Name: imaging intranuclear actin rods in live heat stressed drosophila embryos
Uploaded: 2020-05-15T14:30:00
Description: Watch this Scientific Journal Video about Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos at JoVE.com

Författarna erkänner tacksamt Liuliu Zjengs och Zenghui Xues arbete, som hjälpte till att bana väg för denna teknik i Sokac-labbet, liksom Hasan Seede som hjälpte till med analysen. Arbetet med denna studie finansieras genom ett bidrag från NIH (R01 GM115111).

1. Förbered embryosamlingskoppar och äppeljuiceagarplattor
<ol><li>Fem dagar före injektionsexperimentet,konstruera 28 eller skaffa minst två små embryosamlingskoppar. Gör färska 60 mm äppeljuice agarplattor som ska användas med små uppsamlingskoppar28. Förvara tallrikar i plastlådor täckta med fuktiga pappershanddukar vid 4 °C. OBS: Små embryosamlingskoppar, befolkade med flugnummer enligt beskrivningen i steg 1.3, kommer att ge tillräckligt antal embr...

<ol>
	<li>Figard, L., 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=Cofilin-mediated+Actin+Stress+Response+is+maladaptive+in+heat-stressed+embryos.">Cofilin-mediated Actin Stress Response is maladaptive in heat-stressed embryos.</a> Cell Reports. 26 (49), 3493-3501 (2019).</li><li>Ashworth, S. L., 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=ADF/cofilin+mediates+actin+cytoskeletal+alterations+in+LLC-PK+cells+during+ATP+depletion.">ADF/cofilin mediates actin cytoskeletal alterations in LLC-PK cells during ATP depletion.</a> American Journal of Physiology Renal Physiology. 284 (4), 852-862 (2003).</li><li>Vandebrouck, 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=In+vitro+analysis+of+rod+composition+and+actin+dynamics+in+inherited+myopathies.">In vitro analysis of rod composition and actin dynamics in inherited myopathies.</a> Journal of Neuropathology and Experimental Neurology. 69 (5), 429-441 (2010).</li><li>Minamide, L. S., Striegl, A. M., Boyle, J. A., Meberg, P. J., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegenerative+stimuli+induce+persistent+ADF/cofilin-actin+rods+that+disrupt+distal+neurite+function.">Neurodegenerative stimuli induce persistent ADF/cofilin-actin rods that disrupt distal neurite function.</a> Nature Cell Biology. 2 (9), 628-636 (2000).</li><li>Bamburg, J. 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=ADF/Cofilin-actin+rods+in+neurodegenerative+diseases.">ADF/Cofilin-actin rods in neurodegenerative diseases.</a> Current Alzheimer Research. 7 (3), 241-250 (2010).</li><li>Bamburg, J. R., Bernstein, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+dynamics+and+cofilin-actin+rods+in+alzheimer+disease.">Actin dynamics and cofilin-actin rods in alzheimer disease.</a> Cytoskeleton. 73 (9), 477-497 (2016).</li><li>Bernstein, B. W., Chen, H., Boyle, J. A., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+actin-ADF/cofilin+rods+transiently+retards+decline+of+mitochondrial+potential+and+ATP+in+stressed+neurons.">Formation of actin-ADF/cofilin rods transiently retards decline of mitochondrial potential and ATP in stressed neurons.</a> AJP: Cell Physiology. 291 (5), 828-839 (2006).</li><li>Munsie, L. N., Desmond, C. R., Truant, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cofilin+nuclear-cytoplasmic+shuttling+affects+cofilin-actin+rod+formation+during+stress.">Cofilin nuclear-cytoplasmic shuttling affects cofilin-actin rod formation during stress.</a> Journal of Cell Science. 125 (17), 3977-3988 (2012).</li><li>Iida, K., Iida, H., Yahara, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+induction+of+intranuclear+actin+rods+in+cultured+mammalian+cells.">Heat shock induction of intranuclear actin rods in cultured mammalian cells.</a> Experimental Cell Research. 165, 207-215 (1986).</li><li>Ohta, Y., Nishida, E., Sakai, H., Miyamoto, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dephosphorylation+of+cofilin+accompanies+heat+shock-induced+nuclear+accumulation+of+cofilin.">Dephosphorylation of cofilin accompanies heat shock-induced nuclear accumulation of cofilin.</a> Journal of Biological Chemistry. 264 (27), 16143-16148 (1989).</li><li>Iida, K., Matsumoto, S., Yahara, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+KKRKK+sequence+is+involved+in+heat+shock-induced+nuclear+translocation+of+the+18-kDa+actin-binding+protein,+cofilin.">The KKRKK sequence is involved in heat shock-induced nuclear translocation of the 18-kDa actin-binding protein, cofilin.</a> Cell Structure and Function. 17 (1), 39-46 (1992).</li><li>Minamide, L. 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=Isolation+and+characterization+of+cytoplasmic+cofilin-actin+rods.">Isolation and characterization of cytoplasmic cofilin-actin rods.</a> Journal of Biological Chemistry. 285 (8), 5450-5460 (2010).</li><li>Masurovsky, E. B., Benitez, H. H., Kim, S. U., Murray, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin,+development,+and+nature+of+intranuclear+rodlets+and+associated+bodies+in+chicken+sympathetic+neurons.">Origin, development, and nature of intranuclear rodlets and associated bodies in chicken sympathetic neurons.</a> The Journal of Cell Biology. 44 (7), 172-191 (1970).</li><li>Feldman, M. L., Peters, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranuclear+rods+and+sheets+in+rat+cochlear+nucleus.">Intranuclear rods and sheets in rat cochlear nucleus.</a> Journal of Neurocytology. 1 (2), 109-127 (1972).</li><li>Nishida, E., 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=Cofilin+is+a+component+of+intranuclear+and+cytoplasmic+actin+rods+induced+in+cultured+cells.">Cofilin is a component of intranuclear and cytoplasmic actin rods induced in cultured cells.</a> Cell Biology. 84 (8), 5262-5266 (1987).</li><li>Ono, S., Abe, H., Nagaoka, R., Obinata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colocalization+of+ADF+and+cofilin+in+intranuclear+actin+rods+of+cultured+muscle+cells.">Colocalization of ADF and cofilin in intranuclear actin rods of cultured muscle cells.</a> Journal of Muscle Research and Cell Motility. 14 (2), 195-204 (1993).</li><li>Xue, Z., 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=Back-to-back+mechanisms+drive+actomyosin+ring+closure+during+Drosophila+embryo+cleavage.">Back-to-back mechanisms drive actomyosin ring closure during Drosophila embryo cleavage.</a> The Journal of Cell Biology. 215 (3), 335-344 (2016).</li><li>Cao, J., Albertson, R., Riggs, B., Field, C. M., Sullivan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuf,+a+Rab11+effector,+maintains+cytokinetic+furrow+integrity+by+promoting+local+actin+polymerization.">Nuf, a Rab11 effector, maintains cytokinetic furrow integrity by promoting local actin polymerization.</a> Journal of Cell Biology. 182 (2), 301-313 (2008).</li><li>Spracklen, A. J., Fagan, T. N., Lovander, K. E., Tootle, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pros+and+cons+of+common+actin+labeling+tools+for+visualizing+actin+dynamics+during+Drosophila+oogenesis.">The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.</a> Developmental Biology. 393 (2), 209-226 (2014).</li><li>Chen, Q., Nag, S., Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formins+filter+modified+actin+subunits+during+processive+elongation.">Formins filter modified actin subunits during processive elongation.</a> Journal of Structural Biology. 177 (1), 32-39 (2012).</li><li>Iordanou, E., Chandran, R. R., Blackstone, N., Jiang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+interference+by+dsRNA+injection+into+Drosophila+embryos.">RNAi interference by dsRNA injection into Drosophila embryos.</a> Journal of Visualized Experiments. (50), 2477 (2011).</li><li>Juarez, M. T., Patterson, R. A., Li, W., McGinnis, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+wound+assay+and+in+vivo+localization+of+epidermal+wound+response+reporters+in+Drosophila+embryos.">Microinjection wound assay and in vivo localization of epidermal wound response reporters in Drosophila embryos.</a> Journal of Visualized Experiments. 81, 50750 (2013).</li><li>Carreira-Rosario, 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=Recombineering+homologous+recombination+constructs+in+Drosophila.">Recombineering homologous recombination constructs in Drosophila.</a> Journal of Visualized Experiments. (77), 50346 (2013).</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> Journal of Visualized Experiments. (31), 1382 (2009).</li><li>Catrina, I. E., Bayer, L. V., Omar, O. S., Bratu, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+and+tracking+endogenous+mRNAs+in+live+Drosophila+melanogaster+egg+chambers.">Visualizing and tracking endogenous mRNAs in live Drosophila melanogaster egg chambers.</a> Journal of Visualized Experiments. (148), 58545 (2019).</li><li>Wessel, A. D., Gumalla, M., Grosshans, J., Schmidt, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+properties+of+early+Drosophila+embryos+measured+by+high-speed+video+microrheology.">The mechanical properties of early Drosophila embryos measured by high-speed video microrheology.</a> Biophysical Journal. 108 (8), 1899-1907 (2015).</li><li>Mollinari, C., Gonz&#225;lez, A., Cid-Arregui, A., Garc&#237;a-Carranc&#225;, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+and+transgenesis.">Microinjection and transgenesis.</a> Microinjection and Transgenesis: Strategies and Protocols. , 587-603 (1998).</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> Journal of Visualized Experiments. (49), 2503 (2011).</li><li>Oesterle, 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> Pipette Cookbook 2018: P-97 and P-1000 Micropipette Pullers. , (2018).</li><li>Bownes, 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+photographic+study+of+development+in+the+living+embryo+of+Drosophila+melanogaster.">A photographic study of development in the living embryo of Drosophila melanogaster.</a> Journal of Embryology and Experimental Morphology. 33 (3), 789-801 (1975).</li><li>Powsner, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+temperature+on+the+durations+of+the+developmental+stages+of+Drosophila+melanogaster.">The effects of temperature on the durations of the developmental stages of Drosophila melanogaster.</a> Physiological Zoology. 8 (4), 474-520 (1935).</li><li>Hunter, M. V., Willoughby, P. M., Bruce, A. E. E., Fernandez-Gonzalez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+Stress+Orchestrates+Cell+Polarity+to+Promote+Embryonic+Wound+Healing.">Oxidative Stress Orchestrates Cell Polarity to Promote Embryonic Wound Healing.</a> Developmental Cell. 47 (3), 377-387 (2018).</li></ol>

Betydelsen av denna metod är att den använder det väletablerade protokollet för mikroinjektion i Drosophila-embryon 21,22,23,24,25,26,27 för att möjliggöra ny forskning om ASR och medföljande aktinstångsmontering. En stor fördel med att injicera G-aktinRött i l...

Detta protokoll beskriver hur man injicerar G-aktinRöd för att visualisera monteringen av intranukleära aktinstavar i värmestressade embryon som genomgår ett inducerbart Actin Stress Response (ASR)1. Vi utvecklade detta protokoll för att hjälpa studier av ASR, som i embryon leder till störd morfogenes och minskad livskraft, och hos vuxna mänskliga celltyper är associerad med patologier inklusive njursvikt2,muskel myopatier3, och Alzheimers och Huntingtons sjukdom4,5,6,7,8. Denna ASR framkallas av många cellulära påfrestningar, inklusivevärmechock 9,10,11,oxidativ stress4,6,minskad ATP-syntes12, och onormal Huntingtin eller β-amyloid oligomerization4,5,6,7,9,13,14,15,16. Ett kännetecken för ASR är monteringen av avvikande aktinstavar i antingen cytoplasman eller kärnan i drabbade celler, som drivs av stressinducerad hyperaktivering av ett aktin interagerande protein, Cofilin1,5,6,10. Tyvärr kvarstår viktiga kunskapsluckor när det gäller ASR. Till exempel är funktionen hos aktinstavarna inte känd. Vi förstår inte varför stavar bildas i cytoplasman hos vissa celltyper, men andras kärna. Det är inte heller klart om ASR är skyddande eller maladaptivt för celler eller embryon som genomgår stress. Slutligen känner vi fortfarande inte till de detaljerade mekanismerna bakom Cofilin hyperaktivering eller actin stång montering. Således ger detta protokoll en snabb och mångsidig analys för att undersöka ASR genom att visualisera aktinstångsbildning och dynamik i det mycket lätthanterliga experimentella systemet hos det levande fruktflugaembryon.
Protokollet för att mikroinjektera G-aktinrött i levande Drosophila-embryon utvecklades ursprungligen för att studera dynamiken i normala cytoplasmaiska aktinstrukturer 17 under vävnadsuppbyggnadshändelser. I dessa studier fann vi att G-aktinRöd injektion inte negativt påverkade tidiga utvecklingsprocesser i embryot, inklusive cytokines eller gastrulation17,18. Vi ändrade sedan protokollet, anpassning av embryohanteringen och G-aktinRöd injektion för att möjliggöra avbildning av aktinstavar i värmestressade embryon som genomgår ASR1. Andra metoder förutom G-aktinRöd injektion kan användas för att visualisera aktin i embryon. Dessa metoder är beroende av att uttrycka fluorescerande proteiner (FPs) märkta för att fungera eller till domäner av aktinbindande proteiner, såsom Utrophin-mCherry, Lifeact, F-tractin-GFP och Moesin-GFP (granskas19). Att använda dessa FP-sonder kräver dock försiktighet eftersom de kan stabilisera eller störa vissa aktinstrukturer, inte lika märka alla aktinstrukturer20, och när det gäller actin-GFP, är mycket överuttryckta - problematiska för analysen av stångenheten som inte bara är stressberoende utan också aktinkoncentrationsberoende1. Således är G-aktinRöd den föredragna sonden för stavstudier i flugembryon, och embryots stora storlek möjliggör sin enkla injektion.
Arbetsflödet för detta protokoll liknar andra väletablerade mikroinjektionstekniker som har använts för att injicera proteiner, nukleinsyror, droger och fluorescerande indikatorer i Drosophila-embryon 21,22,23,24,25,26,27. Men efter mikroinjektionen av G-aktinRöd här utsätts embryon för mild värmestress för att inducera ASR och intranukleär aktinstångsuppsättning. För laboratorier med tillgång till flugor och en injektionsrigg bör denna metod vara lätt genomförbar och anpassningsbar för specifika studielinjer med avseende på ASR, inklusive induktion av olika påfrestningar eller modulering i distinkt genetisk bakgrund.

<table border="0" cellpadding="0" cellspacing="0" style="width:837px;" width="838">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Adenosine triphosphate (ATP)</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">A23835G</td>
			<td style="width:299px;">Component of G buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Apple juice, Mott&#39;s, 64 fl oz</td>
			<td style="width:195px;">Mott&#39;s</td>
			<td style="width:121px;">014800000344</td>
			<td>Component of apple juice plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Bacto Agar</td>
			<td style="width:195px;">BD</td>
			<td style="width:121px;">214010</td>
			<td>Component of apple juice plates</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Bleach, PureBright Germicidal, 6.0% sodium hypochlorite</td>
			<td style="width:195px;">KIK International</td>
			<td style="width:121px;">059647210020</td>
			<td>For dechorionating embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Calcium chloride</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">C1016500G</td>
			<td>Component of G buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cell strainer, 70 &#956;m</td>
			<td style="width:195px;">Falcon</td>
			<td style="width:121px;">352350</td>
			<td>For collecting dechorionated embryos</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Confocal microscope, LSM 880 34-channel with Airyscan</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">0000001994956</td>
			<td>For imaging intranuclear actin rods</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Desiccant</td>
			<td style="width:195px;">Drierite</td>
			<td style="width:121px;">24001</td>
			<td>For desiccating embryos</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Dissecting microscope, Stemi 508 Stereoscope with 8:1 zoom</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">4350649000000</td>
			<td>For arranging embryos on agar wedge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Dissecting needle, 5 in</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">08965A</td>
			<td>For arranging embryos on agar wedge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Dithiothreitol (DTT)</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">BP1725</td>
			<td>Component of G buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Double-sided Tape, Scotch Permanent, 0.5 in x 250 in</td>
			<td style="width:195px;">3M</td>
			<td>021200010323</td>
			<td>For making embryo glue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Embryo collection cage</td>
			<td style="width:195px;">Genessee Scientific</td>
			<td style="width:121px;">59100</td>
			<td>For housing adult flies and collecting embryos</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Fine tip tweezers, Dumont Tweezer, Style 5</td>
			<td style="width:195px;">Electron Microscopy Sciences</td>
			<td style="width:121px;">72701D</td>
			<td>For arranging embryos on agar wedge</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Glass capillaries, Borosillicate glass, thin 1 mm x 0.75 mm</td>
			<td style="width:195px;">World Precision Instruments, Inc.</td>
			<td style="width:121px;">TW1004</td>
			<td>For microneedles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Halocarbon oil 27</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">H8773100ML</td>
			<td>For hydration of embryos</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:223px;">Heated stage incubator</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">4118579020000, 4118609020000, 4118609010000</td>
			<td>For confocal imaging</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:223px;">Lab Tissue Wipers, KimWipes</td>
			<td style="width:195px;">Kimberly-Clark</td>
			<td style="width:121px;">34155</td>
			<td>Lab tissue wipers</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Light microscope, Invertoskop 40C Inverted Phase contrast microscope, refurbished</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">Discontinued</td>
			<td>Injection microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Methyl-4-hydroxybenzoate</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">H36471KG</td>
			<td>Component of apple juice plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Microinjector, FemtoJet4x</td>
			<td style="width:195px;">Eppendorf</td>
			<td>5253000025</td>
			<td>Microinjector</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Micro loader tips, epT.I.P.S. 20 &#956;L</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:121px;">5242956003</td>
			<td>For loading microneedles</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Micromanipulator and injection stage with x,y,z dials for needle adjustment</td>
			<td style="width:195px;">Bernard Instruments, Inc (Houston, TX)</td>
			<td style="width:121px;">Custom</td>
			<td>For performing microinjections</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Micropipette puller, Model P-97, Flaming/Brown</td>
			<td style="width:195px;">Sutter Instruments</td>
			<td style="width:121px;">P97</td>
			<td>For pulling capillary tubes to make microneedles</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Microscope cover glass 24x50-1.5</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">12544E</td>
			<td>For mounting embryos</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Microscope slides, Lilac Colorfrost, Precleaned, 25 x 75 x 1mm</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">22037081</td>
			<td>For mounting embryos for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">n-Heptane</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">H3601</td>
			<td>Component of embryo glue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Objective, 10x</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">Discontinued</td>
			<td>10x objective for injection microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Objective, C-Apochromat 40x/1,2 W Korr. FCS</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">4217679971711</td>
			<td>40x water objective for confocal</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:223px;">Objective, LD LCI Plan-Apochromat 25x/0.8 Imm Cor DIC M27 for oil, water, silicone oil or glycerine immersion (D=0-0.17mm) (WD=0.57mm at D=0.17mm)</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">4208529871000</td>
			<td>25x mixed immersion objective for confocal</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Objective, Plan-Apocrhomat 63x/1.40 Oil DIC f/ELYRA</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">4207829900799</td>
			<td>63x oil objective for confocal</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Paintbrush, Robert Simmons Expression E85 Pointed Round size 2</td>
			<td style="width:195px;">Daler-Rowney</td>
			<td style="width:121px;">038372016954</td>
			<td>For transferring embryos</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Paper towels, Kleenex C-fold paper towels, white</td>
			<td style="width:195px;">Kimberly-Clark</td>
			<td style="width:121px;">884266344845</td>
			<td>For blotting cell strainer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Pasteur pipette, 5 3/4 in</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">1367820A</td>
			<td>For covering embryos with oil</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Petri dish, glass, 100 x 20 mm</td>
			<td style="width:195px;">Corning</td>
			<td style="width:121px;">3160102</td>
			<td>For humid incubation chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Petri dish, plastic, 60 x 15 mm</td>
			<td style="width:195px;">VWR</td>
			<td style="width:121px;">25384092</td>
			<td>For apple juice plates</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Pipette, Eppendorf Reference 0.5-10 &#956;L</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:121px;">2231000604</td>
			<td>For loading the microneedle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Pipette tip, xTIP4 250 &#956;L</td>
			<td style="width:195px;">Biotix</td>
			<td style="width:121px;">63300006</td>
			<td>For adding embryo glue to coverslip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Razor blade</td>
			<td style="width:195px;">VWR</td>
			<td style="width:121px;">55411050</td>
			<td>For cutting agar wedge, tape, pipette tips</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Rhodamine-conjugated globular actin, human platelet (non-muscle; 4x10 &#956;g)</td>
			<td style="width:195px;">Cytoskeleton, Inc.</td>
			<td style="width:121px;">APHR-A</td>
			<td>G-actin^Red</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Scintillation vial, 20 mL Glass borosillicate with polyethylene liner and urea caps</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">033377</td>
			<td>For making embryo glue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Screw top jar, 16 oz</td>
			<td style="width:195px;">Nalgene</td>
			<td style="width:121px;">000194414195</td>
			<td>For desiccating embryos</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Stage micrometer</td>
			<td style="width:195px;">Electron Microscopy Sciences</td>
			<td style="width:121px;">602104PG</td>
			<td>For calibrating volume of G-actin injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Sucrose</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">840971KG</td>
			<td>Component of apple juice plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Trizma base</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">T15031KG</td>
			<td>Component of G buffer</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Yeast, Lesaffre Yeast Corporation Yeast, Red Star Active Dry, 32 oz</td>
			<td style="width:195px;">Lesaffre Yeast Corporation</td>
			<td style="width:121px;">117929157002</td>
			<td>Component of yeast paste</td>
		</tr>
	</tbody>
</table>

imaging intranuclear actin rods in live heat stressed drosophila embryos

Syftet med detta protokoll är att visualisera intranukleära aktinstavar som monteras i levande Drosophila melanogaster-embryon efter värmestress. Actin stavar är ett kännetecken för en bevarad, inducerbar Actin Stress Response (ASR) som åtföljer mänskliga patologier, inklusive neurodegenerativ sjukdom. Tidigare visade vi att ASR bidrar till morphogenesis misslyckanden och minskad livskraft att utveckla embryon. Detta protokoll möjliggör fortsatt studier av mekanismer som ligger till grund för actin rod assembly och ASR i ett modellsystem som är mycket mottagligt för avbildning, genetik och biokemi. Embryon samlas in och monteras på ett täckglas för att förbereda dem för injektion. Rhodaminkonjugerat klotformigt aktin (G-aktinrött) späds ut och lastas i en mikroneedel. En enda injektion görs till mitten av varje embryo. Efter injektionen inkuberas embryon vid förhöjd temperatur och intranukleära aktinstavar visualiseras sedan genom konfokal mikroskopi. Fluorescensåterhämtning efter försök med fotoblekning (FRAP) kan utföras på aktinstavarna. och andra aktinrika strukturer i cytoplasman kan också avbildas. Vi finner att G-aktinRöd polymeriserar som endogent G-aktin och inte på egen hand stör normal embryoutveckling. En begränsning av detta protokoll är att försiktighet måste vidtas under injektionen för att undvika allvarlig skada på embryot. Men med övning, injicera G-aktinRöd i Drosophila embryon är ett snabbt och tillförlitligt sätt att visualisera aktin stavar och kan lätt användas med flugor av någon genotyp eller med införandet av andra cellulära påfrestningar, inklusive hypoxi och oxidativ stress.

Ett schematiskt arbetsflöde för embryohantering avbildas i figur 1, och en tidsplan för ett typiskt experiment presenteras i tabell 1. En uppskattning för ett bra experimentellt resultat är att för varje 10 embryon som injiceras kommer minst hälften av de embryon som ses att vara i rätt utvecklingsstadium, oskadade och uppvisa en robust ASR med värmestress vid 32 °C. Denna ASR kommer att bevisas genom montering av intranukleära aktinstavar som visas i den represe...

Watch this Scientific Journal Video about Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos at JoVE.com

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

Målet med detta protokoll är att injicera Rhodamine-konjugerat klotformigt aktin i Drosophila embryon och bild intranuclear actin rod montering efter värmestress.

Imaging Intranuclear Actin Rods i levande värme stressade Drosophila embryon

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...

Research

JoVE Journal

Developmental Biology

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

Live-imaging of the Drosophila Pupal Eye

Live-imaging of the Drosophila Pupal Eye

Live-avbildning av<em&gt; Drosophila</em&gt; PUPP Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Ploiditet Manipulering av zebrafiskembryon med Heat Shock 2 Behandling

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

Biosensing Motor Neuron Membran Potential i Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Long-term Live Imaging of Drosophila Eye Disc

Long-term Live Imaging of Drosophila Eye Disc

Långsiktig Live avbildning av<em&gt; Drosophila</em&gt; Ögon Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In Vitro Fröämnet odling för Live-cell Imaging av zygot polarisering och Embryo mallning i Arabidopsis thaliana.

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Levande avbildning och analys av muskelsammandragningar i Drosophila embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Dissektion och live-avbildning av den sena embryonala Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...

Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility in Drosophila

Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility Coupled with Measurement of Epithelial Tension in Drosophila Embryos

Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility Coupled with Measurement of Epithelial Tension in Drosophila Embryos (Video) | JoVE 

Optogenetisk hämning av rho1-medierad aktomyosinkontraktilitet i kombination med mätning av epitelspänning i Drosophila-embryon

Contractile forces generated by actin and non-muscle myosin II ("actomyosin contractility") are critical for morphological changes of cells and tissues at ...

Imaging Live Brain Explants from Drosophila melanogaster Third Instar Larvae

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Investigating Asymmetric Cell Division Dynamics: A Protocol for Live-Imaging of Drosophila Larval Brain Explants (Video) | JoVE

Levande cellavbildning av Drosophila melanogaster Tredje Instar Larval Brains

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...