Gli autori desiderano ringraziare FI Ataullakhanov per aver contribuito a progettare e realizzare camere di flusso riutilizzabili, N. Dashkevich, N. Gudimchuk e A. Korbalev per la fornitura di immagini per i personaggi, N. Gudimchuk e P. Zakharov per lo sviluppo di un protocollo e fornendo reagenti preparare semi microtubuli digossigenina, A. Potapenko aiuto con l&#39;editing del testo e gli altri membri del Grishchuk laboratorio per le punte e discussioni. Questo lavoro è stato sostenuto in parte dal NIH concedere GM R01-098.389 e una borsa di studio pilota della Pennsylvania Muscle Institute a ELG, che è un Kimmel Scholar, da RFBR concede 12-04-00111-una proprietà di 13 04-40.190-H e 13 -04-40188-H, Accademia Russa delle Scienze Presidio Grants (Meccanismi di sistemi molecolari di integrazione e programmi di biologia cellulare molecolare e molecolari) per FI Ataullakhanov, NIH concedere GM R01 GM033787 a JR McIntosh, e una borsa di studio della Dynasty Dmitry Zimin Fondazione VAV

Gli autori non hanno nulla da rivelare.

Molti saggi singola molecola al giorno d&#39;oggi usano abitualmente coprioggetto appositamente trattata per ridurre drasticamente aspecifica di proteine ​​attaccare. La procedura che descriviamo qui è una modifica del protocollo originale sviluppato in laboratorio Howard 32, e troviamo che silanizzante i coprioggetti è valsa la pena, anche con saggi di perline basati DIC, che non utilizzano la fluorescenza. Chambers assemblati con tali coprioggetto mostrano superfici più puliti, ed i risultati ottenut...

I microtubuli sono altamente conservate strutture citoscheletriche che sono importanti per l&#39;architettura cellulare, la motilità cellulare, divisione cellulare, e trasporto intracellulare 1. Questi polimeri dinamici assemblare da tubulina in presenza di GTP, e si passa spontaneamente tra crescita e accorciando 2. I microtubuli sono molto sottili (solo 25 nm di diametro) quindi particolari tecniche ottiche per migliorare il contrasto dovrebbe essere utilizzato per osservare microtubuli con un microscopio ottico. Il lavoro precedente di questi polimeri esaminato il comportamento dinamico con contrasto di interferenza differenziale (DIC) 3. Questo ed analoghi studi in vitro hanno rivelato che, in condizioni tipiche sperimentali, i microtubuli subiscono catastrofe e passare alla depolimerizzazione solo raramente, una volta ogni 5-15 minuti (questa frequenza è di 7-15 mM concentrazione di tubulina solubile esaminati a 28-32 ° C ) 4. Diverse tecniche sono state quindi proposto di induzionee depolimerizzazione dei microtubuli in modo controllato. Microtubulo accorciamento può essere attivato da lavando via tubulina solubile 5,6, il taglio microtubuli con un raggio laser 7, oppure utilizzando microtubuli segmentati 8, come descritto qui. Impieghi precedenti utilizzando microtubuli segmentati, così come polimeri stocasticamente commutazione, ha trovato che i piccoli carichi intracellulari, come cromosomi, vescicole, e perle rivestite con proteine, possono muoversi alle estremità dei microtubuli accorciamento 9-13. Questo fenomeno è pensato per avere un&#39;implicazione diretta per i movimenti cromosomiche nelle cellule in mitosi, ed i meccanismi di fondo sono attualmente sotto inchiesta attivo 14-16. Recentemente, tecniche fluorescenti-based, tra la fluorescenza totale interna riflessione (TIRF) microscopia, sono stati impiegati per studiare la motilità con microtubuli dinamica finisce 17-24. Il vantaggio di questo approccio è che consente l&#39;esame di interaziones tra i microtubuli e proteine ​​microtubuli vincolante in tempo reale utilizzando proteine ​​marcate con differenti fluorofori. Diversi complessi proteici sono stati trovati a muoversi processively con allungamento e / o accorciando le estremità del microtubulo. Essi comprendono le proteine ​​associate ai microtubuli Dam1 10,12,18, Ska1 19, e XMAP215 20, così come i motori chinesina Kif18A 21,22, MCAK 23 e CENP-E 24. Queste proteine ​​mostrano processive tip-tracking, che è fondamentalmente diversa da quella dei classici proteine ​​punta inseguimento come EB1 25. Anche se le molecole EB1 ei partner associati sembrano rimanere stabilmente associata a microtubuli dinamica finisce, le singole molecole rimangono vincolati alla punta microtubulo solo ~ 0,8 secondi, rapido scambio con la piscina solubile 26. Al contrario, processive punta-trackers, come Dam1, viaggiano con microtubuli finisce per molti micron, e la loro associazione con le punte dei microtubuli può durare per meventuali secondi. La punta associazione tempo, così come la velocità di inseguimento risultante, dipende fortemente dal numero di molecole che formano la punta inseguimento complesso 27. Ensemble di proteine ​​più grandi sono di solito molto migliori tip-trackers. Ad esempio, tali insiemi complessi come isolate cinetocori lievito possono rimanere agganciati al microtubulo estremità per 28 ore. Alcune proteine ​​microtubuli vincolante, ad esempio Ndc80 complesso proteico cinetocoro, sono stati trovati per essere in grado di monitorare con microtubuli termina a livello di singola molecola, ma Ndc80 è molto efficiente nel accoppiando il movimento del tallone carico 19,29-31. Così, per comprendere il meccanismo di ribaltamento inseguimento da diversi complessi proteici, così come i loro ruoli biologici, è importante esaminare punta-tracking in funzione del numero di molecole del complesso-punta inseguimento, nonché per determinare la capacità di questi complessi di esporre motilità collettiva sulla superficie del cordone carico. (Figura 1A). In primo luogo, i vetrini disponibili in commercio sono modificati per inserire tubo polietilene breve (protocollo 1). La camera di flusso microscopia riutilizzabile viene assemblato da un tale scorrimento e il plasma puliti e coprioggetto silanizzata (protocollo 2) 32-34 o chimicamente. Il volume della camera risultante è solo il 20-25 microlitri (o piccolo come 15 microlitri, vedere Nota 3 nel protocollo 1), compreso il volume del tubo di ingresso. Camere di flusso commercialmente disponibili possono anche essere utilizzati, ma il loro volume è di solito più grande, che porta alla inutile spreco di proteine. Se una camera più grande è impiegato, il volume di tutte le soluzioni nei protocolli indicati deve essere scalata proporzionalmente. Semi di microtubuli vengono poi preparati, ad esempio utilizzando lentamente idrolizzabile GTP analogico, GMPCPP (5&#39;-[(α, β)-methyleno] trifosfato) (protocollo 3, vedi anche Hyman &lt;em&gt; et al. 35). I semi vengono immobilizzati su un vetrino pulito e la superficie viene successivamente bloccate per impedire l&#39;assorbimento aspecifico di altre proteine ​​32 (protocollo 4 descrive semi immobilizzazione utilizzando digossigenina). I microtubuli segmentati possono quindi essere preparati utilizzando il protocollo 5. La motivazione principale di questo approccio è che i polimeri microtubuli dinamiche, che formano in presenza di GTP, possono essere stabilizzati temporaneamente aggiungendo le corte &quot;tappi&quot; di segmenti tubulina stabili, che contengono GMPCPP. Questi tappi contengono anche tubulina rodamina marcato, in modo che possano essere rimossi semplicemente illuminando il campo visivo con un laser 530-550 nm o una lampada a vapori di mercurio (Protocollo 6) 36. Intensità di fluorescenza del segnale punta-tracking può quindi essere utilizzato per stimare il numero di molecole che viaggiano con lo smontaggio estremità del microtubulo, tenendo conto della disuniformità del campo di illuminazione microscopio (Protocollo 7). Un approccio simile può essere utilizzatoper studiare le interazioni tra microtubuli depolimerizzazione e perline rivestite con proteine, preparato come descritto in 27 (protocollo 8). Alcune proteine ​​si legano facilmente alle pareti dei microtubuli segmentati, ma pinzette laser può anche essere utilizzato per contenere il tallone vicino alla parete del microtubulo, promuovendo in tal modo il suo legame.

<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 1: Microscopy and other equipment.</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss 
			Nikon</td>
			<td>Axio Imager 2 
			Eclipse Ti</td>
			<td>other microscope models capable of DIC and epi-fluorescence-imaging can be used</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Zeiss 
			Nikon</td>
			<td>420490-9900-000 
			CFI Apo 100x Oil 1.49</td>
			<td>100X, DIC, 1.3-1.49 NA</td>
		</tr>
		<tr>
			<td>Objective heater</td>
			<td>Bioptechs</td>
			<td>150803, 150819-19</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent filter cube</td>
			<td>Chroma</td>
			<td>49004 or 49008 
			41017 or 49020</td>
			<td>optimized for Rhodamine fluorescence&#160; 
			optimized for GFP fluorescence</td>
		</tr>
		<tr>
			<td>Acquisition software</td>
			<td>freeware MicroManager 
			Molecular Devices</td>
			<td>not applicable 
			MetaMorph 7.5</td>
			<td>http://valelab.ucsf.edu/~MM/MMwiki/ 
			other software can be used to acquire images and for a particle tracking</td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor</td>
			<td>iXon3, DU-897E-cs0-#BV</td>
			<td>Highly sensitive EMCCD camera</td>
		</tr>
		<tr>
			<td>Trapping laser</td>
			<td>IPG Photonics</td>
			<td>YLR-10-1064-LP</td>
			<td>1064 nm laser, 10 W</td>
		</tr>
		<tr>
			<td>Fluorescence excitation lasers</td>
			<td>Coherent, Inc. 
			Coherent, Inc.</td>
			<td>Sapphire 488 LP 
			Sapphire 552 LP</td>
			<td>excitation of green fluorophores 
			excitation of red fluorophores</td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial flow chambers</td>
			<td>Warner Instruments</td>
			<td>RC-20 or RC-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion pump</td>
			<td>Cole Palmer 
			Harvard Apparatus</td>
			<td>Masterflex 77120-52 
			Pico Plus</td>
			<td>Both pumps provide the required rate of liquid flow but a peristaltic pump may pulse at very slow speed. The flow with a syringe pump is more consistent for a wide range of rates but this pump has inertia.&#160;</td>
		</tr>
	</tbody>
</table>
<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 2: Microscopy chamber preparation.</td>
		</tr>
		<tr>
			<td>Modified microscope slides for reusable chambers</td>
			<td>Precision Glassblowing of Colorado</td>
			<td>Custom order www.precisionglassblowing.com</td>
			<td>Sonic slots in slides using schematics in Figure 1</td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Intramedic</td>
			<td>427410</td>
			<td>I.D. 0.58 mm, O.D. 0.965 mm; use these tubes to connect assembled chamber to the pump and waste container</td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Intramedic</td>
			<td>427400</td>
			<td>I.D. 0.28 mm, O.D. 0.61 mm; use these tubes to make the reusable chamber</td>
		</tr>
		<tr>
			<td>Regular microscope slides</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td>Other similar slides can be used</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>VWR</td>
			<td>48393-150, 48366-067</td>
			<td>Other similar coverslips can be used</td>
		</tr>
		<tr>
			<td>Silicon sealant</td>
			<td>World Precision Instruments</td>
			<td>KIT, SILICON SEALANT 5 MIN CURE</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy glue</td>
			<td>Loctite</td>
			<td>83082</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate adhesive</td>
			<td>Scotch 3M</td>
			<td>AD114</td>
			<td>Or cyanoacrylate adhesive from other manufacturers</td>
		</tr>
	</tbody>
</table>
<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 3: Coverslips cleaning and coating.</td>
		</tr>
		<tr>
			<td>Molecular Sieves, Grade 564</td>
			<td>Macron</td>
			<td>4490-04</td>
			<td width="200px"></td>
		</tr>
		<tr>
			<td>Coverglass Staining Jar</td>
			<td>Ted Pella, Inc.</td>
			<td>21036</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip Ceramic Holder</td>
			<td>Thomas Scientific</td>
			<td>8542e40</td>
			<td></td>
		</tr>
		<tr>
			<td>PlusOne Repel Silane</td>
			<td>GE Healthcare Biosciences</td>
			<td>17-1332-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-digoxigenin AB</td>
			<td>Roche applied science</td>
			<td>11093274910</td>
			<td></td>
		</tr>
	</tbody>
</table>
<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 4: Preparation of seeds and segmented microtubules.</td>
		</tr>
		<tr>
			<td>Tubulin</td>
			<td>purified from cow brains 
			Cytoskeleton, Inc</td>
			<td>&#160; 
			T238P</td>
			<td>For purification protocols see 49&#8211;51 
			Unlabeled porcine tubulin</td>
		</tr>
		<tr>
			<td>Labeled tubulin</td>
			<td>Cytoskeleton, Inc 
			Invitrogen 
			Invitrogen</td>
			<td>TL590M 
			C1171 (Rhodamine) 
			A-2952 (Digoxigenin)</td>
			<td>Rhodamine-labeled porcine tubulin 
			Tubulin can be labeled with any amine-reactive dye as in reference52.</td>
		</tr>
		<tr>
			<td>GMPCPP</td>
			<td>Jena Biosciences</td>
			<td>NU-405</td>
			<td>Aliquot and store at -70 &#176;C</td>
		</tr>
		<tr>
			<td>VALAP</td>
			<td>vaseline, lanolin, and paraffin at 1:1:2 by mass</td>
			<td></td>
			<td>see ref. 9</td>
		</tr>
	</tbody>
</table>

preparation of segmented microtubules to study motions driven by the disassembling microtubule ends

Microtubulo depolimerizzazione in grado di fornire la forza di trasportare diversi complessi proteici e perline proteine ​​rivestite in vitro. I meccanismi sottostanti sono pensati per svolgere un ruolo vitale nel cromosoma movimenti microtubuli-dipendente durante la divisione cellulare, ma le proteine ​​interessate ei loro ruoli esatti sono mal definiti. Quindi, vi è una crescente necessità di sviluppare saggi con cui studiano tale motilità in vitro utilizzando componenti purificati e ambiente biochimico definito. I microtubuli, tuttavia, sono intrinsecamente instabili polimeri, la loro commutazione tra crescita e l&#39;accorciamento è stocastico e difficile da controllare. I protocolli che descriviamo qui approfittano dei microtubuli segmentati che sono fatti con il photoablatable stabilizzazione caps. Depolimerizzazione di tali microtubuli segmentati può essere attivato con alta risoluzione temporale e spaziale, aiutando in tal modo studi di motilità presso lo smontaggio estremità del microtubulo. Questa tecnica può essere utilizzata per caRRY un&#39;analisi quantitativa del numero di molecole in complessi proteici fluorescenza marcata, che si muovono con processively microtubuli dinamica termina. Per ottimizzare il rapporto segnale-rumore in questo ed altri saggi fluorescenti quantitative, coprioggetto devono essere trattati per ridurre l&#39;assorbimento aspecifico di proteine ​​solubili fluorescenza marcata. Protocolli dettagliati vengono forniti per tenere conto della disuniformità di illuminazione fluorescente, e determinare l&#39;intensità di un singolo fluoroforo utilizzando equidistante forma gaussiana. Infine, si descrive l&#39;uso dei microtubuli segmentati per studiare i movimenti microtubuli-dipendente delle microsfere rivestite di proteine, fornendo approfondimenti nella capacità di diverse proteine ​​motrici e non motori a coppia microtubuli depolimerizzazione al movimento merci processive.

Proteine ​​inseguimento con depolimerizzante microtubuli finisce. Lievito cinetocoro componente Dam1 è di gran lunga il miglior consiglio-tracker del microtubulo depolimerizzante finisce 14. Questo complesso di 10 subunità marcato con GFP può essere facilmente espresso e purificato da cellule batteriche 18,38, quindi si raccomanda di usarlo come controllo positivo per il test-punta inseguimento. Una proteina fluorescente che tiene traccia con la fine depolimerizzante di un m...

Watch this Scientific Journal Video about Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends at JoVE.com

Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

I microtubuli sono intrinsecamente polimeri instabili, e il loro passaggio tra la crescita e l&#39;accorciamento è stocastico e difficile da controllare. Qui si descrivono i protocolli utilizzando microtubuli segmentato con tappi di stabilizzazione photoablatable. Depolimerizzazione dei microtubuli segmentati può essere attivato con alta risoluzione temporale e spaziale, aiutando in tal modo l&#39;analisi delle proposte di risoluzione con lo smontaggio estremità del microtubulo.

Preparazione di segmentate microtubuli allo Studio Proposte guidato dal Smontaggio microtubuli Ends

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are ...

preparation-segmented-microtubules-to-study-motions-driven

Research

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Biology

14.3K Views.  Russian Academy of Sciences.  I microtubuli sono intrinsecamente polimeri instabili, e il loro passaggio tra la crescita e l'accorciamento è stocastico e difficile da controllare. Qui si descrivono i protocolli utilizzando microtubuli segmentato con tappi di stabilizzazione photoablatable. Depolimerizzazione dei microtubuli segmentati può essere attivato con alta risoluzione temporale e spaziale, aiutando in tal modo l'analisi delle proposte di risoluzione con lo smontaggio estremità del m...

Video: Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test di sensibilità visiva alla velocità e direzione del movimento in lucertole

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Ricerca

Biologia

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Computer-Generated modello animale Stimoli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Isolamento e purificazione di Drosophila Neuroni periferici da Magnetic Ordinamento Bead

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Produzione di<em&gt; Xenopus tropicalis</em&gt; Uovo Estratti per identificare RNA associata ai microtubuli

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the Mgm101 protein is involved in the recombinational repair of mitochondrial DNA. Recent investigations have indicated that Mgm101 is related to the Rad52-type recombination protein family. These proteins form large oligomeric rings and promote the annealing of homologous single stranded DNA molecules. However, the characterization of Mgm101 has been hindered by the difficulty in producing the recombinant protein. Here, a reliable procedure for the preparation of recombinant Mgm101 is described. Maltose Binding Protein (MBP)-tagged Mgm101 is first expressed in Escherichia coli. The fusion protein is initially purified by amylose affinity chromatography. After being released by proteolytic cleavage, Mgm101 is separated from MBP by cationic exchange chromatography. Monodispersed Mgm101 is then obtained by size exclusion chromatography. A yield of ~0.87 mg of Mgm101 per liter of bacterial culture can be routinely obtained. The recombinant Mgm101 has minimal contamination of DNA. The prepared samples are successfully used for biochemical, structural and single particle image analyses of Mgm101. This protocol may also be used for the preparation of other large oligomeric DNA-binding proteins that may be misfolded and toxic to bacterial cells.

The yeast mitochondrial nucleoid protein, Mgm101, is a Rad52-type recombination protein that forms large oligomeric rings. A protocol is described to prepare soluble recombinant Mgm101 using the Maltose Binding Protein (MBP)-tagging strategy coupled with cation exchange and size exclusion chromatography.

Preparazione del Mgm101 ricombinazione delle proteine ​​da Strategy Tagging MBP-based

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Utilizzando cellule Caco-2 allo Studio Lipid Trasporto dall&#39;intestino

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

Misurazione della microtubule Dynamics mediante microscopia disco rotante in spindle mitotici monopolari

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement. Here we demonstrate a combination of three of these methodologies. Motility in soft agar is the oldest, offering a strong selection for isolating gain-of-function suppressor mutations in motility-impaired strains, where motility is restored through a second mutation. The cell-tethering technique, first employed to demonstrate the rotary nature of the flagellar motor, can be used to assess the impact of signaling effectors on the motor speed and its ability to switch rotational direction. The &#8220;border-crossing&#8221; assay is more recent, where swimming bacteria can be primed to transition into moving collectively as a swarm. In combination, these protocols represent a systematic and powerful approach to identifying components of the motility machinery, and to characterizing their role in different facets of swimming and swarming. They can be easily adapted to study motility in other bacterial species.

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Many bacteria use flagella-driven motility to navigate their environment and colonize favorable surroundings both individually and as a collective. Demonstrated here is the use of three established methods that exploit motility as a selection tool to identify components/pathways contributing to swimming and swarming motility.

Investigare la motilità guidata dalla Flagella in Escherichia coli applicando tre tecniche consolidate in una serie

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, ...

A Step-by-Step Guide to Mosquito Electroantennography

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.

The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males.

Una guida passo-passo all'elettroantennography delle zanzare

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Un test di chemiotassi migliorato per la rapida identificazione dei chemioattrattivi rizobatterici negli essudati radicali

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...