Bu çalışmada kullanılan C. elegans suşları Ulusal Sağlık Enstitüleri tarafından - Office of araştırma altyapı programları (P40 OD010440) desteklenen Caenorhabditis genetik Merkezi'nden elde edilmiştir. Bu eser NIH tarafından desteklenmiştir: 1R03AG048578-01 Jun Liang, Jun Liang ve PSC CUNY-CCRG 1501-CUNY 66184-00 44 ve 67248-00 45 Jun Liang için. Cathy Savage-Dunn nazikçe onu laboratuvar alanı paylaşımı için teşekkür ederiz. Tüm floresans görüntüleri Queens Koleji çekirdek tesisinde toplanmıştır. William J. Rice el yazması üzerine onun yorum için teşekkür.

1. çözümler
<ol><li>
NGM plakaları
	<ol>
<li>Bir 2 L Erlenmeyer şişesi, NaCl, 3 g, agar 17 g, pepton 2.5 g ve dH2O. kapak alüminyum folyo ile balonun ağzı 975 mL ekleyin. Otoklav şişesi 50 dk. 20-30 dakika sakin ol.</li>
		<li>Sonra steril çözümleri ekleyin: 1 mL 1 M CaCl2, 5 mg/mL kolesterol etanol içinde 1 mL, 1 M MgSO41 mL ve 1 M KPO4 (pH 6.0) arabelleği 25 mL. Onları iyice karıştırın çözümleri girdap.</li>
		<li>Sıvı Sabunluk kullanarak, 60 mm Petri plakaları NGM çözümü dağıtmak. Ağar kaplamalar 2/3 tam dolgusuna. Onları ileride kullanmak üzere 4 ° C'de hava geçirmez bir kap içinde saklayın.</li>
	</ol>
</li>
	<li>
1 M KPO4 arabellek (pH 6.0)
	<ol>
<li>KH2PO4 10.83 g ve 3.56 g K2HPO4 ' te dH2O çalışma geçiyoruz, o zaman 100 ml toplam ses seviyesini. Basınçlı kap 15dk için çözüm.</li>
	</ol>
</li>
	<li>
M9
	<ol>
<li>3 g KH2PO4, 6 g Na2HPO dağıtılması4ve 5 gram NaCl dH2O, 1 M MgSO41 mL ekleyin ve 1 m'ye toplam ses seviyesini</li>
	</ol>
</li>
	<li>
10 µg/mL DAPI
	<ol>
<li>10 mg/ml DAPI dH2O. 999 µL içine 1 µL Ekle</li>
	</ol>
</li>
	<li>
% 95 alkol (v/v)
	<ol>
<li>95 mL %100 alkol ölçmek ve ses seviyesini 100 ml distile su ekleyin.</li>
	</ol>
</li>
	<li>
% 30 aseton (v/v)
	<ol>
<li>30 mL aseton dH2için O ekleyin ve toplam ses seviyesini 100 ml.</li>
	</ol>
</li>
</ol>2. ısı stres
<ol><li>Hedef genlerin9,10translasyonel bir yapı ile bir extrachromosomal dizi satırı oluşturmak. Alternatif olarak, bu satırları üzerinden Caenorhabditis genetik Merkezi ( C. elegans araştırma için bir kamu kaynağı olan CGC), veya daha önce yayımlanmış araştırma laboratuvarı alabilirsiniz.</li>
	<li>Extrachromosomal satırları solucanlar 3.800 Rad γ-radyasyon9açarak genom entegre. Sonra her hayvan bir işaretleyici protein GFP veya silindir gibi ifade eder stabil ifade çizgileri seçin.</li>
	<li>Radyasyon sırasında oluşturulan mutasyonlar kaldırmak için çizgiler en az 2 outcross x. Protein ifade deseni değişiklikleri stres altında transgenik bu cümleyi.</li>
	<li>NGM solucan plakaları 1.1. adımda açıklandığı şekilde hazırlayın. Bir OP50 klon çizgi ve sıvı LB suyu bakterilerde gecede kültür. Solucanlar için bir besin kaynağı olarak bir bakteri çim büyümeye sıvı OP50 NGM pilakalar kültür ve 37 ° C kuluçka plakaları kuluçkaya tohum gecede. Plakalar için kullanmadan önce en az 30 dakika oda sıcaklığında sakin.</li>
	<li>20 ° C ısı stres tahlil öncesinde en az 2 nesiller için açlık olmadan transgenik satırında büyümek.</li>
	<li>8-10 genç gravid yetişkin (rahim yumurta ile dolu) bir yeni solucan plaka, sonra onları yumurtlamak için yaklaşık 3-5 h let için almak ve tüm yetişkin plakalar kaldırın.</li>
	<li>Solucanlar eşitlemek için yumurta yumurtadan ve larvaları yaklaşık 48 saat için dördüncü larva (L4) sahne alanı'na büyümeye izin. L4 sahne (Şekil 1, ok)11tanımlamak için onların vulva yapısı (yarım ay yapısı) inceleyin.</li>
	<li>Isı stres ulaşmak 2-5 h için 35 ° C kuluçka L4 larvaları ile solucan tabak koyun. Bir termometre doğru ısısını ölçmek için kuluçka makinesine koyun.</li>
</ol>3. DAPI boyama
<ol><li>
Etanol fiksasyon
	<ol>
<li>10 µL M9 arabelleği bir cam slayt ortasına ekleyin.</li>
		<li>Isı şok solucanlar adım 2.8 toplar ve onları koyardım M9 arabelleğine cam slayt üzerinde.<ol>
<li>Alternatif olarak, M9 ile plaka uzakta yıkama, 1000 x g oda sıcaklığında 1 dk. için de santrifüj kapasitesi ve süpernatant atmak. Solucanlar için cam slayt ekleyin.</li>
		</ol>
</li>
		<li>Herhangi bir aşırı sıvı boşaltmak için yumuşak bir doku kullanın. Diseksiyon mikroskop altında bu adıma dikkat et.</li>
		<li>10 µL % 95 alkol solucanlar üzerine eklemek ve onları kurutma. Diseksiyon mikroskop altında süreci izlemek ve çok kuru olsun hayvan izin vermeyin. Sonra hemen etanol hayvanlardan buharlaşıp vardır, sonraki adıma geçin. Not: Etanol çok hızlı buharlaşır.</li>
		<li>Adımı yineleyin 3.1.4 iki kez böylece solucanlar sabit. Not: Bu hayvanlar için karanlık ve çarpık bakmak normaldir.</li>
		<li>DAPI 10 µL ekleyin (10 µg/mL) solucanlar için. Hemen onları bir coverslip ile karşılamak ve şeffaf oje jel içeren slaydı mühür.</li>
		<li>Floresan mikroskop üzerinde yaklaşık 10 dakika sonra hayvanlar görüntüleyin.</li>
	</ol>
</li>
	<li>
Aseton fiksasyon (alternatif bir yaklaşım)
	<ol>
<li>Adım 2.8 M9 ile ısı şok plaka uzakta yıkama, 1000 x g oda sıcaklığında 1 dk. için de santrifüj kapasitesi ve süpernatant atmak. Pelet dH2O 1 mL ile yıkayın, Pelet toplamak ve süpernatant atın.</li>
		<li>1000 x g için oda sıcaklığında 1 dk 400 µL % 30 aseton 15 dk. santrifüj ekleyin ve süpernatant atın. Hayvanlar 2 yıkamak için dH2O 500 µL kullanın x. Süpernatant atmak.</li>
		<li>DAPI 200 µL ekleyin (10 µg/mL), veya 4 x tüp 15 dakika içinde toplam solucanlar birim miktarı.</li>
		<li>1000 x g 1 dk. için de santrifüj kapasitesi ve süpernatant atın. Pelet 2 yıkama x 500 µL dH2O. ile solucanlar cam slaytlarda yer, onları coverslips ile karşılamak, şeffaf oje jel içeren slaydı mühür ve floresan mikroskop altında hayvanlar gözlemlemek.</li>
	</ol>
</li>
</ol>4. mikroskobik görüntüleme
<ol><li>UV ışık kaynağı ve floresan mikroskop açın. Mikroskop için bağlı olduğu bilgisayarda açın.</li>
	<li>Floresan mikroskop slaytta yükleyin. Solucanlar bulmak, 400 X büyütme gücünü arttırın ve numune üzerinde odaklanmak için bir düşük güç objektif lens (10 X) kullanın.</li>
	<li>Mikroskop ile sağlanan yazılımını açın. "Alma" menüsünde'ı tıklatın; "Kamera" tıklayın ve mikroskop için bağlı kamera seçin; Bu yazılım ile iletişim kurmak seçili kamera sağlar. Not: Farklı mikroskoplar işleminde değişiklik gösterebilir.</li>
	<li>Altında aynı "alma", "Çok boyutlu edinme"'ı tıklatın. Bu yeni bir "Çok boyutlu edinme" gezinti penceresi açar. "Çok kanallı" düğmesini ve tüm kanallar görüntülenir.</li>
	<li>Mavi, yeşil ve numune gözlemlemek için DIC (diferansiyel girişim kontrast) seçin. "Çok boyutlu edinme" Gezinti penceresinde "Mavi-DAPI", "Yeşil-GFP" ve "Gri-DIC", "Nomarski" kanallarda bırakın; "Kırmızı-rodamine" ve "beyaz parlak alan", gibi diğer kanallar sağ tıklayın onları kapatın.</li>
	<li>
İlk olarak, pozlama süresi her kanal için tedbir:
	<ol>
<li>Seçin ve sonra otomatik çekim hızı ile DAPI kanalın altında canlı bir görüntü çekmek için "Ölçü" tıklayın "Mavi-DAPI" kanal sol tıklatın. Canlı bir görüntü ile yeni bir pencere açılacaktır.</li>
		<li>Canlı görüntü penceresinin sol tarafında el ile istediğiniz gibi resim yapmak gerekirse çekim hızı ayarlayın.</li>
		<li>Son olarak "Tamam tıklayın" canlı görüntü penceresinde. Bu çekim hızı DAPI kanalın altında ayarlar.</li>
	</ol>
</li>
	<li>Diğer kanallar, "Yeşil-GFP" ve "Gri-pozlama süreleri bu kanallar için ayarlamak için DIC", "Nomarski" için 4,6 arasındaki adımları yineleyin.</li>
	<li>"Çok boyutlu edinme" Gezinti penceresinde otomatik olarak 3 resim sipariş (bkz. yukarıda) belirtildiği gibi belirli pozlama süreleri ile 3 farklı kanalların altında toplamak için "Başlat" tıklayın.</li>
	<li>Kurtarmak belgili tanımlık eğe için ana menüye gidin, "Dosya" ve sonra "Kaydet"'i tıklatın. Dosya adını ve klasör adını girin. İleride detaylı görüntüleme bilgileri korumak için varsayılan dosya biçimi olarak kaydedin.</li>
	<li>
Diğer biçimlerde dosyasını dışa aktarmak için:
	<ol>
<li>Gitgidmek "Eğe" ve tıkırtı üstünde "ihracat yapmak". Bu bir dışa aktarma ayarı pencere açılacaktır.</li>
		<li>Dosya adı ve klasör ayarlayın. "Bir proje klasörü için daha iyi oluşturmak" verileri düzenlemek kontrol edin. Yazılım aynı zamanda 4 diğer seçenekleri belirtmek kullanıcı sağlar: "Generate birleştirilmiş görüntüler"kanal görüntülerde renk kullanın",", "Kanal adları kullan" ve "Yalnızca birleştirilmiş resim".</li>
		<li>Açılan menüden "TIF", "BMP", "PSD", veya "JPG" ve diğer türleri gibi istediğiniz dosya türünü seçin. O zaman tıkırtı üstünde "ihracat Başlat".</li>
	</ol>
</li>
</ol>

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J., Shen, Z. Z., Cunha, A., Emmons, S. W., Leroi, 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=Somatic+polyploidization+and+cellular+proliferation+drive+body+size+evolution+in+nematodes.">Somatic polyploidization and cellular proliferation drive body size evolution in nematodes.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (10), 5285-5290 (2000).</li><li>Nagamatsu, Y., Ohshima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+the+control+of+body+size+by+a+G-kinase+and+a+downstream+TGFbeta+signal+pathway+in+Caenorhabditis+elegans.">Mechanisms for the control of body size by a G-kinase and a downstream TGFbeta signal pathway in Caenorhabditis elegans.</a> Genes to Cells. 9 (1), 39-47 (2004).</li></ol>

Yazarlar bildirmek için bir şey yok.

Bu makale için çekirdek sitoplazma protein hücre altı değişiklikleri doğrulamak için hızlı ve etkili bir yöntem mevcuttur. Çekirdek yapı (Şekil 3) boyama DAPI tarafından doğrulandı ise protein ifade GFP füzyon tarafından gösterildi. İmmunostaining C. elegans proteinler zor olduğundan, çoğu C. elegans protein hücre altı yerelleştirmeler GFP, Laz, mCherry ve diğerleri gibi işaret proteinler ile etiketleyerek karakterizedir18</sup...

Protein hücre altı yerelleştirme değişikliği yanıt olarak dahili veya harici sinyaller ısı stres, açlık, oksidatif stres, apoptozis, protein fosforilasyon ve diğerleri gibi ortak bir mekanizmadır. Örneğin, ısı stres FOXO üye DAF-16 nükleer translocation1,2ve3,4sinyal alıcı ölümü üzerine mitokondri için teklif translocates pro-apoptotik BCL-2 protein neden olmaktadır. Çeşitli teknikler bu değişiklikleri algılamak kullanılabilir. Western blot ve biyokimyasal olarak izole hücre altı yapıları (Örneğin, mitokondri veya çekirdek) bir arada de hedef3elde edebiliriz. Ancak, spesifik bir antikor protein ilgi karşı gerektirir. Böylece, köklü bir antikor başarı için anahtar olur. Farklı hücre altı yapıları veya organelleri yeşil floresans protein (GFP), kırmızı floresans protein (RFP), sarı floresans protein (YFP) ve mCherry gibi çeşitli işaretçileri olan etiket ve bu arada protein etiket için alternatif bir yaklaşımdır ilgi diğer işaretleri ile. O zaman, onları hedef5,6yerelleştirmek için confocal mikroskop altında gözlemlemek. Radyoaktif izotoplar hedef proteinler etiketleme ve onların hücre altı yerelleştirme7algılama için alternatif bir seçimdir. Ancak, bu yöntem uygun eğitim ve radyoaktif atıkların işlenmesi gerekir. Spesifik bir antikor eksikliği, uygun bir işaretleyici yokluğu veya ekipman confocal mikroskop gibi scarceness gibi bu şartlar altında başka bir yaklaşım dikkate alınması gerekiyor. Protein nükleer translocation tanımlamak için sadece bir işaretleyici ile hedef proteinler etiketlemek için ve çekirdeklerin Kimyasal reaktif 4', 6-diamidino-2-phenylindole (Bu sadece normal floresan mikroskop gerektirdiğinden DAPI) ile leke için çekici.
Immunolabeling C. elegans antikorlar ile yumurta kabuğu veya hayvan çevreleyen kolajen kütikül düşük geçirgenliği nedeniyle meydan okuyor. Bu arada, C. elegans proteinler--dan onların omurgalı orthologs önemli ölçüde farklı olduğu için birkaç ticari şirketler ile belirli ürünler C. elegans sağlar. C. elegans antikorlar kendileri için üretmek küçük bir laboratuvar için zordur. Araştırmacılar toplumda sık sık tagged proteinler protein yerelleştirme veya gen ifade göstermek için imleyicileri kullanın. Bu makale EXL-1::GFP bir protein nükleer translocation ısı stres8altında izlemek için örnek olarak kullanılmıştır. Bir entegre translasyonel exl-1::gfp içine hayvan genom stabil gen gfp füzyon ile ifade etmek için kullanılır. Araştırma exl-1 bağırsak, vücut duvar kas ve diğer hücre altı yapıları8ifade edilir gösterdi. Bu iletişim kuralı, solucanlar dördüncü larva (L4) sahneye eşleştirir, ısı stres deney ve davranış DAPI boyama, etanol fiksasyonu, aseton fiksasyon ve görüntüleme düzenli floresan mikroskop altında gerçekleştirmek gösterilmiştir.

<table><tbody><tr><td>DAPI</td><td>Biotium</td><td>40043</td><td>both Hoechst 33258 and Hoechst 33342 also works well</td></tr><tr><td>OP50</td><td>CGC</td><td>OP50</td><td>https://cgc.umn.edu/</td></tr><tr><td>Caenorhabditis Genetics Center (CGC)</td><td></td><td></td><td>https://cgc.umn.edu/</td></tr><tr><td>Kim Wipe</td><td>Kimberly-Clark Corporation</td><td></td><td>soft tissue</td></tr><tr><td>Zeiss&amp;nbsp; AX10</td><td>Zeiss</td><td></td><td>fluorescence microscope</td></tr><tr><td>Axiovision Rel 4.8</td><td>Zeiss</td><td></td><td>microscope software</td></tr><tr><td>AxioCam MR Rev3</td><td>Zeiss</td><td></td><td>digital camera</td></tr><tr><td>Incubator&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Micro centrifuge</td><td></td><td></td><td></td></tr><tr><td>Transparent nail polish gel</td><td></td><td></td><td></td></tr><tr><td>60 mm petri dishes</td><td></td><td></td><td></td></tr><tr><td>Glass slides</td><td></td><td></td><td></td></tr><tr><td>Glass coverslips</td><td></td><td></td><td></td></tr><tr><td>1-20 &amp;micro;L pipettor</td><td></td><td></td><td></td></tr><tr><td>20-200 &amp;micro;L pipettor</td><td></td><td></td><td></td></tr><tr><td>200-1000 &amp;micro;L pipettor</td><td></td><td></td><td></td></tr><tr><td>1-200 &amp;micro;L pipet tips</td><td></td><td></td><td></td></tr><tr><td>200-1000 &amp;micro;L pipet tips</td><td></td><td></td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>Platinum wire worm pick</td><td></td><td></td><td></td></tr></tbody></table>

detecting protein subcellular localization by green fluorescence protein tagging and 4',6-diamidino-2-phenylindole staining in caenorhabditis elegans

Bu iletişim kuralı, bir yeşil floresans protein (GFP) füzyon protein ve 4', 6-diamidino-2-phenylindole (DAPI) boyama protein hücre altı yerelleştirme değişiklikleri; izlemek için kullanılır Özellikle, bir ısı altında nükleer translocation stres durumu. Proteinler buna bağlı olarak dış ve iç sinyalleri tepki. Onun hücre altı yerelleştirme değiştirmek için ortak bir mekanizmadır. Bu makalede bir antikor, radyoaktif etiketleme veya confocal mikroskop gerektirmez protein yerelleştirme izlemek için bir iletişim kuralı. Bu makalede, GFP memeli CLIC4 de dahil olmak üzere klorür hücre içi kanal proteinleri (CLICs) ailesinin C. elegans, üye EXL-1 hedef protein etiketlemek için kullanılır. Bir entegre translasyonel exl-1::gfp transgenik satır (bir organizatörü ve tam gen sıralaması) dönüştürme ve γ-radyasyon tarafından oluşturulmuş ve stabil gfpve gen ifade eder. Son araştırma ısı stres, değil oksidatif stres, EXL-1::GFP çekirdeğinde biriken gösterdi. GFP sinyal çekirdek yapısı ve DAPI ile örtüşen sinyalleri stres altında EXL-1 hücre altı yerelleştirme değişiklikleri onaylar. Bu iletişim kuralı DAPI boyama için iki farklı fiksasyon yöntemi sunar: etanol fiksasyon ve aseton fiksasyon. Bu makalede sunulan DAPI boyama iletişim kuralı hızlı ve verimli ve GFP sinyal hem protein hücre altı yerelleştirme değişiklikleri korur. Bu yöntem yalnızca Nomarski, FITC filtre ve DAPI filtre ile floresan mikroskop gerektirir. Bir küçük laboratuvar ayarı, lisans öğrencisi araştırma, lise öğrenci araştırma ve biyoteknoloji derslik için uygundur.

Klorür hücre içi kanal proteinler (klik) türler12arasında son derece korunmuş çok fonksiyonlu proteinlerdir. CLICs hücresel stres, autophagy, apoptozis, karsinojenezis, anjiogenez ve makrofaj doğuştan gelen bağışıklık yanıtı memeli sistem13,14,15,16 düzenleyen çok araştırma gösterir ,17<...

Watch this Scientific Journal Video about Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans at JoVE.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

Bu iletişim kuralı bir protein nükleer translocation ısı stres altında bir yeşil floresans protein (GFP) füzyon proteini bir işaretleyici olarak ve 4', 6-diamidino-2-phenylindole (DAPI) kullanarak izlemek gösterilmiştir boyama. İletişim kuralı boyama DAPI hızlı ve GFP ve protein hücre altı yerelleştirme sinyalleri korur.

Protein hücre altı yerelleştirme yeşil floresans Protein etiketleme ve 4', 6-Diamidino-2-phenylindole Caenorhabditis elegans içinde boyama tarafından tespit

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

detecting-protein-subcellular-localization-green-fluorescence-protein

Research

JoVE Journal

Biology

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

9.5K Görüntüleme.  Borough of Manhattan Community College/CUNY. Bu iletişim kuralı bir protein nükleer translocation ısı stres altında bir yeşil floresans protein (GFP) füzyon proteini bir işaretleyici olarak ve 4', 6-diamidino-2-phenylindole (DAPI) kullanarak izlemek gösterilmiştir boyama. İletişim kuralı boyama DAPI hızlı ve GFP ve protein hücre altı yerelleştirme sinyalleri korur.

Video: Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

C. elegans Excretory kanal hücre içi Lumen morfogenez ve In Vivo polarize membran Biyogenez tek bir hücrede için bir Model olarak: etiketleme GFP-füzyon, RNAi etkileşimi ekran ve görüntüleme

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

Gelişim Biyolojisi

C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining

The C. elegans germline makes an excellent model for studying meiosis, in part due to the ease of conducting cytological analyses on dissected animals. Whole mount preparations preserve the structure of meiotic nuclei, and importantly, each gonad arm contains all stages of meiosis, organized in a temporal-spatial progression that makes it easy to identify nuclei at different stages. Adult hermaphrodites have two gonad arms, each organized as a closed tube with proliferating germline stem cells at the distal closed end and cellularized oocytes at the proximal open end, which join in the center at the uterus. Dissection releases one or both gonad arms from the body cavity, allowing the entirety of meiosis to be visualized. Here, a common protocol for immunofluorescence against a protein of interest is presented, followed by DAPI staining to mark all chromosomes. Young adults are immobilized in levamisole and quickly dissected using two syringe needles. After germline extrusion, the sample is fixed before undergoing a freeze crack in liquid nitrogen, which helps permeabilize the cuticle and other tissues. The sample can then be dehydrated in ethanol, rehydrated, and incubated with primary and secondary antibodies. DAPI is added to the sample in the mounting medium, which allows reliable visualization of DNA and makes it easy to find animals to image under a fluorescent microscope. This technique is readily adopted by those familiar with handling C. elegans after a few hours spent practicing the dissection method itself. This protocol has been taught to high-schoolers and undergraduates working in a research lab and incorporated into a course-based undergraduate research experience at a liberal arts college.

C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining

This is a commonly used method for C. elegans gonad dissection followed by freeze crack, which produces germline samples for immunofluorescence via antibody staining, or for simple DAPI staining to visualize DNA. This protocol has been successful for undergraduates in a research lab and in a course-based undergraduate research experience.

C. elegans · İmmünofloresan ve DAPI Boyama için Gonad Diseksiyonu ve Dondurucu Çatlak

The C. elegans germline makes an excellent model for studying meiosis, in part due to the ease of conducting cytological analyses on dissected animals. ...

Genetik

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate&#160;C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

This method describes a protocol for high-throughput protein extract preparation from Caenorhabditis elegans samples and subsequent co-immunoprecipitation.

Caenorhabditis elegans'tan Protein Özü Hazırlama ve Eş-immün premobpriktiasyon

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Görüntüleme C. elegans Embriyolar Epifluorescent Mikroskop ve Açık Kaynak Yazılım

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Biyoloji

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Erken Embriyo Oluşumu Zaman atlamalı Mikroskopi Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

Caenorhabditis elegans Bağırsaklarında Mikrobiyal Kolonizasyon ve Enfeksiyonu Görselleştirmek için RNA Floresan in situ Hibridizasyon (FISH)

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

İmmünoloji ve Enfeksiyon

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Yöntemler içinde Muscle hücrealtı Kompartmanları DeğerlendirecekÜSKÜP<em&gt; ° C. elegans</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

Caenorhabditis elegans'ta Agrega Niceliğinin Otomatikleştirilmesi

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

Mitofajinin Ölçülmesi C. elegans ve Karaciğer Kanseri Hücre Hattı

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, regulated cell death, and the generation of reactive oxygen species (ROS). ROS are vital for key biological processes. However, when uncontrolled, they can lead to oxidative injury, including mitochondrial damage. Damaged mitochondria release more ROS, thereby intensifying cellular injury and the disease state. A homeostatic process named mitochondrial autophagy (mitophagy) selectively removes damaged mitochondria, which are then replaced by new ones. There are multiple mitophagy pathways, with the common endpoint being the breakdown of the damaged mitochondria in lysosomes. 
Several methodologies, including genetic sensors, antibody immunofluorescence, and electron microscopy, use this endpoint to quantify mitophagy. Each method for examining mitophagy has its advantages, such as specific tissue/cell targeting (with genetic sensors) and great detail (with electron microscopy). However, these methods often require expensive resources, trained personnel, and a lengthy preparation time before the actual experiment, such as for creating transgenic animals. Here, we present a cost-effective alternative for measuring mitophagy using commercially available fluorescent dyes targeting mitochondria and lysosomes. This method effectively measures mitophagy in the nematode Caenorhabditis elegans and human liver cells, which indicates its potential efficiency in other model systems.

Yazar Gündemi: Organele Özgü Boyalar Kullanılarak Caenorhabditis elegans ve Memeli Hücrelerinde Mitofajinin Tespiti  

Exploring mitophagy through electron microscopy, genetic sensors, and immunofluorescence requires costly equipment, skilled personnel, and a significant time investment. Here, we demonstrate the efficacy of a commercial fluorescence dye kit in quantifying the mitophagy process in both Caenorhabditis elegans and a liver cancer cell line.

Caenorhabditis elegans ve Memeli Hücrelerinde Mitopajinin Organele Özgü Boyalar Kullanılarak Saptanması

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, ...

Protein hücre altı yerelleştirme yeşil floresans Protein etiketleme ve 4', 6-Diamidino-2-phenylindole Caenorhabditis elegans içinde boyama tarafından tespit

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Yöntemler içinde Muscle hücrealtı Kompartmanları DeğerlendirecekÜSKÜP

Caenorhabditis elegans'ta Agrega Niceliğinin Otomatikleştirilmesi

Caenorhabditis elegans ve Memeli Hücrelerinde Mitopajinin Organele Özgü Boyalar Kullanılarak Saptanması