Patrick Cafferty Kanada Multipl Skleroz Derneği bir doktora sonrası burs tarafından desteklenmektedir.

Bölüm 1: Ön-deneysel set-up. <ol><li> Bir hafta önceden arkadaşı larva oluşturmak için uçar ifade GFP kontrolü altında özel bir glial organizatörü. Deneme için bir nükleer yerelleştirme sırası ile ters polarite (repo) organizatörü (3, 4) glial hücreleri kullanarak ifade etiketli GFP görüntülendi. </li><li> % 1 gecede kuru poli-L-lisin çözüm ve hava 10 dakika boyunca 18 mm yuvarlak kapak fişleri iliklerine tarafından en az bir gün önceden fişleri kapak hazırla. </li><li> Deney bir gün önce, bir gecede% 70 etanol bileşenleri iliklerine Chamlide manyetik kültür odası temiz. </li><li> Deney günü, Schneider böcek orta aseptik teknik kullanılarak 10 ml fetal sığır serumu, penisilin-streptomisin çözüm, ve insülin ekleyerek kültür ortamı hazırlamak. Çalışma konsantrasyonları: 1X fetal sığır serum; 100U/ml penisilin, 0.1mg/ml streptomisin ve 0.2 mg / ml Schneider böcek ortamda insülin (değiştirilmiş (5)). </li><li> Kültür odasının bir hücre kültürü kaputu kurumaya bırakın. Hazırlanan kültür ortamı ile durulama kültür odası artık etanol yıkayınız. </li></ol> Bölüm 2: Drosophila göz-beyin kompleks Diseksiyon. <ol><li> Birkaç dakika süreyle buz üzerinde soğutulmuş kültür ortamı bir petri açılan bir sinek şişe ve yerin yan tarafında bir üçüncü instar dolaşıp larva seçin. Larva Chilling diseksiyon kolaylığı için peristaltik vücut kasılmaları yavaş tavsiye edilir. </li><li> Diseksiyon mikroskop altında Sylgard kaplı çanak kültür ortamı bir damla soğuk larva yerleştirin. </li><li> Diseksiyon mikroskobu altında, larva arka ucundan yolu yaklaşık üçte birini sıkıca kavramak için Dumont ince forseps bir çift kullanın. Larva ön sonundan itibaren ağzında kanca extrudes kadar bekleyin. Ağız tam uzantısı üzerine ikinci bir çift forseps ile kanca kavrayın. Larva incelemek için, forseps, iki çift zıt yönlerde yavaşça çekin. Göz-beyin kompleksi, tükürük bezleri, yağ organları, ve hayali doku ile birlikte, larva vücuttan uzakta çeker. Ağız, göz-beyin kompleks yırttınız hızla aksi takdirde çok kanca çekmeyin. </li><li> Tükürük bezleri, yağ organları, ve ultra-ince clipper makas kullanılarak hayali diskler uzak kesin. Beyin ve göz-diskler iki yarımküresinin ventral sinir kablosu ve ağız çengelleri eklenecektir. </li><li> 18 mm kapak kayma bir cam slayt üzerine yerleştirin. Sonra kapak kayma pick up için slayt kenarına asılı kapak kayma bir kenar bırakın. 18 mm kapak kayma kültür ortamı üzerine bir damla ekleyin. Forseps ile ağız kanca tutarak, kültür ortamı içine göz-beyin kompleksi aktarın. Forseps veya doku hasar ile doğrudan göz-beyin kompleksi kavramak etmeyin. </li><li> Ince diseksiyon makas kullanmak, ağız, göz-beyin kompleksi ve atın kanca keserek. Ağız kancaları, doku mikroskopi sırasında taşımak için neden kültür ortamı sözleşme devam edecek gibi ağız kanca kaldırılması canlı görüntüleme için önemlidir. </li><li> Göz hayali disk içinde glial göç görselleştirmek için, dikkatlice kesilmiş ve optik sapı, beyin ve göz disk bağlayan ince bir doku (Şekil 1A) beyin uzağa itmek ya da atın. Optik sapı içindeki glia görselleştirmek için, beyin, optik sapı ve sağlam göz hayali disk (Şekil 1E) bırakın. </li><li> Forseps kullanarak lamel Pick up ve kalıcı bir kalem ile ilgi doku etrafında altında bir daire çizin. Bu daire Bölüm 4 mikroskobu için doku yerini yardımcı olacaktır. </li></ol> Bölüm 3: Manyetik bir kültür odasında göz hayali disk montajı. <ol><li> Forseps kullanarak, lamel asma kenarında kavrayın. Doku kültürü bozmadan Chamlide manyetik odasının alt plaka lamel aktarın. </li><li> Chamlide odasının ana gövde üzerine silikon O-ring yerleştirin. Alt plakası ana gövde üstüne yükleyin. </li><li> Odaya kültür ortamı yavaş yavaş ekleyin. Kültür ortamında çok hızlı ya da ilgi doku rahatsız olacak katmayın. Kapağın altında gaz değişimi için izin odası tamamen doldurmayın. </li><li> Chamlide odasının üstünde görünmez kapağını yavaşça yerleştirin. </li></ol> Bölüm 4: göz disk glial hücreleri göç Görselleştirme. <ol><li> Konfokal veya floresan mikroskobu aşamasında kültür odasına yerleştirin. Düşük büyütme bir referans olarak lamel daire kullanarak örnek bulun. </li><li> 40x bir lens kullanarak örnek üzerinde odaklanın. Doku lamel üzerine yerleşmek için izin verin. Göz disk veya optik sapı 3-4 saat boyunca her 10-15dk görüntüler yakalayın. Doku seğirme ve odak dışında hareket edebilir. El görüntü yakalama önce doku odaklanarak inci hafifletmekbir sorundur. </li></ol> Bölüm 5: Temsilcilik sonuçları: Doğru şekilde gerçekleştirilmemesi, protokol bize göz hayali disk (Şekil 1 BD) optik sapı göç GFP etiketli glial hücreleri görüntüler bir dizi toplamak için izin verdi. 60 dakikalık bir süre için canlı görüntüleme, vahşi bir tür göz hayali disk içinde glial çekirdeğinin pozisyonlarda değişiklikleri gözlemlemek için yeterli iken, glial hücre geçiş için gerekli bir genin mutant glial çekirdekleri tamamen optik sapı (oklar çıkmak için başarısız Şekil 1 FH). Biz kültür dokusunun bozulmasına gözlemleyerek önce 240 dakika gibi uzun süreler için kültürlü göz-beyin kompleksleri var. 240 dakikalık zaman noktası, GFP pozitif hücrelerin kültürlü göz-beyin kompleksler (ok Şekil 2 C) çevreleyen kültür ortamında gözlemlemek için başlar. Ayrıca GFP boyunca diffüz noktalar bir arıza doku bütünlüğü düşündüren dokularda birikir. <img src="/files/ftp_upload/1155/figure1.png" alt="" width="589" height="241" /> Şekil 1: gelişmekte olan yabani tip ve mutant ve görüntü sistemleri GFP etiketli glial çekirdekleri Canlı görüntüleme. A, E) Görüntüler diferansiyel girişim kontrast (DIC), kültürlü yabani tip ve mutant göz hayali diskler (ED) mikroskopi kullanılarak çekilen . Vahşi tip, glial hücreler doğumlu ve göz-disk, optik sapı (OS) göç. Beyin, düzleşme ve göz hayali disk görüntüleme kolaylaştırmak için kaldırılmıştır. BD) (A) 0, 30 ve 60 dakikalık zaman noktalarında GFP etiketli glial çekirdekleri göz disk içinde göç ortaya koymaktadır vahşi tip kültür göz hayali disk Floresan mikroskopi. FH) (E) 0, 30 ve 60 dakikalık zaman içinde GFP etiketli glial çekirdekleri durdurduklarını optik sapı (ok) gösteren mutant göz-beyin kompleks Floresan mikroskopi. (BR) beyin sapı içinde glia görüntüleme kolaylaştırmak için optik sapı bağlı bırakılmıştır. <img src="/files/ftp_upload/1155/Figure_2.png" alt="" width="600" height="156" /> Şekil 2: Canlı görüntüleme vahşi bir tür göz hayali disk GFP etiketli glial membranlarda. Biz başarıyla 240 dakikalık süreler için göz-beyin kültür kompleksleri var. Doku kültürü 240 dakikadan daha uzun yıkmak için başlar. 300 dakikalık bir zaman noktası (C) ile karşılaştırıldığında göz hayali disk çevresindeki kültür ortamı (ED), optik sapı (OS) ve 0 (A) ve 150 dakika (B) beyin lobu (BR), ücretsiz GFP pozitif hücreler, bir ok ile gösterilir.

<ol>
	<li>Aigouy, B., Lepelletier, L., Giangrande, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+chain+migration+requires+pioneer+cells.">Glial chain migration requires pioneer cells.</a> J. Neurosci. 28, 11635-11641 (2008).</li><li>Silies, M., Yuva, Y., Engelen, D., Aho, A., Stork, T., Klambt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cell+migration+in+the+eye+disc.">Glial cell migration in the eye disc.</a> J. Neurosci. 27, 13130-13139 (2007).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Sepp, K. J., Auld, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+lacZ+enhancer+trap+lines+to+GAL4+lines+using+targeted+transposition+in+Drosophila+melanogaster.">Conversion of lacZ enhancer trap lines to GAL4 lines using targeted transposition in Drosophila melanogaster.</a> Genetics. 151, 1093-1101 (1999).</li><li>Gibson, M. C., Patel, A. B., Nagpal, R., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emergence+of+geometric+order+in+proliferating+metazoan+epitheia.">The emergence of geometric order in proliferating metazoan epitheia.</a> Nature. 442, 1038-1041 (2006).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+(MARCM)+for+Drosophila+neural+development.">Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development.</a> Trends Neurosci. 24, 251-254 (2001).</li></ol>

The authors have nothing to disclose.

Bu protokol canlı mikroskopi kullanılarak göz hayali disk glial hücre göç gözlem açıklar. Vahşi tip örnek (Şekil 1 MS), bir saat boyunca göz disk glial hücre hareketi gözlemlemek için bir nükleer GFP işareti kullanılır. Şu anda bizim laboratuvarda çalışma kapsamında, glial hücre göçü için gerekli bir aday gen için bir mutant, biz (Şekil 1 EH) bir saatlik bir süre boyunca bir optik sapı içinde glial hücre çekirdeğinin durdurduklarını gözlemlenmiştir. Bizim stratejimiz ilgi gen tarafından düzenlenen hücresel davranışı daha detaylı görselleştirmek için adapte edilebilir. Örneğin, bir zar gibi mCD8-GFP, GFP molekülün hedeflenen hücre süreçlerini görselleştirmek için glia ifade edilebilir. Mutant glial hücreler filopodia gibi hücresel süreçleri göz disk içine uzanan yeteneğine sahip olup olmadığını belirlemek için izin verecek bir membran bağlı GFP marker kullanın. Benzer şekilde, GFP ile etiketlenen veya tubulin GFP ile etiketlenmiş aktin hücre iskeletinin glial hücre göçü sırasında değişiklikler görselleştirmek için gelişmekte olan glia ifade edilebilir. Ayrıca, tek GFP etiketli mutant glial hücrelerin incelenmesi canlı mikroskobu (6) kullanılarak görüntülendi olabilir. Bu teknik bize daha doğru göç glial hücreler düzenleyen ilgi gen gereği tanımlamak için izin verecektir.

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Poly-L-lysine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P8920</td>
<td>&nbsp;</td>
<tr>
<td>Schneider's Insect Media</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0146</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P4458</td>
<td>&nbsp;</td>
<tr>
<td>Insulin solution from bovine pancreas</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I0516</td>
<td>&nbsp;</td>
<tr>
<td>Chamlide Magnetic chamber</td>
<td>Tool</td>
<td>Live cell Instrument</td>
<td>CM-R-10</td>
<td>35 mm dish type chamber for 18 mm coverslip</td>
</tr>
<tr>
<td>Ultra fine clipper scissors</td>
<td>Tool</td>
<td>Fine scientific tools</td>
<td>15200-00</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 forceps</td>
<td>Tool</td>
<td>Fine scientific tools</td>
<td>11251-20</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscope</td>
<td>Microscope</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>Any fluorescent imaging system that has the necessary filters and excitation for GFP can be used. </td>
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live imaging of glial cell migration in the drosophila eye imaginal disc

Omurgalı ve omurgasız organizmaların hem de glial hücreleri ensheath için nihai hedef bölgelere göç ve ilişkili nöronlar desteği gerekir. Son ilerleme gelişmekte olan pupa kanat (1), çalışmaları glial hücreler canlı göç tanımlamak için yapılmış olsa da<em&gt; Drosophila</em&gt; Glial hücre göçü genellikle sabit dokusunun incelenmesi yer var. Canlı hareketli hücrelerin mikroskobik inceleme, oranı ve büyüme yönünde değişiklikler belirlenmesi de dahil olmak üzere göçmen süreci boyunca hücresel davranışlarını incelemek için yeteneği sunar. Genetik araçlarının kullanımı ile eşleştirilmiş, canlı görüntüleme, kalkınma sürecinde belirli genler için daha hassas rollerini belirlemek için kullanılabilir. Silies tarafından Önceki çalışma<em&gt; Ve ark.</em&gt; (2), sabit dokusunda göz hayali disk, optik sapı, gelişmekte olan beyin ve göz bağlayan bir yapı kaynaklanan göç glia tanımladı. Burada, glial hücrelerin canlı göç incelenmesi için bir protokol taslağını<em&gt; Drosophila</em&gt; Göz hayali disk. Biz gelişmekte olan glia yabani tip ve mutant hayvanlar glial hücre ilerlemesini takip etmek, GFP ifade eden Drosophila hattı yararlanmak.

Watch this Scientific Journal Video about Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc at JoVE.com

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

Burada, glial hücreler göç GFP etiketli glial hücreler ile eşleştirilmiş canlı mikroskobik analizi ile gelişmekte olan Drosophila göz içine incelemek için bir protokol açıklar.

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 ...

live-imaging-glial-cell-migration-drosophila-eye-imaginal

Universidad San Sebastian

Research

JoVE Journal

Biology

10.7K Görüntüleme.  University of British Columbia - UBC. Burada, glial hücreler göç GFP etiketli glial hücreler ile eşleştirilmiş canlı mikroskobik analizi ile gelişmekte olan Drosophila göz içine incelemek için bir protokol açıklar.

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

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

3. Instar Drosophila Larva hayali Disklerin Diseksiyon

Biyoloji

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Canlı görselleştirme Drosophila</emFloresan Boyalar> Glial nöromüsküler Kavşağı

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

Göz hayali Disk Görselleştirme Yardım Roman RFP Reporter Drosophila</em

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

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 ...

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 ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

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 ...

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.

Canlı Hücrelerde Protein Aggregation 4D Görüntüleme

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 ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Apoptotik hücresi Temizliği görüntülenmesi sırasında Canlı<em&gt; Drosophila</em&gt; Embriyo

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Erken Otofaji Etkinlikler Hücre Görüntüleme Canlı: Omegasomes ve Ötesi

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

Mozaik Yetişkin Canlı Görüntüleme Domates / GFP-FLP / FRT Yöntemi<em&gt; Drosophila</em&gt; Fotoreseptör Hücreler

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...