Yazar Dr. Asako Sugimoto (Tohoku Üniversitesi) derinden yararlı onun tartışma için teşekkürler. wyIs251 Doktor Kang Shen (Stanford Üniversitesi) cömert bir hediye etmişti. mnIs17 araştırma altyapı programları (P40 OD010440) NIH ofisi tarafından finanse edilen CGC tarafından sağlandı. Bu eser JSP'ler KAKENHI grant #17 H 05010 ve #16 H 06536 ve Daiichi Sankyo Vakfı, beyin bilim Vakfı ve Naito Vakfı tarafından desteklenmiştir.

1. numune hazırlama <ol><li>Generate transgenik C. elegans zorlanma transgenik metodolojisi 18 kullanarak ilgi. Alternatif olarak, uygun suşların Caenorhabditis genetik Merkezi (CGC) dan elde edilir. Sinaptik vezikül öncüleri aksonal taşımacılığının görselleştirmek için transgenik çizgi wyIs251 kullanın [Pmig-13::gfp::rab-3] 7. LFT gözlemlemek için transgenik çizgi mnIs17 [osm-6::gfp] 19 edinin. Bu protokol için bu suşlar kullanılır. 
	Not: Ya extrachromosomal dizi, genomik entegrasyonu veya bile GFP çakma çalışır. Arka plan sinyalleri azaltmak için pan-nöronal rehberleri yerine hücre özel Organizatör kullanın. 
	Not: Bakım Worms diğer protokolleri 20 dakika içinde açıklanmıştır. Suşları tutulan Escherischia coli OP50 besleyici nematodunun büyüme üzerinde çimenler üzerinde orta (NGM) levhalar (%1,7 (w/v) özel, 50 mM NaCl, %0.25 (w/v) pepton, 1 mM CaCl 2, 5 mg/mL kolesterol, 25 mM KH 2 PO 4, 1 mM MgSO 4 60 mm çap plastik yemekleri) 20 ° C.</li>
	<li>Grow solucanlar 20 ° C'de NGM Tabaklarda solucanlar herhangi bir yaşam döngüsü aşaması için. 
	Not: Bir aksonal transport ve LFT herhangi bir aşamada gözlemleyebilirsiniz. Genç Yetişkin geç L4 çünkü iyi bir pozitif kontrol birden çok yayın-si olmak kullanılmış bu aşamaları ve görüntülenmeyecektir kaçakçılığı olayları 14 , 21 , 22 , 23.</li>
</ol> 2. % 10 hazırlanması özel Not: pek çok satıcı agar agar tozlar ve özel gibi benzer ürünler sağlar. Elektroforez sınıf özel kullanın (jel gücü &#62; 1200 g/cm 2). Ucuz agar tozlar işe yaramaz çünkü elde edilen jel solucanlar hareketsiz yeterince güçlü değil.
<ol><li>Mix 0.4 g özel 4 mL distile su (DW) bir cam tüp (1.5 cm x 10,5 cm). Buharlaşma önlemek için cam tüp üzerinde bir kapak koyun.</li>
	<li>90 dk için 95 ° C ısı bloğu cam tüp yerleştirilir. Buna ek olarak, DW ile dolu başka bir cam tüp (1.5 cm x 10,5 cm) hazırlamak ve 95 tutmak ° C. koymak Pasteur damlalıklı 95 ° C'de DW ( şekil 1 A). Bir sürü küçük kabarcıklar özel tüp içinde görülecektir ve çözüm bulanık olmalıdır. Çözüm açık hale gelinceye kadar bırakın. Sonra karıştırmak özel çözüm homojen hale gelinceye kadar yukarı ve aşağı tarafından pipetting. 
	Not: Mikrodalga fırın DW ve özel yapılmış zaman % 10 özel çözüm hazırlamak için kullanılamaz. Küçük kabarcıklar mikrodalga tarafından neden olduğu özel erime önlemek. Ancak, katılaşmış % 10 özel jel kolayca kullanarak yeniden bir mikrodalga erimiş olabilir. Cam tüpler % 10 özel jel içeren önceden hazırlanan ve 4 ° C'de en az 6 ay süreyle depolanır. Böylece, gerektiğinde bir mikrodalga kullanarak stok eritmek ve bu iletişim kuralı 3 adımda başlayabilirsiniz. 
	Not: Özel yavaş yavaş uzun bir süre veya sonra tekrarlanan katılaşma ve erime 95 ° C'de inkübe Eğer kuvvetlendirmek için yetenek kaybeder. Bu durumda, yeni % 10 özel hazırlayın.</li>
</ol> 3. Hazırlık özel ped <ol><li>Adım 2.2, hazırlanan ısıtılan Pasteur damlalıklı kullanarak 76 x 22 mm slayt üzerinde 2 damla % 10 özel yerleştirin.</li>
	<li>Hızlı bir şekilde Resim 1 B ve itme aşağı ikinci slaytta özel çözüm düzleştirmek için gösterildiği gibi kapak özel damla önce ikinci 76 x 22 mm 2 kaydıraklı katılaşır. Kalınlığı 0,5-1 olmalıdır mm.</li>
	Özel çözüm yıkanır kadar <li>hızlı bir şekilde Pasteur pipet pipetting 95 ° C DW tarafından yukarı ve aşağı yıkayın. Aksi takdirde, damlalıklı tarafından özel jel haline tıkanmış. Pipet ayakta 95 ° C'de DW bırakın.</li>
	<li>1 dk bekle. Özel defteri oluşturur ve bulutlu olur. Sonra el ile ( şekil 1 C) ayrı slaytları slayt.</li>
</ol> 4. Kurtlardan montaj Not: solucan hareketi bile az miktarda önlemek iyi gözlem. Levamisole solucan hareket özel yastık 24 , 25 önlemek için geleneksel olarak kullanılmıştır. Ancak, Levamisole C. elegans nöronal reseptörleri inhibe ve bu nedenle nöronlar 15 kaçakçılığı olayları etkileyebilir. Polistren lastikteki kullanarak açıklanan burada iyi bir alternatif 17.
<ol><li>~ 30 transgenik solucanlar yeni NGM için uygun veri işaretleyicilerini ifade plaka Platin tel kullanarak bakteri almak olmadan bir sürgülü ayna transillumination ile donatılmış bir stereo mikroskop altında transfer. 
	Not: Platin tel ve Pasteur pipet (5 inç) bir platin tel çekme yapılmış ve platin tel ucu dümdüz. Platin tel alır hazırlanması ve bunlar solucanlarla kullanımı Wormbook (http://www.wormbook.org) açıklanmıştır.</li>
	<li>Plaka terk solucanlar NGM özel jel bakteri olmadan hareket olarak 1 dk. için bakteri otomatik olarak solucanlar yüzeyler arasında kaldırılır.</li>
	<li>Yerine 0,5-1,0 a µL % 2,6 100 nm çapında polistren lastikteki agar pad ( şekil 1 D) üzerine su içinde damla. 10-20 solucanlar bakteri-özgür NGM plaka için lastikteki aktarın. Damla ve platin tel kullanarak solucanlar almak, örtüşen önlemek için forma solucan organları ( şekil 1 E).</li>
	<li>22 x 40 mm 2 coverslip ( şekil 1 F) uygulanır. Hava kabarcıkları kaldırmak, ancak ezici solucanları önlemek için yavaşça itin. Örnek görüntüleme için hazırdır. 
	Not: anestezi yönetilen çünkü solucanlar sadece agar pad, polistren lastikteki ve coverslip 17 tarafından üretilen sürtünme Kuvvetleri tarafından sabitlenir. Besleyici bakteri ve/veya çok fazla çözüm varlığı sürtünme kuvveti zayıf yapacak. 
	Not: Coverslips (örneğin, 22 x 22 mm 2, 18 x 18 mm 2) iş farklı boyutlarda. Ancak, daha büyük coverslips daldırma su veya örnekleri gözlem sırasında sızıntı objektif lens yağdan önleyebilirsiniz.</li>
</ol> 5. Gözlem Not: her mikroskop sistem ve kamera için uygun görüntüleme parametreleri (lazer gücü, kazanç, binning, vb) farklılık gösterir. Burada, bir iplik disk confocal tarayıcı ve dijital bir CCD kamera ile donatılmış widefield mikroskop kullanılmaktadır.
<ol><li>20 ° C sıcaklık kontrolü sahne alanı ayarla veya oda sıcaklığında 20 ayarlamak ° C.</li>
	<li>Örnek slayt mikroskop sahneye koydu. 100 x kullanın (sayısal diyafram = 1.3 veya daha iyi) objektif lens.</li>
	 Şekil 2 A (için wyIs251 ve aksonal transport) veya Resim 2 B (mnIs17 ve LFT için) pozitif denetimleri olarak belirtilen alanlar için <li>Bak . Diğer alanlarda iyi 22 analiz edilebilir.</li>
	<li>Kümesi parametreleri (CCD kazanç = 200, Binning = 1 veya 2, 488 güçte lazer nm = 1.0-1.3 mW). 4 kare/s zaman sukut yazmak ilâ 1 dk. dosyaları Kaydet mutli-TIFF biçimi olarak gerçekleştirmek. 
	Not: GFP sinyalleri yok ve GFP::RAB-3 veya OSM-6::GFP 30 için izlemeye devam etmek zordur eğer s, lazer güç çok güçlü. Bu durumda, lazer gücü düşür. Diğer taraftan, veziküler hiçbir hareket kaydedildiyse, lazer gücü çok zayıftır. Bu durumda, lazer gücü arttır.</li>
	<li>Farklı bir solucan gözlemlemek ve 5.3 ve 5,4 yineleyin. Gözlemler 20 dakikaya kadar sürebilir, ama her solucan p etkilerini önlemek için yalnızca bir kez kaydedilmesi gerekenHo'ya hasar. 
	Not: En az 20 dk olmadan önemli kusurları 7 , 27 , 28 için solucanlar gözlenmiştir. Daha uzun gözlem cenneti mümkün olabilir &#39; t oldu henüz test. Açık bir işareti nöronal ölüm neurite şişme gibi nöronal Morfoloji değişimdir. Zayıf GFP sinyaller akson ve dendrites wyIs251 ve mnIs17 görüldüğü gibi neurite Morfoloji aksonlar veya dendrites sadece bakarak kolayca denetlenebilir. Nöronal Morfoloji Şekil 2 ' de gösterildiği.</li>
	<li>Film dosyaları oluşturmak.
	Fiji yazılımı kullanarak <ol><li>açık çok TIFF dosyasını. Dosya gidin &#62; aç sonra 5.4. adımda kaydettiğiniz çok TIFF dosyası seçin. 
		Not: Fiji-ebilmek var olmak downloaded jttps://fiji.sc. Resmi J ve eklentileri kymographs oluşturmak için kullanılabilir. Böylece, ilgili talimatları için seçilen eklenti.</li> <li>'ı tıklatın &#34; dikdörtgen seçim &#34; düğmesini ( şekil 3 bir, ok) seçeneğini belirleyip bir dikdörtgen çizerek ilgi alanı bir bilgisayar fare ile (sarı dikdörtgen şekil 3 A). Görüntü gidin &#62; yığınları &#62; araçlar &#62; yapmak Substacks ve filmde içerdiği dilim numaralarını seçin. Bir substack oluşturulur.</li>
		<li>Substack pencereyi tıklatarak etkinleştirin ve resim gidin &#62; ayarla &#62; Parlaklık/Kontrast. Parlaklık ve kontrastı ayarlamak için bir pencere gösterir. Pencerede, üst slayt bar (en az) sağa doğru böylece arka plan sinyal kaybolur ve ikinci top bar (maksimum) sola kadar bu hareketli veziküller ve parçacıklar oldukça iyi arka plan üzerinde görülebilir.</li>
		<li>Gitmek için görüntü &#62; yığınları &#62; zaman Stamper. Film 4 kare/s 5,4 adımda kaydedilen zaman aralığını 0.25 olduğu s. Böylece, yazın &#34; 0,25 &#34; aralığındaki.</li>
		<li>Oluşturmak bir film dosyası olarak kaydet seçerek &#62; AVI Dosya menüsünden (film 1 ve 2). 
		Not: uygun eklentileri kullanarak, dosyayı, diğer biçimleri aşağıdaki gibi kaydedebilirsiniz &#34; mpeg4 &#34; veya &#34; mov &#34; dosyaları.</li>
	</ol></li> <li>Kymographs oluşturmak.
	<ol><li>FIji yazılım kullanarak yeniden özgün film dosyaları açın ve tekrar adım 5.6.2 ve parlaklık ve kontrastı ayarlamak için 5.6.3.</li>
		<li>Tıklama &#34; satır parçalara &#34; ( şekil 3 B, ok). Bir fare kullanarak kirpikler veya axon (Sarı Hat, şekil 3 B) boyunca parçalı bir çizgi çizin.</li>
		<li>Gitmek için analiz &#62; çok Kymograph &#62; çok Kymograph ( şekil 3 C). Satır genişliği penceresi ( şekil 3 D) görüntülenir. Türü &#34; 3 &#34; Linewidth pencere ve Tamam'ı tıklatın ( şekil 3 D).</li>
		<li>Kymograph pencere oluşturulur. Resmi TIFF ya da JPEG formatında kaydedin.</li>
	</ol>
	</li>
</ol>

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I., Avery, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microsurgery+in+Caenorhabditis+elegans.">Laser microsurgery in Caenorhabditis elegans.</a> Methods Cell Biol. 107, 177-206 (2012).</li><li>Kumar, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+Kinesin-3+motor+UNC-104/KIF1A+is+degraded+upon+loss+of+specific+binding+to+cargo.">The Caenorhabditis elegans Kinesin-3 motor UNC-104/KIF1A is degraded upon loss of specific binding to cargo.</a> PLoS Genet. 6 (11), 1001200 (2010).</li><li>Zheng, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vesicle+protein+SAM-4+regulates+the+processivity+of+synaptic+vesicle+transport.">The vesicle protein SAM-4 regulates the processivity of synaptic vesicle transport.</a> PLoS Genet. 10 (10), 1004644 (2014).</li><li>Blacque, O. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+WD+repeat-containing+protein+IFTA-1+is+required+for+retrograde+intraflagellar+transport.">The WD repeat-containing protein IFTA-1 is required for retrograde intraflagellar transport.</a> Mol Biol Cell. 17 (12), 5053-5062 (2006).</li><li>Evans, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+modulation+of+IFT+kinesins+extends+the+sensory+repertoire+of+ciliated+neurons+in+Caenorhabditis+elegans.">Functional modulation of IFT kinesins extends the sensory repertoire of ciliated neurons in Caenorhabditis elegans.</a> J Cell Biol. 172 (5), 663-669 (2006).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat Methods. 2 (12), 905-909 (2005).</li><li>Bindels, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mScarlet:+a+bright+monomeric+red+fluorescent+protein+for+cellular+imaging.">mScarlet: a bright monomeric red fluorescent protein for cellular imaging.</a> Nat Methods. 14 (1), 53-56 (2017).</li><li>Prevo, B., Mangeol, P., Oswald, F., Scholey, J. M., Peterman, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+differentiation+of+cooperating+kinesin-2+motors+orchestrates+cargo+import+and+transport+in+C.+elegans+cilia.">Functional differentiation of cooperating kinesin-2 motors orchestrates cargo import and transport in C. elegans cilia.</a> Nat Cell Biol. 17 (12), 1536-1545 (2015).</li><li>Robison, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detyrosinated+microtubules+buckle+and+bear+load+in+contracting+cardiomyocytes.">Detyrosinated microtubules buckle and bear load in contracting cardiomyocytes.</a> Science. 352 (6284), 0659 (2016).</li><li>Hayashi, S., Okada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+superresolution+fluorescence+imaging+with+spinning+disk+confocal+microscope+optics.">Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics.</a> Mol Biol Cell. 26 (9), 1743-1751 (2015).</li></ol>

Yazar ifşa etmek hiçbir şey vardır.

Sınırlama ile ilgili mevcut yöntemler Burada açıklanan yöntemi aksonal transport ve LFT gibi hızlı olayları gözlemlemek için optimize edilmiştir. Böylece, immobilizasyon daha daha uzun kuluçka öncelik. Ise önemli pertürbasyon olmadan en az 20 dk için kaçakçılık olayları gözlemlemek mümkün olabilirdi, bu yöntem her zaman axon uzama ve hücre göç gibi uzun gözlem gerektiren yavaş olayları gözlemlemek uygun olmayabilir. Daha uzun gözlem için bir özel yüzdesini azaltar...

Canlı hücre görüntüleme hücre içi taşıma analiz etmek için önemli bir araçtır. Nöronal hücre Biyolojide canlı hücre görüntüleme ile aksonal taşıma nöronal fonksiyon ve morfogenetik1anlamak için gerekli analizlerdir. Aksonal transport kusurları birkaç nörodejeneratif hastalıklar2altında yatan. Sırasıyla kinesin süper proteinler ve dinein aksonal transport anterogradely ve retrogradely,1,2taşır.
Kirpikler Mikrotubul ağ ve kaçakçılığı makine gelişmiş3olan başka bir hücresel yuvası vardır. Protein sentezi makine kirpikler, silier proteinler kirpikler ipuçları sitoplazma gönderilmesi gerekir yani yerelleştirilmemiştir. Kirpikler özel kinesin ve dinein, aradım kinesin-2 ve sitoplazmik dinein-2, sırasıyla, kirpikler4, intraflagellar taşıma (LFT)5denilen bir fenomen bileşenleri taşıma. LFT bozukluğu hastalıkları ciliopathies6adı verilen bir spektrum neden olur. Böylece, LFT mekanizması tarafından canlı hücre görüntüleme analizi silier oluşumu ve patogenezinde temel mekanizmaları anlamak için gereklidir.
Caenorhabditis elegans (C. elegans) aksonal transport ve LFT7,8,9eğitim için iyi bir modeldir. LFT gözlemlemek için Chlamydomonas yaygın olarak bir model organizma5,6kullanılmıştır. Chlamydomonas tek hücreli bir organizma olduğu için LFT ilişki ile yaşlanma, nöronal fonksiyon ve davranış analiz etmek zor olacaktır. Buna ek olarak, CRISPR/Cas9 gibi temel genetik teknikleri Chlamydomonasiçin uygulanmadı. Çünkü aksonal transport ve LFT morfogenez ve homeostasis hayvanlar 10için gerekli olan fare ve Drosophila, gibi daha yüksek model organizmalar bozulma aksonal transport ve LFT kez ölümcül fenotipleri neden olur, 11. Fare olması durumunda, hücre kültürü ve transfection genellikle aksonal transport ve birçok beceri ve geniş zaman12,13gerektirir LFT gözlemlemek için gereklidir. Buna ek olarak, önemli fizyolojik bağlam çok kültürlü hücreleri ve hücre satırlarını kaybetmiş. Sinir sisteminin solucanların yaşam için gerekli olduğundan, C. elegans mutantlar hangi aksonal taşıma veya LFT kesintiye öldürücü7,9,14değil çoğu zaman. Doğrudan aksonal transport ve LFT vivo içinde diseksiyon olmadan C. elegans saydamdır ve bu nedenle GFP etiketli işaretleri gözlemlemek kolaydır çünkü görülebilmektedir.
C. elegans, anestezi16veya lastikteki17bir mikrosıvısal aygıt15, özel kablolar kullanarak gibi hareketsiz için çeşitli protokoller vardır. Anestezi eklenmesi nöronlar15kaçakçılığı olayları inhibe. Mikrosıvısal-aygıt yöntemin açık bir dezavantajı bir mikrosıvısal aygıt hazırlanıyor her zaman kolay değildir. Bunun yerine, özel yastıkları ve lastikteki immobilizasyon zaman atlamalı görüntüleme C. elegansiçinde gerçekleştirmek için rahat ve kolay bir yöntem var. Burada, C. elegans hareketsiz ve aksonal transport ve LFT in vivo olarak görselleştirmek için bu temel iletişim kurallarını açıklamak C. elegans. Diğer yöntemlerine göre burada açıklanan yöntemi özel ekipman gerektirmez ve çok daha ucuz ve daha kolay gerçekleştirmek.

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			<td height="21" style="height:21px;width:263px;">Slideglass (76&#160; x 26 mm)</td>
			<td style="width:167px;">Matsunami</td>
			<td style="width:139px;">S1111</td>
			<td style="width:271px;"></td>
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			<td height="21" style="height:21px;width:263px;">Coverglass (22&#160; x 40 mm)</td>
			<td style="width:167px;">Matsunami</td>
			<td style="width:139px;">C024401</td>
			<td></td>
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			<td height="21" style="height:21px;width:263px;">Agarose</td>
			<td style="width:167px;">Wako</td>
			<td style="width:139px;">318-01195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Polystylene microbeads 0.1 micron</td>
			<td style="width:167px;">Polysciences</td>
			<td style="width:139px;">#00876</td>
			<td></td>
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			<td height="21" style="height:21px;width:263px;">Heat block</td>
			<td style="width:167px;">TAITEC</td>
			<td style="width:139px;">0063288-000</td>
			<td>CTU-mini</td>
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			<td height="21" style="height:21px;width:263px;">Microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:139px;">IX-71</td>
			<td>widefield microscope</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Spinning disk Scanner</td>
			<td style="width:167px;">Yokogawa</td>
			<td style="width:139px;">CSU-X1</td>
			<td>spinning disc confocal scanner&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Digital CCD camera</td>
			<td style="width:167px;">Hamamatsu Photonics</td>
			<td style="width:139px;">C10600-10B</td>
			<td>ORCA-R2 degital CCD camera</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Objective lens (x100, NA1.4)</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:139px;">UPLSAPO 100XO</td>
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			<td height="21" style="height:21px;width:263px;">Pasteur pipette (5 inch)</td>
			<td style="width:167px;">IWAKI</td>
			<td style="width:139px;">IK-PAS-5P</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Glass tube (1.5 cm diameter x 10.5 cm)</td>
			<td style="width:167px;">IWAKI</td>
			<td style="width:139px;">9820TST15-105NP</td>
			<td></td>
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			<td height="21" style="height:21px;width:263px;">TV9211: wyIs251</td>
			<td style="width:167px;">Laboratory of Kang Shen</td>
			<td>N/A</td>
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			<td height="21" style="height:21px;width:263px;">OTL11: mnIs17</td>
			<td style="width:167px;">Ref. 27, 29</td>
			<td style="width:139px;">N/A</td>
			<td>SP2101 was backcrossed with wild type for 6 times</td>
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			<td height="21" style="height:21px;width:263px;">Stereo microscope</td>
			<td style="width:167px;">Carl Zeiss</td>
			<td style="width:139px;">435064-9000-000</td>
			<td>STEMI 508&#160;</td>
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			<td height="21" style="height:21px;width:263px;">Mirror transillumination unit</td>
			<td style="width:167px;">Carl Zeiss</td>
			<td>435425-9010-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">platinum wire (0.2 mm)</td>
			<td style="width:167px;">Nilaco Corporation</td>
			<td style="width:139px;">m78483501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm plastic dish</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:139px;">#351007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Fiji</td>
			<td style="width:167px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td>https://fiji.sc/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">nematode growth medium (NGM)&#160;&#160;</td>
			<td style="width:167px;"></td>
			<td style="width:139px;"></td>
			<td>1.7% (w/v) agarose, 50mM NaCl, 0.25% (w/v) Peptone, 1 mM CaCl2, 5 mg/mL Cholesterol, 25 mM KH2PO4, 1 mM MgSO4&#160;</td>
		</tr>
	</tbody>
</table>

immobilization of caenorhabditis elegans to analyze intracellular transport in neurons

Aksonal taşıma ve intraflagellar taşıma (LFT) akson ve kirpikler morfogenez ve işlevi için gerekli olan. Kinesin süper proteinler ve dinein anterograd ve retrograd taşıma, sırasıyla düzenleyen moleküler motorlar vardır. Bu motorlar Mikrotubul ağları raylar kullanır. Caenorhabditis elegans (C. elegans) aksonal transport ve LFT vivo içindeçalışmaya bir güçlü model organizmadır. Burada, aksonal transport ve LFT yaşam gözlemlemek için bir protokol tarif C. elegans. Taşınan kargo kargo protein flüoresan proteinler yeşil flüoresan protein (GFP) gibi kullanarak etiketleyerek görüntülenmeyecektir. C. elegans şeffaf ve kargo GFP öğesini proteinler hücre özel Organizatör altında belirli hücrelerdeki ifade edilebilir. Yaşayan solucanlar lastikteki % 10 özel jel üzerinde öldürmek veya solucanlar anesthetizing tekrarlanmasıyla çözülebilir. Bu koşullar altında kargo hareket doğrudan akson ve yaşam kirpikler gözlenen C. elegans diseksiyon olmadan. Bu yöntem hedef proteinler değiştirerek herhangi bir hücre ve/veya onlar ifade edilir hücreleri herhangi bir kargo molekül gözlenmesi için uygulanabilir. Moleküler motorlar gibi en temel proteinler ve aksonal transport ve LFT katılmaktadırlar adaptör protein C. elegansiçinde korunmuş. Diğer model organizmalar için karşılaştırıldığında, mutantlar elde edilen ve daha kolay C. elegansiçinde saklanır. Bu yöntem çeşitli C. elegans mutantlarla birleştirerek aksonal transport ve LFT moleküler mekanizmaları açıklığa kavuşturmak.

Aksonal DA9 nöron nakliyesi Anterograd ve retrograd aksonal transport GFP::RAB-3 wyIs251 satırını kullanarak aynı anda DA9 motor nöron kaydedilebilir. Anterograd ve retrograd DA9 nöron proksimal dorsal axon taşımacılığında ortalama hızı yaklaşık 1,8 ve 2.6 mikron/s, sırasıyla22var. 0.03 ve akson s başına mikron başına 0,018 hakkında hareketli veziküller sayısıdır. Böylece, bir 30 s gözlem DA9 akson 100 x le...

Watch this Scientific Journal Video about Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons at JoVE.com

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) aksonlar ve hücre içi taşıma çalışma için iyi bir modeldir. Burada, vivo içinde kayıt ve analiz C. elegansaksonlar ve intraflagellar taşıma için bir protokol açıklayın.

Caenorhabditis elegans nöron hücre içi taşıma analiz immobilizasyon

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

The overall goal of this time lapse recording is to observe the intracellular transport events in Caenorhabditis elegans neurons.This method can help answer key questions in the cell biology field such as axonal transport and interflagellar transport.The main advantage of this technique is that one can observe intracellular transport in C.Elegans without dissection.Start this experiment by growing transgenic C.elegans strain of interest at 20

immobilization-caenorhabditis-elegans-to-analyze-intracellular

Research

JoVE Journal

Developmental Biology

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

7.9K Görüntüleme.  Tohoku University. The overall goal of this time lapse recording is to observe the intracellular transport events in Caenorhabditis elegans neurons.This method can help answer key questions in the cell biology field such as axonal transport and interflagellar transport.The main advantage of this technique is that one can observe intracellular transport in C.Elegans without dissection.Start this experiment by growing transgenic C.elegans strain of interest at 20

Video: Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

Nematod kullanma Germline Kimyasal zehirlilik Kapsamlı Değerlendirme<em&gt; Caenorhabditis elegans</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

Gelişim Biyolojisi

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

Aktif ERK Mekansal ve Zamansal Analizi<em&gt; C. elegans</em&gt; Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments. The first protocol provides a method to assess the spatial distribution of stable and dynamic MTs and perform a quantitative analysis of these populations with image-processing software. This approach complements existing tools to image microtubule dynamics and distribution in real-time, using transgenic lines or transient expression of tagged constructs. Indeed, such tools are very useful, however they do not readily distinguish between dynamic and stable MTs. The ability to image and analyze these distinct microtubule populations has important implications for understanding mechanisms underlying cell polarization and morphogenesis. The second protocol outlines a technique to analyze nascent MTs specifically. This is accomplished by capturing the de novo growth properties of MTs over time, following microtubule depolymerization with the drug nocodazole and a recovery period after drug washout. This technique has not yet been applied to the study of MTs in zebrafish embryos, but is a valuable assay for investigating the in vivo function of proteins implicated in microtubule assembly.

Immunolabeling methods to analyze distinct populations of microtubules in the developing zebrafish brain are described here, which are broadly applicable to other tissues. The first protocol outlines an optimized method for immunolabeling stable and dynamic microtubules. The second protocol provides a method to image and quantify nascent microtubules specifically.

Zebra balığı embriyo, dinamik, kararlı ve doğmakta olan mikrotübüller analiz etmek için Immunolabeling kullanımı

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Diseksiyon ve mantar vücut immünfloresan boyama ve yetişkin Drosophila melanogaster beynindeki nöronlar Photoreceptor

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Uygulama için yeniden programlamak Germ hücreleri için C. elegans nöronlarda RNAi ve transkripsiyon faktörü ifade ısı şok bağlı

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

Bağırsak hayatın Caenorhabditis elegans lizozomal Alkalinization değerlendirilmesi

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Çekirdeklerin dağıtım, proteinler ve sitoiskeleti çalışmaya Caenorhabditis elegans Germline sayısal analizi

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

Oksidatif stres nedeniyle Mitis grubu streptokok Caenorhabditis elegans içinde eğitim

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

İzotropik hafif levha mikroskobu ve otomatik hücre Lineage analizleri kataloğa Caenorhabditis elegans Embriyogenesis Subcellular çözünürlük ile

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

Ölçme sperm rehberlik ve motilite içinde Caenorhabditis elegans hermafrodit üreme yolu

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...