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 hjelp. Dette arbeidet støttes av 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 av henholdsvis la Ligue contre le cancer og Labex DevWeCan fellowships.

1. Kloning strategi for å merke PlexinA1 reseptor med pHluorin
<ol><li>Velg en passende uttrykksvektor som ryggrad (f.eks. musreseptoren plexinA1 som uttrykker vektor, en slags gave av Dr. Andreas Puschel11). Merk: Denne plexinA1-vektoren ble utviklet for å oppnå effektiv HA- eller VSV-merket reseptorinnsetting i plasmamembranen.</li>
	<li>Forsterke av PCR den ekliptiske pHluorin-kodesekvensen ved hjelp av tilstrekkelig plasmid som mal (f.eks. pHluorin-merket GABA A-reseptor, en slags...

<ol>
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Denne protokollen gir en trinnvis prosedyre for å følge dynamikken i en axonveiledningsreseptor både i cellekultur og i utviklingskonteksten til kyllingembryoens ryggmarg.
For å designe et de novo pHluorin-merket protein, må to punkter vurderes angående kloningsstrategien. For det første bør pHluorin-taggen eksponeres for lumen av de sure endosomene, og følgelig til det ekstracellulære rommet for å visualisere plasmamembranreseptorbassenget. Dermed er riktig posisjonering a...

Under navigasjonen integrerer axoner flere miljøsignaler som veileder dem mot målet. Disse signalene aktiverer veiledningsreseptorer på overflaten av axonterminaler, vekstkjeglene, som igjen starter en passende signalvei. Dermed er den tidsmessige og romlige reguleringen av celleoverflatefordelingen av reseptorene avgjørende for å sette følsomheten til vekstkjeglen1. I denne sammenhengen er midtlinjekryssing av kommissurale axoner en utmerket modell for å undersøke reguleringen av reseptorcelleoverflatenivåer. I den utviklende ryggmargen tiltrekkes kommissurale aksoner i utgangspunktet mot ventral gulvplate der de krysser midtlinjen. Etter kryssing mister de sin respons på gulvplatens tiltrekningsmidler og får respons på gulvplateavstøtende midler slik at de kan gå ut av gulvplaten og navigere mot deres endelige destinasjon i den kontralaterale siden av nervesystemet2,3. Regulering av reseptortilgjengelighet på vekstkjegleoverflaten er en av mekanismene som ligger til grunn for overgangen av respons til midtlinjesignaler4,5. Dermed er selektiv overvåking av reseptorene som er tilstede ved plasmamembranen av vekstkjegler av største betydning. Vi beskriver her en metode basert på den pH-avhengige fluorescensegenskapen til en grønn fluorescerende protein (GFP) -variant for å visualisere axonveiledningsreseptorene som er adressert til plasmamembranin vitro og in vivo, i den utviklende kyllingens ryggmarg.
Rothman og kolleger konstruert av punktmutasjoner pH-sensitive varianter av GFP inkludert ecliptic pHluorin6. Ecliptic pHluorin har egenskapen til å være ikke-påvirkende når den utsettes for sur pH (<6), samtidig som den er fluorescerende ved nøytral pH. Dette gjør det mulig å skille nonfluorescent reseptorer lokalisert i intracellulære sure rom (dvs. endosomer, trafficking vesicles) fra fluorescerende reseptorer innlemmet i plasmamembranen og dermed utsatt for den ekstracellulære nøytrale pH7. Vi benyttet oss av dette for å overvåke plasmamembranlokaliseringen av plexinA1, en axonveiledningsreseptor som formidler vekstkjegleresponsen til midtlinjeavstøtende semaphorin 3B5 (figur 1A). Vi beskriver her in vitro karakterisering av en pHluorin-plexinA1 konstruksjon, sammen med i ovo elektroporasjon8-10 av denne konstruksjonen i den utviklende kylling ryggmargen etterfulgt av mikroskopisk analyse av kryoseksjoner som gjør det mulig å følge axon veiledning reseptor dynamikken in vivo med både romlige og temporale oppløsninger.

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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>
		</tr>
		<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>
		</tr>
		<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>
		<tr height="21">
			<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 utviklingen spiller axonveiledningsreseptorer en avgjørende rolle i å regulere axonenes følsomhet for både attraktive og frastøtende signaler. Faktisk er aktivering av veiledningsreseptorene det første trinnet i signalmekanismene som tillater axonspisser, vekstkjeglene, å svare på ligandene. Som sådan er moduleringen av deres tilgjengelighet på celleoverflaten en av mekanismene som deltar i å sette vekstkjeglefølsomheten. Vi beskriver her en metode for å visualisere den romlige-temporale celleoverflatedynamikken til en axonveiledningsreseptor både in vitro og in vivo i den utviklende kyllingens ryggmarg. Vi benyttet oss av den pH-avhengige fluorescensegenskapen til en grønn fluorescerende proteinvariant (GFP) for å spesifikt oppdage brøkdelen av axonveiledningsreseptoren som er adressert til plasmamembranen. Vi beskriver først in vitro-valideringen av slike pH-avhengige konstruksjoner, og vi beskriver ytterligere deres bruk in vivo, i kyllingens spinal akkord, for å vurdere den romlige-temporale dynamikken i axonveiledningsreseptoren av 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 med pHluorin-plexinA1 fluorescensegenskapene i en cellulær kontekst. PHluorin er ikke-påvirkende i intracellulære rom der pH er sur (<6) som i menneskehandel vesicules eller i endosomer og er fluorescerende når den utsettes for det ekstracellulære mediet der pH er nøytral. Dette g...

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 bruken av en pH-sensitiv grønn fluorescerende proteinvariant, pHluorin, for å studere den romlige-temporale dynamikken i axonveiledningsreseptorer som smugler på celleoverflaten. Den pHluorin-taggede reseptoren uttrykkes både i cellekultur og in vivo, ved hjelp av elektroporasjon av kyllingembryoet.

Bruk av pHluorin for å vurdere dynamikken i Axon-veiledningsreseptorer i cellekultur og i chick-embryoet

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 av Proprioceptors 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

Electroporation av hindbrain å spore Aksonal Trajectories og Synaptic Targets 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

Inducing Plasticity av astrocytic Reseptorer av Manipulering av nevronal utløsning 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

Hel-mount Imaging av Mouse Embryo Sensory Axon Anslag

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 fotoreseptor Axon terminaler 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

Utnytte Kombinert Metoder for å definere rollen til plasmamembranen Levering Under Axon Forgrening og neuronal morphogenesis

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

Bruk av spor Eyeblink klassisk betinging å vurdere hippocampus dysfunksjon i en rotte modell av Fetal Alcohol Spectrum Disorders

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 merking av pyramidale nevroner i hjerneskiver og i primær cellekultur

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