</td></tr><tr><td>BSS (10 mM NaCl, 10 mM KCl, 2.5 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, pH 6.5)</td><td></td><td></td><td>self made</td></tr><tr><td>0.45-&amp;mu;m Filtropure S filter</td><td>Sarstedt</td><td>83.1826</td><td></td></tr><tr><td>Falcon 24-well Tissue culture plate</td><td>Fisher Scientific</td><td>08-772-1H</td><td></td></tr><tr><td>Cellobserver microscope</td><td>Zeiss</td><td></td><td>custom built</td></tr><tr><td>AxioVision software</td><td>Zeiss</td><td></td><td></td></tr><tr><td>IPC Microprocessor&amp;ndash;controlled dispensing pump</td><td>ISMATEC</td><td>ISM 931</td><td></td></tr><tr><td>Axiovert 135M microscope</td><td>Zeiss</td><td>491237-0001-000</td><td></td></tr><tr><td>Incubation Shaker</td><td>Inforst HT Minitron</td><td></td><td></td></tr></tbody></table>

investigating the function of coronin a in the early starvation response of dictyostelium discoideum by aggregation assays

אמבת discoideum Dictyostelium נמצאת באדמה, האכלה על חיידקים. כאשר מקורות המזון הופכים נדירים, הם מפרישים גורמים ליזום תוכנית פיתוח רב-תאיים, במהלכה תאים בודדים chemotax לעבר מרכזי צבירה 1-4. תהליך זה תלוי בשחרור של monophosphate אדנוזין המחזורי (cAMP) 5. cAMP מופק בגלים דרך הפעולה המתואמת של cyclase adenylate ו phosphodiesterases, ונקשר לקולטנים cAMP G- חלבון בשילוב 6,7. Assay בשימוש נרחב כדי לנתח את המנגנונים מעורבים מעגל ההתפתחות של discoideum Dictyostelium eukaryote הנמוך מבוסס על התצפית של צבירת תא בתנאים שקועים 8,9. פרוטוקול זה מתאר את הניתוח של התפקיד של coronin במחזור התפתחותי ברעב צלחות רקמות תרבות שקועה תמיסת מלח המאוזן (BSS) 10. Coronin A הוא חבר pr נשמרת נרחבהמשפחה otein של coronins כי היו מעורבים במגוון רחב של פעילויות 11,12. תאים Dictyostelium חסר coronin A הם מסוגלים ליצור אגרגטים רב-תאיים, פגם זה יכול להינצל על ידי אספקת פולסים של cAMP, דבר המצביע על כך coronin מתפקדת במעלה הזרם של cAMP מפל 10. הטכניקות המתוארות מחקרים אלה מספקים כלים חזקים לחקור פונקציות של חלבונים בשלבים הראשונים של מעגל ההתפתחות של discoideum Dictyostelium במעלה הזרם של מפל cAMP. לכן, ניצול assay אגרגציה זה עשוי לאפשר מחקר נוסף של coronin פונקציה ולקדם את הבנתנו ביולוגיה coronin.

תאים חסרים coronin מופע פגם בהתפתחות מוקדמת (איור 2). בהעדר coronin תאים אינם מסוגלים ליצור אגרגטים רב-תאיים, המהווה את הצעד הראשוני במהלך מעגל ההתפתחות של discoideum Dictyostelium. לכן, coronin ינוגן תפקיד במהלך התגובה הרעבה מוקדם ו / או איתות cAMP. אכן, חוסר הי?...

Watch this Scientific Journal Video about Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays at JoVE.com

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

חקירת הפונקציה של Coronin במענה הרעב מוקדם של<em&gt; Discoideum Dictyostelium</em&gt; באמצעות אגרגציה מבחנים

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular ...

investigating-function-coronin-early-starvation-response

Research

JoVE Journal

Biology

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

7.2K Views.  University of Basel. The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

Video: Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

הנדסה גנטית של Dictyostelium discoideum תאים בהתבסס על הבחירה וצמיחה על חיידקים

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Genetics

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein aggregation. The accumulation of protein aggregates in bacterial cells can lead to significant alterations in the cellular phenotypic behavior, including a reduction in growth rates, stress resistance, and virulence. Several experimental procedures exist for the examination of these stressor-mediated phenotypes. This paper describes an optimized assay for the extraction and visualization of aggregated and soluble proteins from different Escherichia coli strains after treatment with a silver-ruthenium-containing antimicrobial. This compound is known to generate reactive oxygen species and causes widespread protein aggregation. 
The method combines a centrifugation-based separation of protein aggregates and soluble proteins from treated and untreated cells with subsequent separation and visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. This approach is simple, fast, and allows a qualitative comparison of protein aggregate formation in different E. coli strains. The methodology has a wide range of applications, including the possibility to investigate the impact of other proteotoxic antimicrobials on in vivo protein aggregation in a wide range of bacteria. Moreover, the protocol can be used to identify genes that contribute to increased resistance to proteotoxic substances. Gel bands can be used for the subsequent identification of proteins that are particularly prone to aggregation.

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

This protocol describes the extraction and visualization of aggregated and soluble proteins from Escherichia coli after treatment with a proteotoxic antimicrobial. Following this procedure allows a qualitative comparison of protein aggregate formation in vivo in different bacterial strains and/or between treatments.

מיצוי והדמיה של אגרגטים חלבון לאחר טיפול של Escherichia coli עם מתח פרוטאוטוקסי

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein ...

Biochemistry

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

ניטור הצטברות חלבונים קינטיקה ב Vivo באמצעות ספירת הכללה אוטומטית ב Elegans Caenorhabditis

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.

We present a freely available workflow built for rapid exploration and accurate analysis of cellular bodies in specific cell compartments in fluorescence microscopy images. This user-friendly workflow is designed on the open-source software Icy and also uses ImageJ functionalities. The pipeline is affordable without knowledge in image analysis.

מנתח מבנה משנה: זרימת עבודה ידידותית למשתמש לחקר מהיר וניתוח מדויק של גופים סלולריים בתמונות מיקרוסקופ פלואורסצנטית

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in ...

Bioengineering

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

התפוקה הגבוהה-שיתוף תרבות החברה לחקירת אינטראקציות מחיידקים

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

Immunology and Infection

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

לימוד חלבון מערכת בקרת איכות של<em&gt; D. discoideum</em&gt; שימוש הדמית תא חי בקרת טמפרטורה

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

הערכה של Dictyostelium discoideum בתגובה חריפה גירוי מכני

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

שיטות מחקר לשינויים הגלום צבירת חלבון עם הגיל ב Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

מדידת תפוקה גבוהה Dictyostelium discoideum Macropinocytosis על ידי Cytometry זרימה

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...

חקירת הפונקציה של Coronin במענה הרעב מוקדם של

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הנדסה גנטית של Dictyostelium discoideum תאים בהתבסס על הבחירה וצמיחה על חיידקים

מיצוי והדמיה של אגרגטים חלבון לאחר טיפול של Escherichia coli עם מתח פרוטאוטוקסי

ניטור הצטברות חלבונים קינטיקה ב Vivo באמצעות ספירת הכללה אוטומטית ב Elegans Caenorhabditis

מנתח מבנה משנה: זרימת עבודה ידידותית למשתמש לחקר מהיר וניתוח מדויק של גופים סלולריים בתמונות מיקרוסקופ פלואורסצנטית

התפוקה הגבוהה-שיתוף תרבות החברה לחקירת אינטראקציות מחיידקים

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

לימוד חלבון מערכת בקרת איכות של

הערכה של Dictyostelium discoideum בתגובה חריפה גירוי מכני

שיטות מחקר לשינויים הגלום צבירת חלבון עם הגיל ב Caenorhabditis elegans

מדידת תפוקה גבוהה Dictyostelium discoideum Macropinocytosis על ידי Cytometry זרימה