L'autore ringrazia profondamente Dr. Asako Sugimoto (Università di Tohoku) per la sua discussione utile. wyIs251 era un dono generoso da Dr. Kang Shen (Stanford University). mnIs17 è stato fornito dalla CGC, che è finanziato dall'ufficio di NIH di programmi di ricerca dell'infrastruttura (P40 OD010440). Questo lavoro è stato supportato da grant JSPS KAKENHI #17 H 05010 e #16 H 06536 e Fondazione di Daiichi Sankyo, Brain Science Foundation e Naito Foundation.

1. preparazione del campione <ol><li>genera un transgenico c. elegans ceppo di interesse utilizzando metodologie transgeniche 18. In alternativa, ottenere ceppi appropriati da Caenorhabditis Genetics Center (CGC). Per visualizzare il trasporto assonale dei precursori delle vescicole sinaptiche, utilizzare la linea transgenica wyIs251 [Pmig-13::gfp::rab-3] 7. Per osservare IFT, ottenere la linea transgenica mnIs17 [osm-6::gfp] 19. Questi ceppi sono utilizzati in questo protocollo. 
	Nota: Una matrice extracromosomico, integrazione genomica o anche GFP knock-in works. Per ridurre i segnali di fondo, utilizzare promotori di cellula-specifico, piuttosto che pan-neuronale promotori. 
	Nota: Manutenzione di worms è descritto in altri protocolli 20. Ceppi sono stati mantenuti su prati dell'alimentatore Escherischia coli OP50 sulla crescita del nematode piastre di terreno (NGM) (agarosio 1,7% (p/v), 50 mM NaCl, 0,25% (p/v) di peptone, 1 mM CaCl 2, 5mg/mL colesterolo, 25mm KH 2 PO 4, 1mm MgSO 4 su piatti di plastica di diametro 60 mm) a 20 ° C.</li>
	<li>Crescere i vermi a 20 ° C sulle piastre di NGM a qualsiasi fase del ciclo di vita dei vermi. 
	Nota: Si può osservare sia trasporto assonale e IFT in qualsiasi fase. L4 tardivo a giovane adulto è un buon controllo positivo perché più pubblicazioni hanno utilizzato queste fasi e visualizzato in quel momento 22 eventi traffico 14 , 21 , , 23.</li>
</ol> 2. Preparazione del 10% dell'agarosi Nota: molti fornitori offrono prodotti simili, quali polveri di agar agar e agarosio. Utilizzare agarosio per elettroforesi (resistenza del gel &#62; 1200 g/cm 2). Polveri di agar a buon mercato non funzionano perché il gel risultante non è abbastanza forte per immobilizzare worm.
<ol><li>Mix 0,4 g agarosio in 4 mL di acqua distillata acqua (DW) in un tubo di vetro (1,5 x 10,5 cm). Mettere un coperchio sul tubo di vetro per evitare l'evaporazione.</li>
	<li>Mettere il tubo di vetro su un blocco di calore di 95 ° C per 90 min. Inoltre, preparare un altro tubo di vetro (1,5 x 10,5 cm) riempito con DW e tenerlo a 95 ° C. mettere una pipetta Pasteur a 95 ° C DW ( Figura 1 A). Un sacco di piccole bolle sarà visibili nel tubo di agarosio e la soluzione deve essere torbida. Lasciare fino a quando la soluzione diventa chiara. Quindi, mescolare pipettando su e giù fino a quando la soluzione di agarosio diventa omogenea. 
	Nota: Forno a microonde non può essere utilizzato per preparare la soluzione di agarosio 10% quando effettuata in DW e dell'agarosi. Piccole bolle causate dalle microonde evitare la fusione dell'agarosi. Tuttavia, gel di agarosio solidificato 10% facilmente possono essere fusi nuovamente utilizzando un forno a microonde. Tubi di vetro contenenti gel di agarosio al 10% possono essere preparati in anticipo e conservati a 4 ° C per almeno 6 mesi. Se in questo modo, sciogliere il brodo utilizzando un forno a microonde quando necessario e iniziare dal passaggio 3 in questo protocollo. 
	Nota: Agarosio perde gradualmente la capacità di solidificare se incubate a 95 ° C per un tempo lungo o dopo ripetute solidificazione e fusione. Se questo accade, preparare nuove 10% agarosio.</li>
</ol> 3. Preparazione di agarosio Pad <ol><li>usando la pipetta di Pasteur riscaldata preparata al punto 2.2, inserire 2 gocce di agarosio al 10% in una diapositiva di 76 x 22 mm.</li>
	<li>Rapidamente coprire con una seconda diapositiva 2 76 x 22 mm prima della caduta dell'agarosi si solidifica come mostrato in Figura 1 B e spingere verso il basso della seconda diapositiva per appiattire la soluzione di agarosio. Lo spessore deve essere di 0,5-1 mm.</li>
	<li>Rapidamente svuotare la pipetta di Pasteur di pipettaggio 95 ° C DW su e giù fino a quando la soluzione di agarosio è lavata fuori. In caso contrario, la pipetta sarà ostruirsi di gel di agarosio. Lasciare la pipetta a 95 ° C DW.</li>
	<li>Attesa per 1 min. Il pad di agarosio forma e diventa torbida. Quindi, far scorrere le diapositive apart a mano ( Figura 1 C).</li>
</ol> 4. Montaggio di vermi Nota: anche piccole quantità di movimento verme prevenire buona osservazione. Levamisole è stato tradizionalmente usato per impedire il movimento della vite senza fine sull'agarosio pad 24 , 25. Tuttavia, Levamisole inibisce i recettori neuronali in c. elegans e pertanto può influenzare eventi di traffici in neuroni 15. Utilizzando microsfere di polistirolo descritto qui una buona alternativa 17.
<ol><li>Sotto un microscopio stereo equipaggiato con una transilluminazione specchio scorrevole, trasferire ~ 30 vermi transgenici che esprimono gli indicatori appropriati per un nuovo NGM piastra senza batteri utilizzando un filo di platino pick. 
	Nota: Un filo di platino pick è costituito da filo di platino e pipetta Pasteur (5 pollici), e la punta del filo platino è stata appiattita. La preparazione dei picconi filo di platino e la manipolazione di vermi con questi sono descritti in Wormbook (http://www.wormbook.org).</li>
	<li>Lasciare la piastra per 1 min. batteri vengono rimossi automaticamente dalle superfici di vermi come i vermi muoversi su gel di agarosio NGM senza batteri.</li>
	<li>Put a 0,5-1,0 µ l goccia del 2,6% 100 microsfere di polistirolo del diametro nm in acqua sul blocchetto di agar ( Figura 1 D). Consente di trasferire le microsfere 10-20 vermi dalla piastra di NGM esente da batteri. Goccia e la diffusione di worm usando il legare di platino pick per evitare la sovrapposizione di verme corpi ( Figura 1 E).</li>
	<li>Applicare un vetrino coprioggetti 2 di 22 x 40 mm ( Figura 1, F). Spingere delicatamente per rimuovere le bolle d'aria, ma evitare di schiacciare le viti senza fine. Il campione è pronto per l'imaging. 
	Nota: Poiché non è amministrata anestesia, vermi vengono risolti solo dalla forza di attrito generata da agar pad, microsfere di polistirolo e coprioggetto 17. La presenza di batteri di alimentatore e/o troppo soluzione renderà la forza di attrito più debole. 
	Nota: Diverse dimensioni del lavoro di vetrini coprioggetti (ad es., 22 x 22 mm 2, 18 x 18 mm 2). Tuttavia, le lamelle più grandi possono impedire immersione acqua o olio dalla lente dell'obiettivo che perde in campioni durante l'osservazione.</li>
</ol> 5. Osservazione Nota: appropriati parametri di imaging (potenza laser, guadagno, binning, ecc.) saranno diverso per ogni sistema di microscopio e la fotocamera. Qui, viene utilizzato un microscopio widefield equipaggiato con uno scanner confocale del disco rotante e una macchina fotografica digitale del CCD.
<ol><li>Impostare il controllo della temperatura della fase a 20 ° C, o regolare la temperatura a 20 ° C.</li>
	<li>Mettere il vetrino sul tavolino del microscopio. Utilizzare x 100 (apertura numerica = 1.3 o superiore) dell'obiettivo.</li>
	<li>Cercare le aree indicate in Figura 2 A (per wyIs251 e trasporto assonale) o Figura 2 B (per mnIs17 e IFT) come controlli positivi . Altre zone possono essere analizzati come ben 22.</li>
	<li>Set di parametri (CCD guadagno = 200, Binning = 1 o 2, potenza laser a 488 nm = 1.0-1.3 mW). Eseguire la registrazione continua a 4 fotogrammi/s per fino a 1 min Salva file come formato mutli-TIFF. 
	Nota: Se GFP segnali scomparire ed è difficile continuare a osservare GFP::RAB-3 o OSM-6::GFP per 30 s, la potenza del laser è troppo forte. In tal caso, ridurre la potenza del laser. Al contrario, se non è registrato nessun movimento vescicolare, la potenza del laser è troppo debole. In tal caso, aumentare la potenza del laser.</li>
	<li>Osservare un worm diverso e ripetere 5.3 e 5.4. Le osservazioni possono durare fino a 20 min, ma ogni verme deve essere registrati una sola volta per evitare gli effetti di pdanni di Hoto. 
	Nota: I vermi sono stati osservati per almeno 20 min senza difetti significativi 7 , 27 , 28. Osservazione più lunga può essere possibile, ma porto &#39; t stato ancora testato. Un chiaro segno della morte neuronale è il cambiamento della morfologia neuronale come gonfiore del neurite. Segnali deboli di GFP possono essere visto negli assoni e dendriti sia wyIs251 e mnIs17, morfologia del neurite può essere facilmente controllato da solo guardando gli assoni o dendriti. Morfologia neuronale è illustrato nella Figura 2.</li>
	<li>Generare filmati.
	<ol><li>Apri il file Multi-TIFF file utilizzando software di Fiji. Vai a File &#62; Open quindi selezionare il file Multi-TIFF salvato al passaggio 5.4. 
		Nota: Fiji può essere scaricato presso jttps://fiji.sc. Immagine J e plugin può anche essere utilizzato per generare chimografi. Se in questo modo, seguire le istruzioni pertinenti per il plugin scelto.</li> <li>scegliere la &#34; selezione rettangolare &#34; ( Figura 3 A, freccia) e seleziona l'area di interesse disegnando un rettangolo con un mouse del computer (rettangolo giallo in Figura 3 A). Vai a immagine &#62; stack &#62; strumenti &#62; fare Substacks e selezionare i numeri di fetta che sono inclusi nel film. Viene generato un substack.</li>
		<li>Attivare la finestra di substack cliccando e passare all'immagine &#62; regolare &#62; luminosità/contrasto. Una finestra per regolare la luminosità e il contrasto si presenta. Nella finestra, fare scorrere la parte superiore bar (minimo) a destra così che il segnale di fondo sparisce e il secondo bar top (massimo) a sinistra così che le particelle e le vescicole commovente possono essere visto abbastanza bene sullo sfondo.</li>
		<li>Vai a immagine &#62; stack &#62; tempo Stamper. Come il film è registrato a 4 fotogrammi/s al punto 5.4, l'intervallo di tempo è 0,25 s. Quindi, digitare &#34; 0.25 &#34; nell'intervallo di.</li>
		<li>Generare un file di filmato selezionando Salva come &#62; AVI dal menu File (film 1 e 2). 
		Nota: Utilizzando il plugin appropriato, è possibile salvare il file come altri formati come &#34; mpeg4 &#34; o &#34; mov &#34; file.</li>
	</ol></li> <li>Generare chimografi.
	<ol><li>Aprire i file del filmato originale utilizzando software di FIji nuovamente e ripetere i passaggi 5.6.2 e 5.6.3 per regolare la luminosità e il contrasto.</li>
		<li>Clic &#34; segmentato linea &#34; ( Figura 3 B, freccia). Usando un mouse, disegnare una linea segmentata lungo le ciglia o assone (linea gialla, in Figura 3 B).</li>
		<li>Andare a analizzare &#62; Multi Kymograph &#62; Multi Kymograph ( Figura 3 C). Linea larghezza finestra ( Figura 3 D). Tipo &#34; 3 &#34; nella finestra di Linewidth e fare clic su OK ( Figura 3 D).</li>
		<li>Kymograph finestra viene creata. Salvare l'immagine in formato TIFF o JPEG.</li>
	</ol>
	</li>
</ol>

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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>

L'autore non ha nulla di divulgare.

Limitazione per quanto riguarda i metodi esistenti Il metodo qui descritto è ottimizzato per osservare eventi veloci come trasporto assonale e IFT. Così, l'immobilizzazione è più una priorità di incubazione più lungo. Mentre siamo stati in grado di osservare eventi di traffici per almeno 20 min senza perturbazione significativa, questo metodo potrebbe non essere sempre adatto per osservare eventi lenti che richiedono osservazioni più lungo, ad esempio allungamento dell'assone e migrazione cellu...

Live cell imaging è uno strumento essenziale per l'analisi di trasporto intracellulare. Nella biologia delle cellule neuronali, sono essenziali per comprendere la funzione e la morfogenesi neuronale1analisi del trasporto assonale con formazione immagine di cellule vive. Difetti nel trasporto assonale sono alla base di disordini neurodegenerative diversi2. Dineina e chinesina superfamiglia proteine svolgere trasporto assonale anterograda e retrogradely, rispettivamente1,2.
Le ciglia sono un altro compartimento cellulare in cui la rete dei microtubuli e traffico macchine sono altamente sviluppato3. Macchinario di sintesi proteica non è localizzata nelle ciglia, il che significa che ciliare proteine devono essere trasportati dal citoplasma verso le punte di ciglia. Cilia-specifiche chinesina e dineina, chiamato chinesina-2 e citoplasmatici dineina-2, rispettivamente, i componenti di ciglia4, in un fenomeno chiamato trasporto intraflagellare (IFT)5di trasporto. Il danno dell'IFT causa uno spettro di malattie chiamate ciliopatie6. Così, per capire i meccanismi di base della formazione ciliare e la patogenesi sono necessaria analisi del meccanismo IFT da formazione immagine di cellule vive.
Caenorhabditis elegans (C. elegans) è un buon modello per studiare il trasporto assonale e IFT7,8,9. Per osservare IFT, Chlamydomonas è stato ampiamente utilizzato come organismo modello5,6. Essendo un organismo unicellulare Chlamydomonas , il rapporto dell'IFT con invecchiamento, funzione di un neurone e comportamento sarebbe difficile da analizzare. Inoltre, tecniche di genetiche essenziale come CRISPR/Cas9 non siano state applicate a Chlamydomonas. Negli più alti organismi di modello, come topi e Drosophila, interruzione del trasporto assonale e IFT spesso provoca fenotipi letali perché trasporto assonale e IFT sono essenziali per la morfogenesi e l'omeostasi del animali 10, 11. Nel caso di topi, trasfezione e coltura delle cellule è generalmente necessario osservare trasporto assonale e IFT, che richiede molte competenze e tempo estesa12,13. Inoltre, un sacco di contesto fisiologico importante può essere perso in linee cellulari e cellule coltivate. Poiché il sistema nervoso non è essenziale per la sopravvivenza dei vermi, c. elegans mutanti in cui trasporto assonale o IFT sono interrotti non sono spesso letali7,9,14. Trasporto assonale e IFT può essere osservato direttamente in vivo senza dissezione perché c. elegans è trasparente ed è quindi facile osservare GFP-etichettato marcatori.
Esistono diversi protocolli per immobilizzare c. elegans, come l'utilizzo di un dispositivo di microfluidica15, pastiglie di agarosio con anestesia16o microsfere17. L'inclusione dell'anestesia può inibire gli eventi traffici in neuroni15. Un chiaro svantaggio del metodo microfluidici-periferica è che preparando un dispositivo microfluidico non è sempre facile. Invece, l'immobilizzazione di agarosio pastiglie e microsfere è un modo conveniente e facile per eseguire imaging lasso di tempo in c. elegans. Qui, descrivo questo protocollo di base per immobilizzare c. elegans e visualizzare trasporto assonale e IFT in vivo in elegans del c. Rispetto agli altri metodi, il metodo qui descritto non richiede attrezzature speciali ed è molto più economico e più facile da eseguire.

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	<tbody>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">TV9211: wyIs251</td>
			<td style="width:167px;">Laboratory of Kang Shen</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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

Trasporto assonale e trasporto intraflagellare (IFT) sono essenziali per la funzione e la morfogenesi dell'assone e le ciglia. Dineina e chinesina superfamiglia delle proteine sono motori molecolari che regolano anterograda e retrograda trasporto, rispettivamente. Questi motori utilizzano microtubule network come rotaie. Caenorhabditis elegans (C. elegans) è un organismo potente modello per studiare il trasporto assonale e IFT in vivo. Qui, descrivo un protocollo per osservare il trasporto assonale e IFT nella vita di c. elegans. Merce trasportata può essere visualizzato mediante codifica proteine cargo utilizzando proteine fluorescenti come proteina fluorescente verde (GFP). C. elegans è trasparente e proteine GFP-etichettato cargo possono essere espresso in cellule specifiche sotto promotori di cellula-specifico. Viti senza fine viventi possono essere risolto microsfere su gel di agarosio 10% senza uccidere o anestetizzare i vermi. In queste condizioni, movimento di carico può essere osservato direttamente negli assoni e ciglia di vivere c. elegans senza dissezione. Questo metodo può essere applicato per l'osservazione di qualsiasi molecola di carico in tutte le cellule modificando proteine bersaglio e/o le cellule in che si sono espressi. Le proteine più elementari quali motori molecolari e proteine adattatrici che sono coinvolti nel trasporto assonale e IFT sono conservate in c. elegans. Rispetto ad altri organismi di modello, mutanti possono essere ottenute e mantenute più facilmente in c. elegans. Combinando questo metodo con vari mutanti di c. elegans può chiarire i meccanismi molecolari del trasporto assonale e IFT.

Trasporto assonale nel neurone DA9 Utilizzando la riga di wyIs251 , l'anterograda e il trasporto assonale retrogrado di GFP::RAB-3 possa essere registrata simultaneamente nel neurone motore DA9. La velocità media di anterograda e retrograda trasporto nell'assone dorsale prossimale del neurone DA9 è circa 1,8 e 2,6 μm/s, rispettivamente22. Il numero delle vescicole commovente è circa 0,03 e 0,018 a μm di assone a s. Così, per un'os...

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) è un buon modello per studiare il trasporto assonale e intracellulare. Qui, descrivo un protocollo per la registrazione in vivo e l'analisi del trasporto assonale e intraflagellare c.elegans.

Immobilizzazione del Caenorhabditis elegans per analizzare il trasporto intracellulare nei neuroni

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

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Developmental Biology

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

7.9K Views.  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.

Valutazione completa della linea germinale Chemical Tossicità Utilizzando il nematode<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 ...

Ricerca

Biologia dello sviluppo

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.

Spaziale e temporale Analisi di attiva ERK nel<em&gt; C. elegans</em&gt; Linea germinale

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.

Uso di Immunolabeling per analizzare i microtubuli stabili, dinamici e nascenti nell'embrione di pesce zebra

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.

Dissezione e macchiatura immunofluorescente del corpo di fungo e del fotoricettore neuroni nel cervello adulto Drosophila melanogaster

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.

Applicazione di RNAi ed espressione di fattore di trascrizione indotta da calore-shock per riprogrammare le cellule germinali ai neuroni in c. elegans

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)

Valutazione Lysosomal alcalinizzazione l' intestino di 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 ...

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.

Analisi computazionale della linea germinale Caenorhabditis elegans di studiare la distribuzione dei Nuclei, le proteine ed il citoscheletro

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.

Studiando lo Stress ossidativo causato dagli streptococchi di gruppo Mitis in Caenorhabditis elegans

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.

Microscopia isotropica leggera e analisi di lignaggio cellulare automatizzate al catalogo Caenorhabditis elegans embriogenesi con risoluzione subcellulare

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.

Misurare la guida dello sperma e la motilità all'interno del tratto riproduttivo ermafrodita di Caenorhabditis elegans

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