פרוטוקול זה פותח תחת האמון Wellcome גרנט. אנו מודים לגב &#39;מורין סינקלייר לשמירה המניות לטוס והכנת מזון זבוב עם השנים. אנו רוצים גם להודות למר מיכאל אייצ&#39;יסון על עזרתו ותמיכה טכנית בפיתוח של פרוטוקול זה.

<ol>
	<li>Holland, A. J., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boveri+revisited:+chromosomal+instability,+aneuploidy+and+tumorigenesis.">Boveri revisited: chromosomal instability, aneuploidy and tumorigenesis.</a> Nat. Rev. Mol. Cell Biol. 10, 478-4787 (2009).</li><li>Zekanowski, C., Wojda, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aneuploidy,+chromosomal+missegregation,+and+cell+cycle+reentry+in+Alzheimer's+disease.">Aneuploidy, chromosomal missegregation, and cell cycle reentry in Alzheimer's disease.</a> Acta Neurobiol. Exp. (Wars). 69, 232-253 (2009).</li><li>Buffin, E., Emre, D., Karess, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flies+without+a+spindle+checkpoint.">Flies without a spindle checkpoint.</a> Nat. Cell Biol. 9, 565-572 (2007).</li><li>Tang, Z., Shu, H., Oncel, D., Chen, S., Yu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+Cdc20+by+Bub1+provides+a+catalytic+mechanism+for+APC/C+inhibition+by+the+spindle+checkpoint.">Phosphorylation of Cdc20 by Bub1 provides a catalytic mechanism for APC/C inhibition by the spindle checkpoint.</a> Mol. Cell. 16, 387-397 (2004).</li><li>Huang, J., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+disappearance+of+cyclin+B+at+the+end+of+mitosis+is+regulated+spatially+in+Drosophila+cells.">The disappearance of cyclin B at the end of mitosis is regulated spatially in Drosophila cells.</a> EMBO Journal. 18, 2184-2195 (1999).</li><li>Xia, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformation-specific+binding+of+p31(comet)+antagonizes+the+function+of+Mad2+in+the+spindle+checkpoint.">Conformation-specific binding of p31(comet) antagonizes the function of Mad2 in the spindle checkpoint.</a> Embo. J. 23, 3133-3143 (2004).</li><li>Lorca, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fizzy+is+required+for+activation+of+the+APC/cyclosome+in+Xenopus+egg+extracts.">Fizzy is required for activation of the APC/cyclosome in Xenopus egg extracts.</a> Embo. J. 17, 3565-3575 (1998).</li><li>Howell, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+dynein/dynactin+drives+kinetochore+protein+transport+to+the+spindle+poles+and+has+a+role+in+mitotic+spindle+checkpoint+inactivation.">Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.</a> J. Cell Biol. 155, 1159-1172 (2001).</li><li>Foe, V. E., Alberts, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+nuclear+and+cytoplasmic+behaviour+during+the+five+mitotic+cycles+that+precede+gastrulation+in+Drosophila+embryogenesis.">Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis.</a> Journal of cell science. 61, 31-70 (1983).</li><li>Li, D., Morley, G., Whitaker, M., Huang, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+of+Cdc20+to+the+kinetochore+requires+BubR1+but+not+Mad2+in+Drosophila+melanogaster.">Recruitment of Cdc20 to the kinetochore requires BubR1 but not Mad2 in Drosophila melanogaster.</a> Mol. Cell Biol. 30, 3384-3395 (2010).</li><li>Karess, R. E., Glover, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P+element+mediated+germ+line+transformation+of+Drosophila.">P element mediated germ line transformation of Drosophila.</a> DNA cloning: A practical approach. 2, 121-141 (1985).</li><li>Wakefield, J. G., Huang, J. Y., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosomes+have+a+role+in+regulating+the+destruction+of+cyclin+B+in+early+Drosophila+embryos.">Centrosomes have a role in regulating the destruction of cyclin B in early Drosophila embryos.</a> Current Biology. 10, 1367-1370 (2000).</li><li>Huang, J. Y., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+localisation+of+the+Drosophila+APC/C:+evidence+for+the+existence+of+multiple+complexes+that+perform+distinct+functions+and+are+differentially+localised.">The dynamic localisation of the Drosophila APC/C: evidence for the existence of multiple complexes that perform distinct functions and are differentially localised.</a> Journal of Cell Science. 115, 2847-2856 (2002).</li><li>Raff, J. W., Jeffers, K., Huang, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+Fzy/Cdc20+and+Fzr/Cdh1+in+regulating+the+destruction+of+cyclin+B+in+space+and+time.">The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the destruction of cyclin B in space and time.</a> Journal of Cell Biology. 157, 1139-1149 (2002).</li><li>Brust-Mascher, I., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+Techniques+for+Studying+Mitosis+in+the+Drosophila+melanogaster+Syncytial+Embryo.">Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo.</a> J. Vis. Exp. (31), e1382 (2009).</li><li>Figard, L., Sokac, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Cell+Shape+Change+in+Living+Drosophila+Embryos.">Imaging Cell Shape Change in Living Drosophila Embryos.</a> J. Vis. Exp. (49), e2503 (2011).</li></ol>

אין לנו מה למסור.

פרוטוקול המתוארת כאן היא שיטה גנרית עבור הדמיה לטוס עוברים סינסיציאלי באמצעות TCS Leica SP2 confocal מיקרוסקופ לייזר סורק והוא יכול להיות שונה כדי להתאים מערכות מיקרוסקופ אחרים, ויכול גם להיות מותאם ללמוד פונקציות גנים אחרים באמצעות עוברים תסיסנית סינסיציאלי. השתמשנו בפרוטוקול זה ללמוד היבטים רבים של המחסום ציר הרכבה, הדינמיקה חלבון proteolysis חלבון מהונדס באמצעות זבובים או נוגדנים polyclonal לדמיין לוקליזציה חלבונים בדגימות חיים או קבועים 4,6,12-14. היא הפשוטה ביותר לשמור על זבובים ב צלוחיות מזון טרי לטוס בעת איסוף מספר קטן (~ 10-100) של ביצים. פעולה זו מפחיתה את הטרחה של ביצוע והכנת נוספים אגר מיץ תפוחים צלחות איסוף בתאי ביצה כפי שמתואר בפרסומים אחרים 15,16. תוספת של ריבוע קטן של זכוכית שבורה coverslip בקצה אחד של רצועת דבק על coverslip עושה את זה הרבה יותר קלכדי לפתוח את המחט לקבוע את עוצמת הזריקה באמצעות התאמת מערכת הגדרות microinjection תוך המחט הוא לטבול בשמן 10s. מידת התייבשות העובר חשוב להצלחה של הזריקה למניעת דליפות ושמירה על יכולת הקיום עוברים במיוחד עבור אלה ניסויים הדורשים תקופת זמן ארוכה או כאשר העלאת transformants לאחר ההזרקה. עם זאת, אין כלל הזהב עבור בדיוק כמה זמן נדרש התייבשות. פעולה זו שונה בין ניסוי לניסוי וגם תלוי בכמות של פתרון מוזרק והלחות של סביבת העבודה. פונקציות של חלבון מסוים שמעניין ניתן להשפיע על ידי microinjecting מעכבי חלבונים ספציפיים, מונו או פולי משובט נוגדנים נגד חלבון מסוים, RNA שליח או פפטידים שכותרתו fluorescently תחת סוג בר או רקע מוטציה גנטית. רבים מאותם קווים מוטנטים או GFP-מתוייגים קווי מהונדס הם באב פומביתailable מ תסיסנית מניות מרכזי ורובם מפורטים על Flybase ( <a href="http://flybase.org/" target="_blank">http://flybase.org/~~V</a> ), ואפשר לחפש באינטרנט.

ציוד חיוני ריאגנטים: מערכת הדמיה confocal: מערכת הדמיה שתואר בפרוטוקול זה הוא TCS Leica SP2 סריקת לייזר מיקרוסקופ confocal מערכת הפוכה. בשיטה המתוארת מתאימה גם אולי עם כמה שינויים קלים עבור מערכות הדמיה אחרים. לנתח את המיקרוסקופ: אנו משתמשים SZX7 אולימפוס עם DF PLAPO 1X-4 העדשה. מחט חולץ: בוער / חום micropipette חולץ (ממודל כלי סאטר,: P-97). מערכת Microinjection: מערכת microinjection Eppendorf מתואר אבל כל מערכת מתאימה אחרת ניתן להשתמש. טוס מפיץ מזון: Jencons מדעי משאבת בע&quot;מ peristaltic. ריאגנטים: <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> מרכיבים </td><td> משקל<html> של </td><td> משאבים </td><td> הרבה לא. </td></tr><tr><td> תירס ארוחה </td><td> 100.0g </td><td> SUMA, בריטניה </td><td></td></tr><tr><td> סוכר חום </td><td> 50.0g </td><td> Billington של בריטניה </td><td></td></tr><tr><td> שמרים יבשים </td><td> 25.0g </td><td> מת&quot;ק שמרים בע&quot;מ בבריטניה </td><td></td></tr><tr><td> אגר </td><td> 12.5g </td><td> פישר סיינטיפיק </td><td> 106556 </td></tr><tr><td> Sorbic חומצה </td><td> 0.4g </td><td> BDH, VWR אינטרנשיונל בע&quot;מ בבריטניה </td><td> 8829310 </td></tr><tr><td> חומצה בנזואית </td><td> 2.9g </td><td> פישר סיינטיפיק </td><td> 1019599 </td></tr><tr><td> Nipagin (Methyl-4-Hydro xybenzoate) </td><td> 0.9g </td><td> BDH, VWR אינטרנשיונל בע&quot;מ בבריטניה </td><td> K35969015 </td></tr><tr><td colspan="3"> H 2 O עד 1 ליטר </td><td></td></tr></tbody></table>class = &quot;jove_content&quot;&gt; בטבלה 1. טוס רכיבי מזון. Holocarbon 700 ו 27 שמנים שנרכשו סיגמא. שמרים יבשים: תומס אליסון, בריטניה. הכנת דבק heptane: קח כ -1.5 מטרים של סרט דו צדדית ויסקי, הכניסו אותו לתוך צינור פלקון 15 מ&quot;ל עם 5 מ&quot;ל של heptane וסובב במשך 3-5 שעות או לילה. לאחר תקופה מחולקים 4 aliquots שווים 1.5 צינורות מ&quot;ל eppendorf צנטריפוגות ומסובב 5 דקות במהירות מהירה בצנטריפוגה הספסל העליון להסיר כל לכלוך. אחסן את הפתרון דבק heptane בבקבוק 10 מ&quot;ל נפח ולשמור לשימוש. הכוח דבק ישתנה בהתאם ריכוז סכומי דבק בו. בדרך כלל, דק יותר דבק מייצרת רעש רקע נמוך יותר כאשר סרק על ידי מיקרוסקופ confocal. <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Coverslip: BDH, VWR אינטרנשיונל בע&quot;מ בבריטניה </li><li style=";text-align:right;direction:rtl"> שקופיות מיקרוסקופ: BDH, VWR אינטרנשיונל בע&quot;מ בריטניה &lt;/ Li&gt; <li style=";text-align:right;direction:rtl"> Siliconised זכוכית נימים: GC-100-10, הרווארד </li><li style=";text-align:right;direction:rtl"> טוען עצה פיין: 20μl הרבה eppendorf: W215818J </li><li style=";text-align:right;direction:rtl"> מלקטת </li><li style=";text-align:right;direction:rtl"> עט פיין מברשת </li></ul>

studying mitotic checkpoint by illustrating dynamic kinetochore protein behavior and chromosome motion in living drosophila syncytial embryos

הרכבה ציר מחסום (SAC) המנגנון הוא אות הפעיל, העוקב אחר האינטראקציה בין kinetochores כרומוזום microtubules ציר כדי למנוע התפרצות anaphase עד הכרומוזומים מחוברים כראוי. תאים משתמשים מנגנון זה, כדי למנוע חוסר יציבות גנומית או aneuploidy, ולכן סרטן ומחלות אנושיות אחרות, כמו מומים מולדים ואלצהיימר 1. מספר הרכיבים שק כגון Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, רוד ואורורה ב קינאז זוהו והם כל החלבונים דינמיים kinetochore 2. הראיות עולה כי kinetochore הוא שם את האות SAC היא יזמה. המטרה העיקרית היא SAC הרגולציה Cdc20. Cdc20 הוא אחד APC / C (P naphase romoting omplex C או C yclosome) activators חיוניים 3, והוא גם חלבון דינמי kinetochore 4-6. כאשר מופעל, SAC מעכב את הפעילות שלPC / C כדי למנוע את הרס שני מצעים מרכזיים, cyclin B ו-securin, ובכך למנוע metaphase כדי anaphase המעבר 7,8. בדיוק איך האות SAC הוא יזם התאספו על kinetochores את ומסר על הנגמ&quot;ש / C לעכב את תפקידו עדיין נשאר חמקמק. תסיסנית היא מערכת ניסיונית צייתן מאוד, אורגניזם פשוט יותר וטוב יותר מובנות לעומת האדם אבל אף אחד כי תהליכים מניות יסוד משותפים. זהו, אולי, אחד היצורים הטובים ביותר לשימוש עבור ביו הדמיה מחקרים בתאים חיים, במיוחד עבור הדמיה של אירועי mitotic במרחב ובזמן, שכן העובר בשלב מוקדם עובר 13 מחזורים מהירים חטיבת גרעיני באופן סינכרוני (8-10 דקות לכל מחזור של 25 ° C) ולאט לאט מארגנת גרעינים בשכבה יחידה בדיוק מתחת קליפת המוח 9. הנה, אני מציג את שיטת ביו הדמיה באמצעות מהונדס תסיסנית הבעהשרים GFP (חלבון ירוק זרחני) או חלבונים גרסה במיקוד שלה של ריבית TCS Leica SP2 confocal מערכת לייזר מיקרוסקופ סריקה כדי ללמוד את הפונקציה SAC אצל זבובי, על ידי הצגת תמונות של חלבונים GFP שילוב של כמה מרכיבים SAC, Cdc20 ו Mad2 , כמו בדוגמה.

Watch this Scientific Journal Video about Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos at JoVE.com

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

Kinetochore שם SAC יוזם האות שלה ניטור הפרדה mitotic של chromatids אחותו. השיטה מתוארת לדמיין את גיוס מחזור של אחד החלבונים kinetochore ותיאום עם תנועה כרומוזום<em&gt; תסיסנית</em&gt; עוברים באמצעות לייזר Leica מערכת סריקה confocal.

לימוד מחסום mitotic ידי הממחישות התנהגות חלבון דינמי Kinetochore ו Motion כרומוזום חיים<em&gt; תסיסנית</em&gt; עוברים סינסיציאלי

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

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Biology

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

11.3K Views.  University of Newcastle, United Kingdom. Kinetochore שם SAC יוזם האות שלה ניטור הפרדה mitotic של chromatids אחותו. השיטה מתוארת לדמיין את גיוס מחזור של אחד החלבונים kinetochore ותיאום עם תנועה כרומוזום תסיסנית עוברים באמצעות לייזר Leica מערכת סריקה confocal.

Video: Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

יישום של שרף אור נרפא, דבק שיניים אלקטרודות הרכבה או בדיקות Microdialysis בניסויים כרונית

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

ברזולוציה גבוהה מדידה של התנהגות ריח מונחה על הזחלים תסיסנית

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

Microinjection טכניקות לימוד מיטוזה ב תסיסנית melanogaster סינסיציאלי עובריים

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

In-vivo צנטריפוגה של עוברים תסיסנית

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi הפרעה על ידי הזרקה לתוך dsRNA תסיסנית עוברים

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

צורה הדמיה תא שינוי חיים תסיסנית עוברים

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

זיהוי אינטראקציה בין חלבונים ב<em&gt; דרוזופילה</em&gt; ראשי מבוגרים טנדם זיקה טיהור (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

In vivo גישת Crosslinking לבודד חלבון מן מכלולים דרוזופילה עוברים

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

הקרנת הגנום כל חלבונים אינטראקציה ידי חלבון-בר השלמה Assay (PCA) בתאים חיים

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

לימוד מבנה ותפקוד מיטוכונדריאלי<em&gt; תסיסנית</em&gt; שחלות

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...