Bu çalışma NIH ALAN programından hibe GM096076 tarafından desteklenmiştir.

1. Diseksiyon için Sinekler hazırlanması <ol><li> Eski bir haftadan daha kısa olan arzu edilen genotip (örneğin, bir GFP-etiketli protein eksprese eden) sağlıklı sinekleri anestetize. Yuvarlak krem ​​renkli karınları ile 10-15 büyük kadın seçin. </li><li> Yeni hafifçe mayalı gıda ihtiva eden bir şişeye, seçilen sinekler ve birkaç (3-5) erkek aktarın ve sinekler 25 ° C&#39;de iki gün boyunca beslemek izin vermek Drosophila hazırlanması ile ilgili ayrıntılar için Weil ve diğ. 6&#39;ya bakınız. Besi sinekler aşamalarında çeşitli, özellikle aşamaları 8-12, görüntüleme için kullanılabilir olmasını sağlar. Kötü besiye veya unfattened genellikle sadece çok erken dönemlerinde (1-6) ve olgun (evre 14) oosit içeren uçar. </li></ol> 2. Dik Bileşik Mikroskop Kullanarak Görüntüleme için Oosit hazırlanması <ol><li> Silikon greas kullanarak, yaklaşık 1 cm aralıklı, slayt iki lamelleri koymak suretiyle standart bir cam slayt hazırlayıne. Sadece lamel ve slayt arasında iyi bir sızdırmazlık sağlamak için yeterli gres kullanmayın. Her lamel bastırarak ince ve eşit gres yayılacak. Lamelleri ve slayt arasındaki noktada bazı Halokarbon yağı 27 (Sigma) ekleyin. Bunlar sonraki adımlarda izole oosit yaralanmasına önlemek için ayırıcılar olarak hizmet edecektir. No 1.5 lamelleri geç evre oositlerin eşleştiğini ortalama kalınlığı (0,16-0,18 mm) ile, iyi çalışır. </li><li> 22 mm&#39;lik 2 lamel yüzeyi üzerinde yer halokarbon yağ 27. 2-3 damla. Mümkün olan sağlıklı oosit elde etmek için tek başına bir dişi ağız pipetleme ile gıda tüpten çıkarıldı ve hafifçe anestezi olmadan önce halokarbon yağ yatırılır. </li><li> Keskin diseksiyon forseps (örneğin Dumont # 5 gibi) kullanarak, lamel karşı sinek bacağına ve karın ucu çekin. Petrol altında, lamel yüzeyi boyunca sürükleyerek yumurtalık çıkarın. Bu adhesio teşvik edeceğizn lamel oositlerin. </li><li> Yavaşça oogenezisin en az evre 10B olan oositlerin arıyor, yumurtalıklar dışında tease. Bu aşamada Oosit oosit yumurta odasının toplam hacminin en az% 50-60 kapladığı yumurta odaları arayan tarafından tespit edilebilir. Bu noktada hayvanlardaki anda, oosit örten folikül hücreleri kolumnar olur ve hemşire hücrelere bağlantıları kalır küçük bir bölge hariç her tarafta oosit çevreleyen var. Ovariole kılıf bireysel oosit kaldırmak için forseps kullanın. </li><li> 5-8 hasarsız oosit lamel izole edilene kadar, 1-3 dişiler kullanarak, devam edin. Herhangi unuseable doku çıkarın. </li><li> Dikkatlice oosit iki aralayıcı arasında konumlandırılmış şekilde önceden hazırlanmış slayt üzerine oosit içeren lamel ters. Bu oositlerin zarar verecektir ters lamel aşağı basmayın. Gerekirse, t halokarbon yağ az miktarda eklemek&quot;sandviç&quot; arasında o kenarlarında boşluk sağlamak için doldurulur. (Opsiyonel:. Lamel yerleşme için izin vermek için birkaç dakika için slayt dinlendirin) slayt artık görüntüleme için hazırdır. </li></ol> 3. Bir Ters Bileşik Mikroskop Kullanarak Görüntüleme için Oosit hazırlanması <ol><li> Oosit yerine lamelleri arasında kullanılan bir cam alt uç (Mattek, 35 mm) ile bu küçük Petri yemekler hariç, esas olarak Bölüm 2&#39;de anlatılan disseke edilir. Bu örnek kapsayacak şekilde gereksiz ve diseksiyon sonrası, doku yemekler doğrudan yansıması olabilir, ancak bakım oosit yağ ile kaplı ve kurumasına izin verilmez kalmasını sağlamak için alınmalıdır. </li></ol> 4. Görüntüleme Canlı <ol><li> DIC veya faz kontrast optikler kullanılarak odak içine bir yumurta getir ve bu sitoplazma sızıntı veya folikül hücreleri şişkin olarak hasar herhangi bir işaret için oosit inceleyin. Sağlıklı ve hasarsız görünen Resim yalnızca oosit. </li><li> Streasitoplazmada yumurta sarısı veziküllerin 7 autofluorescent olduğundan lama, GFP etiketleme için gerek olmaksızın, doğrudan izlenebilir. Bu sinyal kapsamı FITC aralığında yakalanabilir ama büyük olasılıkla GFP etiketli proteinlerin için kullanılandan daha yüksek kazanç ayarları gerektirir. </li><li> Akışının en iyi görüntü sadece cam lamel / cam petri karşı duran bir yumurtanın yüzeyinin altında olan optik kesit elde edilir. İşte oosit biraz odak daha iyi bir uçak için yapım, düzleştirilir. Görüntüleri 200X de çekilebiliyor - 1,000 X. Lamel oosit ve objektif arasında bir bariyer oluşturur yana düzenleme kullanarak burada açıklanan, hava hedefleri kullanılmalıdır. Büyük ölçüde örnek sürüklenme ve hareketi en aza indirir, çünkü bu en uygunudur. </li><li> Ilgilenilen bölge odak sonra, brighfield optik kapatın ve konfokal görüntüleme geçmek. Yazılım hızlı tarama / önizleme fonksiyonunu kullanarak, odak ayarı ve kazançGerekirse. </li><li> Yakalama ayarları mikroskop mikroskop değişir, ancak genel parametreleri aşağıdaki gibidir: ~ 488 nm dalgaboyu ve ~ 515 nm dalgaboyuna kullanın. Z ekseni Z ekseni çerçeve arasında 10 saniyelik bir gecikme ile sabit kalır, böylece yakalama ayarlayın. Oosit dururken ve odak düzlemi uygun sağlamak için 5-10 kare bir test dizisi yakalayın. </li><li> Tipik bir zaman atlamalı dizisi 20 arasında yakalayacaktır - 50 kare ve video kalitesini değerlendirmek için tamamlanmasından hemen sonra gözden geçirilebilir. Işlem slayt diğer hücreleri için tekrar edilebilir, ya da gerektiğinde ek parçalara dişi olabilir. </li></ol> 5. Sorun Giderme ve Ortak Sorunlar <ol><li> Drift - dik bir mikroskop kullanılarak, hücreler bazen nedeniyle petrol yayılmasına kayması olabilir. Bu lamel altındaki alanı kapsayacak şekilde sadece yeterli yağ kullanılarak minimize edilebilir. Sorun devam ederse, daha az silikon g kullanınslayda yapıştırmayın ayırıcılar için rease. </li></ol> Not: Bazı konfokal yazılım paketleri hücre izleme için izin verir, ama genel olarak sürüklenen oosit görüntüleme terk edip onun yerine başka bir örnek kullanmak daha iyidir. <ol start="2"><li> Hiçbir akarsu belirtileri - akış belirgin değilse, büyük olasılıkla iki nedenlerinden biri nedeniyle, ya resim çekimi için odak düzlemi doğru değil (genellikle yumurtanın içine çok derin) veya oosit hasarlı. Zaman ve pratik hem hataları en aza indirebilirsiniz. </li></ol>

<ol>
	<li>Becalska, A. N., Gavis, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lighting+up+mRNA+localization+in+Drosophila+oogenesis.">Lighting up mRNA localization in Drosophila oogenesis.</a> Dev. 136, 2493-2503 (2009).</li><li>He, L., Wang, X., Montell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shining+light+on+Drosophila+oogenesis:+Live+imaging+of+egg+development.">Shining light on Drosophila oogenesis: Live imaging of egg development.</a> Curr. Opin. Gen. Dev. 21, 612-619 (2011).</li><li>King, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ovarian development Drosophila melanogaster. , (1970).</li><li>Spradling, A. C., Bate, M., Martinez-Arias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+genetics+of+oogenesis.">Developmental genetics of oogenesis.</a> The Development of Drosophila melanogaster. 1, 1-70 (1993).</li><li>Serbus, L. R., Cha, B. -. J., Theurkauf, W. E., Saxton, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynein+and+the+actin+cytoskeleton+control+kinesin-driven+cytoplasmic+streaming+in+Drosophila+oocytes.">Dynein and the actin cytoskeleton control kinesin-driven cytoplasmic streaming in Drosophila oocytes.</a> Dev. 132, 3743-3752 (2005).</li><li>Weil, T. T., Parton, R. M., Davis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+Individual+Drosophila+Egg+Chambers+for+Live+Imaging.">Preparing Individual Drosophila Egg Chambers for Live Imaging.</a> J. Vis. Exp. (60), e3679 (2012).</li><li>Theurkauf, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+microtubule-dependent+cytoplasmic+streaming+in+cappuccino+and+spire+mutant+oocytes.">Premature microtubule-dependent cytoplasmic streaming in cappuccino and spire mutant oocytes.</a> Science. 265, 2093-2096 (1998).</li><li>Morin, X., Daneman, R., Zavortink, M., Chia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protein+trap+strategy+to+detect+GFP-tagged+proteins+expressed+from+their+endogenous+loci+in+Drosophila.">A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila.</a> PNAS. 98, 15050-15055 (2001).</li><li>R&#246;per, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rtnl1+is+enriched+in+a+specialized+germline+ER+that+associates+with+ribonucleoprotein+granule+components.">Rtnl1 is enriched in a specialized germline ER that associates with ribonucleoprotein granule components.</a> J. Cell Sci. 120, 1081-1092 (2007).</li><li>Pokrywka, N. J., Payne-Tobin, A., Raley-Susman, K. M., Swartzman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubules,+the+ER+and+Exu:+New+associations+revealed+by+analysis+of+mini+spindles+mutations.">Microtubules, the ER and Exu: New associations revealed by analysis of mini spindles mutations.</a> Mech. Dev. 126, 289-300 (2009).</li><li>Petri, W. H., Mindrinos, M. N., Lombard, M. F., Margaritis, 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=In+vitro+development+of+the+Drosophila+chorion.ih+a+chemically+defined+organ+culture+medium.">In vitro development of the Drosophila chorion.ih a chemically defined organ culture medium.</a> Wilhelm Roux's Arch., Devl. Biol. 186, 351-362 (1979).</li></ol>

Ben ifşa etmek başka bir şey var.

Drosophila oosit hücreleri ve hücre içi bileşenlerin fonksiyon ve organizasyon ile ilgili çeşitli sorular ele almak için çok yönlü bir model sistem vardır. Protein fonksiyonu tam bir anlayış protein davranış hem mekansal ve zamansal yönleri izlenmesini gerektirir. Drosophilists kullanılabilir görüntüleme sistemleri ve genetik araçları hem ilerlemeler mümkün hatta küçük laboratuvarlarında yaşam oositler yüksek kaliteli görüntüleme deneyler gerçekleştirmek için yapmak. Burada protein işlevi yoklamak için genetik geçmişleri çeşitli ile birlikte kullanılabilir geç oogenez orta-sırasında GFP etiketli veya autofluorescent proteinler ve yapıların davranışını analiz etmek için bir protokol özetlemektedir. Bu protokolde Ben oosit görüntüleme ve floresan proteinleri ve yapıların zaman atlamalı video edinimi sağlayacak temel konfokal ayarları için hazırlanan hangi işlemi açıklar. Ek detaioosit hazırlanması ls Weil ve arkadaşları tarafından tarif edilmiştir. 6 A çeşitli endojen ekson-bindirme ile etiketlenmiş proteinleri ifade sinek suşların 8 mevcuttur ve sonsuz daha fazla protein varyantları mühendislik ve sinekler için yeniden girmesini olabilir. Canlı doku ile çalışma olarak, örneklerin sağlığı büyük önem taşımaktadır. Sahne 10B dan Yumurta odalarına kısa süreli görüntüleme çalışmaları için ideal bir aslında otonom moda 11 oogenezisin tamamlayabilirsiniz. Bakım kaliteli veri almak için birkaç temel adım at alınmalıdır. Diseksiyon sırasında mümkün olduğunca az yumurtalıklar ve oosit işlemek ve diseksiyon ve görüntüleme tamamlanması arasındaki süreyi en aza indirmek için önemlidir. İdeal olarak, küçük bir diseksiyon istasyonu konfokal kapsamı evler aynı odada yer almalıdır ve bir seferde sadece bir kaç yumurta odaları yansıması olmalıdır. Numarası (5-8 burada önerilen) Kaliteli görüntüler elde olasılığını maksimize ederken diseksiyon zamanı minimize edilmesini sağlar. Ben görüntü kalitesini değerlendirmek ve yakalama ayarları daha uzun bir zaman atlamalı dizisi yakalamadan önce, optimize edilmiş onaylamak için görüntüleri bir dizi kısa yakalama öneririz. Çoğu yazılım ayrı ayrı kareler kaydedilmesini sağlar, ve bu görüntü daha sonra ImageJ 12 ya da başka yazılım kullanarak çeşitli şekillerde olarak analiz edilebilir.

<table border="1"><tbody><tr><td> Isim </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Halokarbon yağı 27 </td><td> Sigma-Aldrich </td><td> H8773-100ML </td><td></td></tr><tr><td> # 5SF Forseps </td><td> Güzel Bilim Araçları </td><td> 11252-00 </td><td></td></tr><tr><td> Silikon gres </td><td> Sigma Aldrich </td><td> Z273554-1EA </td><td></td></tr><tr><td> Cam alt petri </td><td> Mattek </td><td> P35G-0-20-C </td><td></td></tr></tbody></table>

live imaging of gfp-labeled proteins in drosophila oocytes

Drosophila oosit gibi sitoskeletal fonksiyonu, hücre organizasyonu ve organel yapısı ve fonksiyonu gibi temel soruları araştırmak için çok yönlü bir sistem olarak kurulmuştur. Çeşitli GFP etiketli proteinlerin durumu birçok hücresel süreçleri dakika veya saat boyunca canlı hücreler ve bu tekniği kullanarak izlenebilir anlamına gelir, böyle RNP ulaşım, epitel morfolojilerinden ve doku remodeling gibi süreçler çok detaylı olarak tarif edilmiştir Drosophila oosit 1,2. Mutantlar zengin repertuarı ile birlikte video görüntüleme gerçekleştirmek için yeteneği genler ve süreçleri muazzam bir çeşitlilik inanılmaz ayrıntılı olarak incelenecektir sağlar. Böyle bir örnek, orta oogenez 3,4 az başlatır ooplazmik akışı, bir süreçtir. Sitoplazmik veziküllerin Bu kuvvetli hareket mikrotübül ve kinesin-bağlı 5 ve yararlı bir sistemi içerirBu aşamada hücre iskeleti fonksiyonlarını araştırmak için tem. Burada hemen hemen her konfokal mikroskopi kurulumu kullanarak yaşam oositlerin zaman atlamalı görüntüleme için bir protokol mevcut.

GFP etiketli proteinlerin Görüntüleme etiketli protein ifade ne kadar güçlü bağlı olarak değişir. Reticulon benzeri 1 (Rtnl-1), ekzon GFP 8,9 ile etiketlendi ifade Bir orta kademe oosit güçlü bir sinyal üretir. Rtnl-1 görünür 10 ooplazmik akışı (Şekil 1A) sırasında mevcut uzun mikrotübül ile işbirliği yerelleştirilmesine. Rtnl-1 akışı gözlemlemek için iyi bir belirteç olduğunu ve aynı zamanda geç evre oositlerinde mikrotübül organizasyonu bir göstergesidir. Tek kare yakalama oranı her 10 sn (Şekil 1B) kullanırken bir yabani-tip yumurta odasında ooplazmik akarsu autofluorescent sarısı veziküllerin belirgin hareketi gibi açıktır. Beş kare bir maksimum hareket eden bir projeksiyon veziküllerin yol işaretlemek için oluşturulabilir. <img alt="Şekil 1" src="/files/ftp_upload/50044/50044fig1.jpg" /> Şekil 1. Autofl hem görüntüleme Canlıuorescent ve GFP etiketli yapılar. 400X az Rtnl1-GFP ifade eden bir sahne 11 oositin A) Tek time-lapse görüntü. Rtnl1-GFP elyaf akışı oosit bir dalga gibi hareket hareket. Autofluorescent sarısı veziküller 400X büyütmede, 50 sn toplam her 10 sn görüntülü edildiği bir sahne 10B yumurta odasının B) beş kare maksimum projeksiyon. Ölçek bar = 30 mikron

Watch this Scientific Journal Video about Live Imaging of GFP-labeled Proteins in Drosophila Oocytes at JoVE.com

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

Bireyin GFP etiketli proteinlerin ya autofluorescent yapıların canlı görüntüleme için bir protokol<em&gt; Drosophila</em&gt; Oositler tarif edilmektedir.

GFP etiketli Proteinlerin Görüntüleme Canlı<em&gt; Drosophila</em&gt; Oosit

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

live-imaging-of-gfp-labeled-proteins-in-drosophila-oocytes

Research

JoVE Journal

Biology

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

11.3K Görüntüleme.  Vassar College. Bireyin GFP etiketli proteinlerin ya autofluorescent yapıların canlı görüntüleme için bir protokol Drosophila Oositler tarif edilmektedir.

Video: Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Drosophila Göz hayali Disk Glial Hücre Göç Canlı Görüntüleme

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Biyoloji

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

Hazırlanması Drosophila</emAsma Bırak Protokolü kullanarak Canlı Görüntüleme> Embriyolar

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Uygulamalarda Canlı Görüntüleme Drosophila melanogaster Embriyonik Hemocyte Göçler

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

Canlı Hücre Görüntüleme için SNAP-tag Fusion Proteinler ifade COS-7 Floresan Etiketleme

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

Geliştirilmesi ve Yetişkin hazırlanması Drosophila Brain ve Canlı Görüntüleme için Retinae

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

VE Dik Görüntüleme Drosophila Embriyolar

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

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.

Living Görüntüleme Hücre Şekli Değiştir Drosophila Embriyolar

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

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Bireysel hazırlanması Drosophila</emCanlı Görüntüleme için> Yumurta Chambers

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

DNA Hasarı Tepki Proteinler İki ve Üç Boyutlu Canlı Hücre Görüntüleme

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.