Finanziamenti forniti dalla National Science Foundation (MF).

Introduzione L&#39;OMT è un adattamento di un sistema di legare magnetica [1] progettato per incorporare un &quot;simulatore virtuale pennacchio&quot;. I seguenti protocolli spiegherà come legare correttamente le mosche (Parte 1) e fornire le strategie per creare i magneti (Parte 2) e la massa del flusso regolato sistema di erogazione degli odori (Part3). Le informazioni qui descritto è ottimizzato per questo particolare sistema e possono variare per i componenti con le altre specifiche tecniche. Parte 1: vola Tethering I passaggi per legare qui descritte sono simili a molti studi precedenti [2], ma adattati per l&#39;uso in OMT [3]. Tethering corretta è fondamentale per garantire solide prove sperimentali e ripetibili. L&#39;importanza di legare attento non può essere sopravvalutata. <ol><li> Raccogliere vola 4-6 giorni dopo l&#39;eclosion e farli morire di fame per 4-6 ore. Posizionare un kimwipe inumidito con acqua nel flacone senza cibo per prevenire l&#39;essiccamento. </li><li> Trasferimento di un lotto di mosche da una bottiglia di una piccola fiala e inserirlo in un blocco di ottone su un palco di raffreddamento Peltier fissato a circa 4 gradi Celsius. La temperatura corretta anestetizza le mosche entro circa 30 secondi. </li><li> Ordina le mosche sulla superficie fredda della fase di raffreddamento. Un Kimwipe umido garantisce un raffreddamento costante e assorbimento dell&#39;umidità dalla cuticola. Selezionare la più grande femmine per il tethering. </li><li> Utilizzando un pennello fine, spingere una mosca in posizione nel custom-built a forma di sarcofago per ospitare un unico selvatico moscerino della frutta femmina tipo. <img src="/files/ftp_upload/old/1063_2008_11_26_2/1063_Figure-01_thumb.png" alt="" /> Figura 1. <a href="https://www.jove.com/files/1063_2008_10_28_1/1063_Figure-01.png">Clicca qui per vedere la versione ingrandita della figura.</a> </li><li> Noi posizione un perno minutien (Multe Strumenti scienza) con una bacchetta magnetica montata su un micromanipolatore. Il perno è allineato in modo tale che l&#39;estremità smussata tocca il torace dorsale appena dietro la testa tra le due ali. Il perno deve essere perfettamente verticale quando viene visualizzato a testa alta verso gli occhi. Anche una piccola quantità di &#39;roll&#39; tra il volare e il pin non può essere tollerato. La vista laterale angolo tra il perno e l&#39;asse longitudinale del corpo deve essere di un naso-up angolo di 30 gradi rispetto all&#39;orizzonte (Fig. 1). Nota: Per un funzionamento ottimale nella pastoia magnete, il pin può essere necessario tagliati a lunghezze. Questo dipenderà interamente dalla distanza tra i magneti sospensione. </li><li> Una volta che il perno minutien è allineato, usare un sottile filo d&#39;acciaio per applicare una piccola goccia di colla UV-attivati ​​(Electrolite Corp) alla fine smussato del perno minutien. </li><li> Manipolare il perno delle gocce di colla in modo che il torace tocca al volo appena sopra la testa. Con la dimensione goccia corretta, la colla deve temprare e il flusso dal perno sul torace. Fare attenzione a non usare troppa colla. </li><li> Cura la colla con due 20 secondi scoppia di luce UV. </li><li> Posizionare il volano fisso da parte e ripetere. Fetta piazzette di Kimwipe e con una pinza dare uno a ciascuno volare legato a frenare volo. </li><li> Dopo un&#39;ora di recupero, le mosche legato sono pronti per gli esperimenti. </li><li> Incollaggio improprio può ostacolare la capacità di una mosca a svolgere nel campo magnetico. Ogni volo deve essere controllato per rotazione regolare all&#39;inizio di ogni esperimento per assicurare le mosche siano incollati. Questo può essere ottenuto dalla rotazione di un modello a strisce con un periodo sufficientemente ampio spazio per ottenere risposte optomotor forte [4]. Se il volo è incollato a l&#39;angolo di inclinazione sbagliata o inclinato sul pin con rispetto all&#39;asse di rollio, la mosca non possono (i) girare a tutti, (ii) hanno una gamma limitata di movimento, o (iii) ruotare a incoerente velocità. Nei casi di cui sopra al volo deve essere eliminata e sostituita con una nuova. Se questo risolve il problema era probabilmente un caso di incollaggio improprio. (Se la sostituzione al volo non risolve il problema, vedere la Parte 2.4) </li></ol> Parte 2: Allineare i magneti Allineando i magneti (terre rare-magnets.com) è necessario per la corretta volare per raggiungere una vasta liscia a 360 gradi di movimento. Attenersi alla seguente procedura per allineare correttamente i magneti. <ol><li> I magneti ad anello inferiore sono tenuti orizzontalmente da un collare in plastica e posto direttamente sopra una camera a vuoto chiaro (Fig. 1, descritta più avanti nella parte 3). Il magnete asta superiore è fissato in verticale di circa 3 / 4 &quot;sopra i magneti più basso. </li><li> Circa allineare il magnete superiore e inferiore visivamente posizionando il magnete superiore asta direttamente sopra il punto centrale dei magneti dell&#39;anello inferiore. E &#39;utile per fissare il magnete superiore ad un micromanipolatore (Siskiyou Inc.) per la regolazione scala fini. </li><li> Epossidica un V-gioiello del cuscinetto (parti piccole) sulla superficie inferiore del magnete superiore per ridurre al minimo l&#39;attrito di rotazione e standardizzare il posizionamento volare. </li><li> Mettere una mosca in campo e regolare la orizzontale e vertical posizione del magnete superiore fino al volo possa agevolmente e in costante rotazione di 360 gradi lungo l&#39;asse di imbardata. Ogni volo deve essere controllato per rotazione regolare all&#39;inizio di ogni esperimento per assicurare i magneti sono correttamente allineati e che singoli pin minutien non siano danneggiati (vedi Parte 1.11). Se i magneti non sono allineati correttamente al volo non possono (i) girare a tutti i (ii) hanno una gamma limitata di movimento (iii) ruotare a velocità incoerente. Se il problema è risolto sostituendo al volo con uno nuovo, è stato molto probabilmente dovuto ad un problema di incollaggio (vedi parte 1). Se questi problemi esistono per ogni volo, è probabile un problema con l&#39;allineamento dei magneti e di ulteriori aggiustamenti sarà richiesto. </li><li> Attenzione: durante il gioco con magneti in terre rare può essere divertente, sono molto pericolosi. L&#39;abuso si traduce in attrezzature danneggiate e / o minori lesioni personali. Prestare la massima attenzione a questi magneti allettante e facilmente sottovalutato. </li></ol> Parte 3: Consegna Odore <ol><li> Il sistema di erogazione odore è basato su quello descritto in precedenza [5] ed è la parte più difficile da ottimizzare (Fig. 1). Si richiede molta pazienza e tentativi ed errori. Seguire questa procedura per ottenere il monitoraggio stabile odore. </li><li> Il set-up iniziale del sistema odore richiede l&#39;esecuzione di stretta misura tubi in Teflon (parti piccole) dal gas multiplexer (Sable Systems International) per l&#39;acqua su misura / odore fiale. La presa di queste fiale si collega direttamente ai tubi porta il ipodermico utilizzando il tubo stesso in Teflon. </li><li> Una chiara camera a vuoto acrilico è posizionato sotto l&#39;arena per supportare i magneti. Usiamo un 4 millimetri tubo di vetro montato sotto il volano collegato alla camera a vuoto per portare il vuoto di apertura più vicino al volo e ridurre il diametro dell&#39;apertura. </li><li> Mettere una mosca legato nell&#39;arena con pinze. </li><li> Accendere un odore attraente (aceto di sidro di mele funziona bene), e verificare visivamente che la mosca tracce l&#39;odore. Se la mosca sembra avere alcuna preferenza per l&#39;odore, seguire i prossimi tre passi. Questo può richiedere vola diversi, e diverse prove. </li><li> Regolare la posizione del porto odore fino a quando il volo sembra seguire l&#39;odore. Abbiamo montato le porte odore di micromanipolatori (Siskiyou Inc.) e posizionati quattro millimetri anteriore dorsale e 3mm a testa della mosca. </li><li> Regolare la portata di massa fino a quando il volo sembra seguire l&#39;odore. Le nostre impostazioni sono 7ml/min (Sable Systems International MFC-4). </li><li> Regolare la portata del vuoto fino a quando il volo sembra seguire l&#39;odore. Abbiamo impostato la portata vuoto a 13l/min utilizzando un regolatore di flusso (Cole Parmer). Nota: Costruzione linee di alimentazione a vuoto possono variare nel tempo causando variazioni nella struttura pennacchio. Può essere utile per collegare una pompa esterna a vuoto per il regolatore di flusso per le condizioni di flusso costante di vuoto. </li><li> Una volta che la posizione del porto e le portate sono impostati, periodicamente passare da una fornitura di vapore acqueo e vapori odore utilizzando una commutabile tramite un multiplexer gas e verificare visivamente che la mosca tracce solo l&#39;odore, quando l&#39;odore è acceso. Occasionalmente, odore residuo all&#39;interno del porto odore può provocare monitoraggio odore è l&#39;odore è spento. Ciò può essere evitato senza rimuovere la porta odore posizionata correttamente compilando i tubi ipodermico con etanolo e raccolta alla fine consegna con un Kimwipe. Con aria compressa, soffiare ogni residuo di etanolo fuori e riprendere gli esperimenti. </li></ol> Rappresentante dei risultati: Quando questi passaggi sono seguiti correttamente due cose devono essere osservati. In primo luogo, sotto costante stimoli visivi a rotazione una mosca legato posto nel OMT dovrebbe girare senza problemi a 360 gradi lungo l&#39;asse di imbardata. &quot;Jerky&quot; giri (chiamati saccadi) sono caratteristici del comportamento spontaneo di svolta in assenza di costanti stimoli visivi. Se le mosche non possono ruotare apparentemente senza problemi, incollaggio improprio (vedi parte 1) e magneti allineati possano esserne la causa (cfr. parte 2). In secondo luogo, quando l&#39;odore è acceso, il volo dovrebbe dirigersi in direzione dell&#39;odore. Quando l&#39;odore è spento, la mosca dovrebbe saccade spontaneamente, senza preferenza per ogni singola posizione in campo. Se vola non attivamente traccia il pennacchio odore, la posizione del porto odore, la portata d&#39;aria e / o la portata di vuoto può avere bisogno di regolazione (cfr. parte 3).

<ol>
	<li>Bender, J. A., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+stimulation+of+saccades+in+magnetically+tethered+Drosophila.">Visual stimulation of saccades in magnetically tethered Drosophila.</a> J. Exp. Biol. 209, 3170-3182 (2006).</li><li>Tammero, L. F., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collision-avoidance+and+landing+responses+are+mediated+by+separate+pathways+in+the+fruit+fly,+Drosophila+melanogaster.">Collision-avoidance and landing responses are mediated by separate pathways in the fruit fly, Drosophila melanogaster.</a> J. Exp. Biol. 205, 2785-2798 (2002).</li><li>Duistermars, B. J., Frye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossmodal+visual+input+for+odor+tracking+during+fly+flight.">Crossmodal visual input for odor tracking during fly flight.</a> Curr. Biol. 18, 270-275 (2008).</li><li>Reiser, M. B., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+display+system+for+insect+behavioral+neuroscience.">A modular display system for insect behavioral neuroscience.</a> J. Neurosci. Methods. 167, 127-139 (2008).</li><li>Frye, M. A., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+output+reflects+the+linear+superposition+of+visual+and+olfactory+inputs+in+Drosophila.">Motor output reflects the linear superposition of visual and olfactory inputs in Drosophila.</a> J. Exp. Biol. 207, 123-131 (2004).</li></ol>

Tethering mosche e suscitando monitoraggio stabile odore in questo campo è relativamente semplice. Si richiede pratica, pazienza e tentativi ed errori. Molte parti di questa arena può essere ulteriormente ottimizzato o sostituiti per produrre variazioni di potenziale sperimentale. Per esempio, le versioni più recenti della camera a vuoto sono molto più piccoli e permettere più spazio sotto l&#39;arena. Inoltre, la forza e il tipo di magneti in terre rare può essere variato leggermente l&#39;odore e le porte aggiuntive potrebbero esser...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cooling Stage</td>
<td>&nbsp;</td>
<td>Teca</td>
<td>LHP-300CP</td>
<td>&nbsp;</td>
<tr>
<td>Brass Cooling Block</td>
<td>&nbsp;</td>
<td>Custom Machined</td>
<td>&nbsp;</td>
<td>~4 x 8 x 2cm</td>
</tr>
<tr>
<td>Sarcophagus</td>
<td>&nbsp;</td>
<td>Custom Machined</td>
<td>&nbsp;</td>
<td>Contact us for details</td>
</tr>
<tr>
<td>UV-Activated Glue</td>
<td>&nbsp;</td>
<td>Kemxert corp</td>
<td>KOA300</td>
<td>1oz bottle lasts years</td>
</tr>
<tr>
<td>Panel System</td>
<td>&nbsp;</td>
<td>Caltech</td>
<td>http://www.dickinson.caltech.edu/panels</td>
<td>[4]</td>
</tr>
<tr>
<td>Minutien Pins</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-20</td>
<td>&nbsp;</td>
<tr>
<td>Lower Ring Magnets</td>
<td>&nbsp;</td>
<td>Rare-earth-magnets.com</td>
<td>NSN0615</td>
<td>5 - 0.75"o.d. x 0.375" i.d. x 0.125"</td>
</tr>
<tr>
<td>Upper Rod Magnet</td>
<td>&nbsp;</td>
<td>Rare-earth-magnets.com</td>
<td>NSN0750</td>
<td>0.125" x 1"</td>
</tr>
<tr>
<td>V-Jewel Bearing</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>VJ-0469-01</td>
<td>&nbsp;</td>
<tr>
<td>MFC-4 Flow Regulators</td>
<td>&nbsp;</td>
<td>Sable Systems International</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gas Multiplexer</td>
<td>&nbsp;</td>
<td>Sable Systems International</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Teflon Tubing</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>STT-20-C</td>
<td>&nbsp;</td>
<tr>
<td>20 Gauge Hypodermic Tubes</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>HTX-20R-06</td>
<td>&nbsp;</td>
<tr>
<td>Glass Tubing</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>4mm o.d.</td>
</tr>
<tr>
<td>Vacuum Flow Regulator</td>
<td>&nbsp;</td>
<td>Cole Parmer</td>
<td>EW-32464-52</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a magnetic tether system to investigate visual and olfactory mediated flight control in drosophila

E &#39;stato chiaro da molti anni che gli insetti utilizzano segnali visivi di stabilizzare la loro voce in un flusso di vento. Molti animali gli odori portata in pista dal vento. Come tale, la stabilizzazione visiva di bolina monitoraggio direttamente aiuti nel monitoraggio degli odori. Ma i segnali olfattivi influenzano direttamente il comportamento visivo di tracciamento indipendente di spunti vento? Inoltre, il diluvio recenti della ricerca sulla neurofisiologia e della genetica neurocomportamentale dell&#39;olfatto in Drosophila ha motivato test comportamentali sempre più tecnicamente sofisticate e quantitativi. Qui, abbiamo modificato un sistema magnetico tether originariamente ideato per gli esperimenti visione dotando l&#39;arena con pennacchi strette a flusso laminare odore. Una mosca è incollata ad un piccolo perno in acciaio e sospesa in un campo magnetico che permette di imbardata liberamente. Di piccolo diametro pennacchi odori degli alimenti sono diretti verso il basso sopra la testa al volo s, suscitando monitoraggio stabile da una mosca fame. Qui ci concentriamo sulla meccanica critica di tethering, allineando i magneti, definire il pennacchio odore, e confermando il monitoraggio stabile odore.

Watch this Scientific Journal Video about A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila at JoVE.com

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

Qui si descrivono come legare una mosca in un apparato olfattivo magnetico-legare (OMT). Noi descriviamo come allineare la magneti in terre rare e le porte degli odori, e come impostare le portate sia per la consegna stimolo e di aspirazione a vuoto per raggiungere il monitoraggio ottimale degli odori.

Un sistema magnetico Tether per Indagare visivo e olfattivo di controllo mediata Volo in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

a-magnetic-tether-system-to-investigate-visual-olfactory-mediated

Research

JoVE Journal

Biology

11.2K Views.  University of California, Los Angeles. Qui si descrivono come legare una mosca in un apparato olfattivo magnetico-legare (OMT). Noi descriviamo come allineare la magneti in terre rare e le porte degli odori, e come impostare le portate sia per la consegna stimolo e di aspirazione a vuoto per raggiungere il monitoraggio ottimale degli odori.

Video: A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Visivamente monitoraggio Odore mediata durante il volo in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

Ricerca

Biologia

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Unicellulari registrazioni di aspirazione da Fotorecettori Cone mouse

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

Un approccio pratico alla mappatura genetica Destino inducibile: una guida visiva a Mark e Pista Cellule In Vivo</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

Rapida rilevazione omogenea dei saggi biologici Utilizzando Magnetic biopercezione sistema di modulazione

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Vescicole lipidiche-mediata Cromatografia di affinità mediante Magnetic separazione delle cellule attivate (LIMACS): un nuovo metodo per l&#39;analisi delle interazioni proteina-lipide

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi di screening per identificare fenotipi postembrionale in C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Misurazione quantitativa della Invadopodia-mediata proteolisi della matrice extracellulare in singoli contesti e pluricellulari

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

Progettazione e analisi del comportamento delle preferenze di temperatura e del suo ritmo circadiano in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Un metodo migliore per precisa e rapida misurazione delle performance in volo<em&gt; Drosophila</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

La convalida di un modello di topo per Disrupt LINC Complessi in modo specifico-Cell

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...