Name: use of phluorin to assess the dynamics of axon guidance receptors in cell culture and in the chick embryo
Uploaded: 2014-01-12T14:00:00
Description: Watch this Scientific Journal Video about Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo at JoVE.com

Vi takker Homaira Nawabi, Frederic Moret og Isabelle Sanyas for deres hjælp. Dette arbejde understøttes af CNRS, Association Francaise contre les Myopathies (AFM), ANR YADDLE, Labex DevWeCan, Labex Cortex, ERC YODA til V.C.; C.D-B og A.J støttes af henholdsvis en La Ligue-contre le cancer og Labex DevWeCan-stipendier.

1. Kloning strategi for tag PlexinA1 receptor med pHluorin
<ol><li>Vælg en passende udtryksvektor som rygrad(f.eks. musereceptoren plexinA1, der udtrykker vektor, en slags gave fra Dr. Andreas Puschel11). Bemærk: Denne plexinA1 vektor blev udviklet til at opnå effektiv HA- eller VSV-mærket receptorindføring i plasmamembranen.</li>
	<li>Forstærk den ekliptiske pHluorin-kodningssekvens ved hjælp af den passende plasmid som skabelon(f.eks. pHluorin-mærket GABA A-receptor, en slags g...

<ol>
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Protoc.. 2012, 213-217 (2012).</li><li>Avraham, O., Zisman, S., Hadas, Y., Vald, L., Klar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+axonal+pathways+of+genetically+defined+groups+of+neurons+in+the+chick+neural+tube+utilizing+in+ovo+electroporation.">Deciphering axonal pathways of genetically defined groups of neurons in the chick neural tube utilizing in ovo electroporation.</a> J. Vis. Exp. (39), 1792-17 (2010).</li><li>Blank, M. C., Chizhikov, V., Millen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+ovo+electroporations+of+HH+stage+10+chicken+embryos.">In ovo electroporations of HH stage 10 chicken embryos.</a> J. Vis. Exp. (9), (2007).</li><li>Wilson, N. H., Stoeckli, E. 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Denne protokol giver en trin-for-trin procedure for at følge dynamikken i en axon vejledning receptor både i cellekultur og i den udviklingsmæssige sammenhæng af chick embryo rygmarven.
For at designe et de novo pHluorin-mærket protein skal der tages hensyn til to punkter vedrørende kloningsstrategien. For det første bør pHluorin-mærket udsættes for lumen af de sure endosomer og dermed for det ekstracellulære rum for at visualisere plasmamembranreceptorpuljen. Den korrekte ...

Under deres navigation integrerer axoner flere miljømæssige signaler, der guider dem mod deres mål. Disse signaler aktiverer styrereceptorer på overfladen af axonterminaler, vækstkeglerne, som igen starter en passende signalvej. Således er den tidsmæssige og rumlige regulering af receptorernes celleoverfladefordeling afgørende for at indstille følsomheden af vækstkeglerne1. I denne sammenhæng er midterlinjeovergang med commissural axoner en fremragende model til at undersøge reguleringen af receptorcelleoverfladeniveauer. I den udviklende rygmarv tiltrækkes commissural axoner i første omgang mod den ventrale gulvplade, hvor de krydser midterlinjen. Efter krydsning mister de deres lydhørhed over for gulvpladens tiltrækningskraft og får respons på gulvpladeafvisende midler, så de kan forlade gulvpladen og navigere mod deres endelige destination i den kontralaterale side af nervesystemet2,3. Regulering af receptortilgængelighed på vækstkegleroverfladen er en af de mekanismer, der ligger til grund for skiftet af reaktionsevne til midterlinjesignaler4,5. Således er selektiv overvågning af de receptorer, der er til stede ved plasmamembranen af vækstkegler, af største betydning. Vi beskriver her en metode baseret på den pH-afhængige fluorescensegenskab af en grøn fluorescerende protein (GFP) variant til specifikt at visualisere axon-vejledningsreceptorerne, der er adresseret til plasmamembranen in vitro og in vivo, i den udviklende chick rygmarv.
Rothman og kolleger manipuleret af punkt mutationer pH-følsomme varianter af GFP herunder ekliptika pHluorin6. Ekliptisk pHluorin har den egenskab, at den ikke er fluorescerende, når den udsættes for sur pH (<6), samtidig med at den er fluorescerende ved neutral pH. Dette gør det muligt at skelne ikkefluorescerende receptorer, der er lokaliseret i intracellulære sure rum(dvs. endosomer, menneskehandelsvesikler) fra fluorescerende receptorer, der er indbygget i plasmamembranen og dermed udsat for den ekstracellulære neutrale pH7. Vi benyttede os af dette til at overvåge plasmamembranlokaliseringen af plexinA1, en axon-vejledningsreceptor, der formidler vækstkeglerresponsen på den midline-afvisende semaphorin 3B5 (Figur 1A). Vi beskriver her in vitro karakterisering af en pHluorin-plexinA1 konstruere, sammen med in ovo elektroporation8-10 af denne konstruktion i udviklingslandene chick rygmarv efterfulgt af mikroskopiske analyse af cryosections som gør det muligt at følge axon vejledning receptor dynamik in vivo med både rumlige og tidsmæssige opløsninger.

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		<tr height="21">
			<td height="21" style="height:21px;width:264px;">COS7 cells</td>
			<td style="width:147px;">ATCC</td>
			<td style="width:219px;">CRL-1651</td>
			<td style="width:379px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">DMEM GlutaMAX</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">61965-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Sodium pyruvate</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">11360-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Amphotericin B</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">A2942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fetal bovine serum</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Penicillin/Streptomycin</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:264px;">Exgen500 reagent</td>
			<td style="width:147px;">Euromedex Fermentas</td>
			<td style="width:219px;">ET0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">PBS -Ca2+ -Mg2+</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fast green dye</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">F7252</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">32% Paraformaldehyde aqueous solution</td>
			<td style="width:147px;">Electron Microscopy</td>
			<td style="width:219px;">15714-S</td>
			<td>Dilute extemporaneously in PBS to achieve a 4% solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Gelatin from cold water fish skin</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">G7041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Sucrose</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">S0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Cryomount</td>
			<td style="width:147px;">Histolab</td>
			<td style="width:219px;">00890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Hoechst 34580</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:219px;">H21486</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Mowiol 4-88</td>
			<td style="width:147px;">Fluka</td>
			<td style="width:219px;">81381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1008px;">Consumables</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Bottom-glass 35 mm dish</td>
			<td style="width:147px;">MatTek</td>
			<td style="width:219px;">P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">5 ml Syringe</td>
			<td style="width:147px;">Terumo</td>
			<td style="width:219px;">SS-05S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Needles 0.9 mm x 25 mm</td>
			<td style="width:147px;">Terumo</td>
			<td style="width:219px;">NN-2025R</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Capillaries</td>
			<td style="width:147px;">CML</td>
			<td style="width:219px;">PP230PO</td>
			<td>capillaries are stretched manually in the flame</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Superfrost Plus Slides</td>
			<td style="width:147px;">Thermo Scientific</td>
			<td>4951PLUS</td>
			<td></td>
		</tr>
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			<td colspan="4" height="21" style="height:21px;width:1008px;">Material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Curved scissors</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">129-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Microscalpel</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">10316-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Forceps</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">Dumont #5 REF#11254</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1008px;">Equipment/software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Time lapse microscope</td>
			<td style="width:147px;">Zeiss</td>
			<td style="width:219px;">Observer 1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">Temp module S</td>
			<td style="width:147px;">PECON for Zeiss</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">CO2 module S</td>
			<td style="width:147px;">PECON for Zeiss</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Metamorph software</td>
			<td style="width:147px;">Metamorph</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">Eggs incubator</td>
			<td style="width:147px;">Sanyo</td>
			<td style="width:219px;">MIR154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Electroporator apparatus</td>
			<td style="width:147px;">Nepa Gene CO., LTD</td>
			<td style="width:219px;">CUY21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Electrodes</td>
			<td style="width:147px;">Nepa Gene CO., LTD</td>
			<td style="width:219px;">CUY611P7-4</td>
			<td>4 mm platinum electrodes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fluorescence stereomicroscope</td>
			<td style="width:147px;">LEICA</td>
			<td style="width:219px;">MZ10F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Cryostat</td>
			<td style="width:147px;">MICROM</td>
			<td style="width:219px;">HM550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Confocal microscope</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:219px;">FV1000, X81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fluoview software</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CLC Main Workbench software</td>
			<td style="width:147px;">CLC Bio</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

use of phluorin to assess the dynamics of axon guidance receptors in cell culture and in the chick embryo

Under udviklingen spiller axon-vejledningsreceptorer en afgørende rolle i reguleringen af axoners følsomhed over for både attraktive og frastødende signaler. Faktisk er aktivering af vejledningsreceptorerne det første trin i signalmekanismerne, der tillader axonspidser, vækstkeglerne, at reagere på liganderne. Som sådan er gradueringen af deres tilgængelighed på celleoverfladen en af de mekanismer, der deltager i fastsættelsen af vækstkeglernes følsomhed. Vi beskriver her en metode til præcist at visualisere spatio-temporal celleoverfladedynamik af en axon-vejledningsreceptor både in vitro og in vivo i den udviklende chick rygmarv. Vi udnyttede den pH-afhængige fluorescensegenskab for en grøn fluorescerende proteinvariant (GFP) til specifikt at detektere den brøkdel af axonvejledningsreceptoren, der er adresseret til plasmamembranen. Vi beskriver først in vitro-valideringen af sådanne pH-afhængige konstruktioner, og vi beskriver yderligere deres anvendelse in vivo, i chick spinal akkord, for at vurdere den spatio-tidsmæssige dynamik i axon-vejledningsreceptoren af interesse.

<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50883/50883fig1highres.jpg" src="/files/ftp_upload/50883/50883fig1.jpg"> Figur 1. A. Ordningen for egenskaberne pHluorin-plexinA1 fluorescens i en cellekontekst. PHluorin er ikke-fluorescerende i intracellulære rum, hvor pH-handelen er sur (<6), f.eks. Dette gør det kun muligt at visualisere celleoverfladepuljen for pHluorin-plexinA1-receptoren. B. Kort over k...

Watch this Scientific Journal Video about Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo at JoVE.com

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

Vi beskriver her brugen af en pH-følsom grøn fluorescerende proteinvariant, pHluorin, til at studere spatio-tidsmæssig dynamik axon vejledning receptorer handel på celleoverfladen. Den pHluorin-mærkede receptor udtrykkes både i cellekultur og in vivoved hjælp af elektroporation af kyllingembryoet.

Brug af pHluorin til at vurdere dynamikken i Axon-vejledningsreceptorer i cellekultur og i chickembryoet

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Research

JoVE Journal

Neuroscience

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualisering af proprioceptorer i<em&gt; Drosophila</em&gt; Larver og pupper

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Elektroporation af baghjerne at spore axonale baner og Synaptic Mål i Chick Embryo

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Inducerende plasticitet astrocytisk receptorer ved manipulation af neuronfyring priser

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

Hele-mount Imaging af museembryo Sensory Axon Fremskrivninger

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Imaging Ca<sup&gt; 2+</sup&gt; Dynamics i Cone Lysmodtageren Axon Terminals i Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

Udnytte Kombineret Metoder til Definer rolle plasmamembranen Delivery Under Axon Forgrening og neuronal Morfogenese

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Brugen af Trace Eyeblink klassisk konditionering at vurdere hippocampus dysfunktion i en rotte Model af føtale alkohol spektrum forstyrrelser

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

Ballistisk mærkning af pyramideformede neuroner i hjerneskiver og i primærcellekultur

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...