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>

<ol>
	<li>Weijer, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenetic+cell+movement+in+Dictyostelium.">Morphogenetic cell movement in Dictyostelium.</a> Semin Cell Dev Biol. 10, 609-619 (1999).</li><li>Devreotes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dictyostelium+discoideum:+a+model+system+for+cell-cell+interactions+in+development.">Dictyostelium discoideum: a model system for cell-cell interactions in development.</a> Science. 245, 1054-1058 (1989).</li><li>Kimmel, A. R., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cAMP+signal+transduction+pathways+regulating+development+of+Dictyostelium+discoideum.">cAMP signal transduction pathways regulating development of Dictyostelium discoideum.</a> Curr Opin Genet Dev. 1, 383-390 (1991).</li><li>Clarke, M., Gomer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PSF+and+CMF,+autocrine+factors+that+regulate+gene+expression+during+growth+and+early+development+of+Dictyostelium.">PSF and CMF, autocrine factors that regulate gene expression during growth and early development of Dictyostelium.</a> Experientia. 51, 1124-1134 (1995).</li><li>Robertson, A., Drage, D. J., Cohen, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Aggregation+in+Dictyostelium+discoideum+by+an+External+Periodic+Pulse+of+Cyclic+Adenosine+Monophosphate.">Control of Aggregation in Dictyostelium discoideum by an External Periodic Pulse of Cyclic Adenosine Monophosphate.</a> Science. 175, 333-335 (1972).</li><li>Konijn, T. M., Chang, Y. Y., Bonner, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+cyclic+AMP+in+Dictyostelium+discoideum+and+Polysphondylium+pallidum.">Synthesis of cyclic AMP in Dictyostelium discoideum and Polysphondylium pallidum.</a> Nature. 224, 1211-1212 (1969).</li><li>Iranfar, N., Fuller, D., Loomis, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+expression+analyses+of+gene+regulation+during+early+development+of+Dictyostelium+discoideum.">Genome-wide expression analyses of gene regulation during early development of Dictyostelium discoideum.</a> Eukaryot Cell. 2, 664-670 (2003).</li><li>Darmon, M., Brachet, P., Da Silva, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotactic+signals+induce+cell+differentiation+in+Dictyostelium+discoideum.">Chemotactic signals induce cell differentiation in Dictyostelium discoideum.</a> Proc Natl Acad Sci U S A. 72, 3163-3166 (1975).</li><li>Mann, S. K., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+regulation+of+early+gene+expression+in+Dictyostelium+discoideum:+mediation+via+the+cell+surface+cyclic+AMP+receptor.">Cyclic AMP regulation of early gene expression in Dictyostelium discoideum: mediation via the cell surface cyclic AMP receptor.</a> Mol Cell Biol. 7, 458-469 (1987).</li><li>Vinet, A. F., 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=Initiation+of+multicellular+differentiation+in+Dictyostelium+discoideum+is+regulated+by+coronin+A.">Initiation of multicellular differentiation in Dictyostelium discoideum is regulated by coronin A.</a> Mol Biol Cell. 25, 688-701 (2014).</li><li>Eckert, C., Hammesfahr, B., Kollmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+holistic+phylogeny+of+the+coronin+gene+family+reveals+an+ancient+origin+of+the+tandem-coronin,+defines+a+new+subfamily,+and+predicts+protein+function.">A holistic phylogeny of the coronin gene family reveals an ancient origin of the tandem-coronin, defines a new subfamily, and predicts protein function.</a> BMC Evol Biol. 11, 268 (2011).</li><li>Pieters, J., Muller, P., Jayachandran, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+guard:+coronin+proteins+in+innate+and+adaptive+immunity.">On guard: coronin proteins in innate and adaptive immunity.</a> Nat Rev Immunol. 13, 510-518 (2013).</li><li>Gatfield, J., Albrecht, I., Zanolari, B., Steinmetz, M. O., Pieters, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+the+Leukocyte+Plasma+Membrane+with+the+Actin+Cytoskeleton+through+Coiled+Coil-mediated+Trimeric+Coronin+1+Molecules.">Association of the Leukocyte Plasma Membrane with the Actin Cytoskeleton through Coiled Coil-mediated Trimeric Coronin 1 Molecules.</a> Mol Biol Cell. 16, 2786-2798 (2005).</li><li>Kammerer, R. A., 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=A+conserved+trimerization+motif+controls+the+topology+of+short+coiled+coils.">A conserved trimerization motif controls the topology of short coiled coils.</a> Proc Natl Acad Sci U S A. 102, 13891-13896 (2005).</li><li>Uetrecht, A. C., Bear, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronins:+the+return+of+the+crown.">Coronins: the return of the crown.</a> Trends Cell Biol. 16, 421-426 (2006).</li><li>Jayachandran, R., 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=Coronin+1+Regulates+Cognition+and+Behavior+through+Modulation+of+cAMP/Protein+Kinase+A+Signaling.">Coronin 1 Regulates Cognition and Behavior through Modulation of cAMP/Protein Kinase A Signaling.</a> PLoS Biol. 12, e1001820 (2014).</li><li>Suo, D., 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=Coronin-1+is+a+neurotrophin+endosomal+effector+that+is+required+for+developmental+competition+for+survival.">Coronin-1 is a neurotrophin endosomal effector that is required for developmental competition for survival.</a> Nat Neurosci. 17, 36-45 (2014).</li><li>Mueller, P., 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=Regulation+of+T+cell+survival+through+coronin-1-mediated+generation+of+inositol-1,4,5-trisphosphate+and+calcium+mobilization+after+T+cell+receptor+triggering.">Regulation of T cell survival through coronin-1-mediated generation of inositol-1,4,5-trisphosphate and calcium mobilization after T cell receptor triggering.</a> Nat Immunol. 9, 424-431 (2008).</li><li>de Hostos, E. L., Bradtke, B., Lottspeich, F., Guggenheim, R., Gerisch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronin,+an+actin+binding+protein+of+Dictyostelium+discoideum+localized+to+cell+surface+projections,+has+sequence+similarities+to+G+protein+beta+subunits.">Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein beta subunits.</a> EMBO J. 10, 4097-4104 (1991).</li><li>Shina, M. C., 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=Redundant+and+unique+roles+of+coronin+proteins+in+Dictyostelium.">Redundant and unique roles of coronin proteins in Dictyostelium.</a> Cell Mol Life Sci. 68, 303-313 (2011).</li><li>Cornillon, S., 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=Phg1p+is+a+nine-transmembrane+protein+superfamily+member+involved+in+dictyostelium+adhesion+and+phagocytosis.">Phg1p is a nine-transmembrane protein superfamily member involved in dictyostelium adhesion and phagocytosis.</a> J Biol Chem. 275, 34287-34292 (2000).</li><li>Gomer, R. H., Yuen, I. S., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+secreted+80+x+10(3)+Mr+protein+mediates+sensing+of+cell+density+and+the+onset+of+development+in+Dictyostelium.">A secreted 80 x 10(3) Mr protein mediates sensing of cell density and the onset of development in Dictyostelium.</a> Development. (112), 269-278 (1991).</li><li>Iijima, N., Takagi, T., Maeda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+proteinous+factor+mediating+intercellular+communication+during+the+transition+of+Dictyostelium+cells+from+growth+to+differentiation.">A proteinous factor mediating intercellular communication during the transition of Dictyostelium cells from growth to differentiation.</a> Zoological science. 12, 61-69 (1995).</li><li>Mehdy, M. C., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+secreted+factor+and+cyclic+AMP+jointly+regulate+cell-type-specific+gene+expression+in+Dictyostelium+discoideum.">A secreted factor and cyclic AMP jointly regulate cell-type-specific gene expression in Dictyostelium discoideum.</a> Mol Cell Biol. 5, 705-713 (1985).</li><li>Jain, R., Yuen, I. S., Taphouse, C. R., Gomer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+density-sensing+factor+controls+development+in+Dictyostelium.">A density-sensing factor controls development in Dictyostelium.</a> Genes Dev. 6, 390-400 (1992).</li><li>Loomis, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+signaling+during+development+of+Dictyostelium.">Cell signaling during development of Dictyostelium.</a> Dev Biol. 391, 1-16 (2014).</li></ol>

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 1L: 5 g proteose peptone, 5 g thiotone E peptone, 10 g glucose, 5 g yeast extract, 0.35 g Na2HPO4&#8727;2H2O, 0.35 g KH2PO4, 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>Na2HPO4&#8727;2H2O</td>
			<td>Fluka</td>
			<td>71643</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</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 &#956;M CaCl2, and l mM MgCl2, 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 CaCl2, pH 6.5)</td>
			<td></td>
			<td></td>
			<td>self made</td>
		</tr>
		<tr>
			<td>0.45-&#956;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><a href="https://www.fishersci.com/shop/products/falcon-tissue-culture-plates-23/087721h">08-772-1H</a></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&#8211;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 ...

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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

Investigating the Microbial Community in the Termite Hindgut - Interview

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes. The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

חקירת הקהילה חיידקים במעי האחורי הטרמיטים - ראיון

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

שימוש בתגובה optokinetic ללמוד לתפקד החזותי של דג הזברה

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

כימות γH2AX מוקדים בתגובה קרינה Ionising

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

קולטן הדמיה G-חלבון יחד (GPCR) בתיווך אירועים איתות כי הפיקוח של Chemotaxis Discoideum Dictyostelium</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

מעקב אחר דפורמציות המוךפו&quot;גנטי שהולך רקמות בעובר צ&#39;יק מוקדם

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

מדידת תחבורת קטיון ידי Na, K-ו-H, K-ATPase ב<em&gt; Xenopus</em&gt; ביציות על ידי spectrophotometry קליטה האטומית: חלופית למבחני radioisotope

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

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 ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

במבחני צבירת מבחנה באמצעות חלבון Hyperphosphorylated טאו

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

ניתוח של הרכב נפרון ותפקוד בכליות דג הזברה למבוגר

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...