Name: real-time imaging of axonal transport of quantum dot-labeled bdnf in primary neurons
Uploaded: 2014-09-15T15:00:00
Description: Watch this Scientific Journal Video about Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons at JoVE.com

Vi vil gerne takke Yue (Pauline) Hu, Rachel SINIT for deres tekniske bistand. Undersøgelsen er støttet af NIH tilskud (PN2 EY016525) og ved finansiering fra Down Syndrom forskning og behandling Fond og Larry L. Hillblom Foundation.

Kirurgiske og dyreforsøg udføres i nøje overensstemmelse med NIH Guide til Pleje og anvendelse af forsøgsdyr. Alle forsøg med brug af dyr er godkendt af UCSD Institutional Animal Care og brug Udvalg. 1. Plasmid Kloning, ekspression og oprensning af Mono-biotinyleret BDNF (mBtBDNF) BEMÆRK: Konstruere præ-proBDNFavi og BirA cDNA ind pcDNA3.1 vektor og coudtrykke i HEK293FT celler 10. Renses mBtBDNF anvendelse af Ni-NTA-perler ifølge tidligere offen...

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I denne undersøgelse rapporterer vi udviklingen af ​​en ny teknik til at fremstille biologisk aktivt monobiotinylated BDNF (mBtBDNF), der kan anvendes til at spore axonal transport af BDNF. Ved konjugering af proteinet til kvantepunktet streptavidin, og ved hjælp af en mikrofluid kammer metoden gør det muligt at detektere axonal transport af BDNF i ubearbejdet neuroner med enkelt molekyle følsomhed, i realtid og med rumlige og tidslige resolutioner. De værktøjer, der anvendes heri, tilvejebringe et middel til ...

Neuroner er stærkt polariserede celler, hvis lange og ofte meget udarbejdet processer er afgørende for etablering og vedligeholdelse af strukturen og funktionen af ​​neurale kredsløb. Axon spiller en afgørende rolle i udførelsen gods til og fra synapser. Proteiner og organeller syntetiseret i cellen soma skal transporteres gennem axoner at nå den præsynaptiske terminal til at understøtte neuronal funktion. Tilsvarende modtager signaler på distale axoner skal transduceres og transporteres til soma. Disse processer er afgørende for neuronal overlevelse, differentiering og vedligeholdelse. I skal gennemføres, at axonal transport i nogle neuroner gennem afstande mere end 1.000 gange diameteren af ​​cellen kroppen, er muligheden for let forestille sig, at selv små underskud markant kan påvirke neuronal og kredsløb funktion. Hjerne-afledt neurotrofisk faktor (BDNF), et medlem af neurotrofinfamilien af ​​vækstfaktorer, er til stede i maNY hjerne regioner, herunder hippocampus, hjernebark og basale forhjerne. BDNF spiller en afgørende rolle i erkendelse og hukommelse dannelse ved at støtte overlevelse, differentiering og funktion af neuroner, der deltager i kognitive kredsløb. BDNF binder til sin receptor, tyrosinkinase TrkB på Axon terminal, hvor det aktiverer TrkB-medierede signalveje herunder mitogenaktiveret proteinkinase / ekstracellulære signal-reguleret protein kinase (MAPK / ERK), phosphatidylinositol-3-kinase (PI3K) og phospholipase C-gamma (PLCγ). De proteiner, der deltager i disse signalveje er pakket på endocytiske vesikulære strukturer til dannelse af BDNF / TrkB signalering endosom 1-6, som derefter retrogradt transporteres til den neuronale soma. Den mikrofluid kultur kammer er en meget nyttig platform for at studere axonal biologi under normale forhold samt i fastlæggelsen af skade og sygdom 7,8. Ved isolerer axonerfra cellelegemerne, enheden har tilladelse til at studere specifikt transporten i axoner 8-10. PDMS baseret mikrofluide platforme med 450 um Microgroove barrierer, der anvendes i denne undersøgelse, blev købt i handlen (se Materialer tabel). At undersøge BDNF transport, vi udviklet en ny teknologi til at producere monobiotinylated BDNF (mBtBDNF). Vi tog fordel af biotin acceptor-peptid, AP (også kendt som AviTag). Det er en 15 aminosyresekvens, der indeholder en lysinrest som specifikt kan ligeret til et biotin af Escherichia coli-enzym biotin-ligase, BirA. Vi fusionerede den AviTag til den C-terminale ende af muse-præ-proBDNF cDNA ved PCR (figur 1A). Konstruktionen blev klonet ind i den mammale ekspressionsvektor pcDNA3.1 myc-sin vektor. Vi klonede også det bakterielle BirA DNA i pcDNA3.1 myc-sin vektor. De to plasmider blev transient cotransficeret ind HEK293FT celler til at udtrykke begge proteiner. BirA katalyserede ligering af bioti specifikt til lysin opholde sig AviTag ved C-terminalen af ​​BDNF ved en 1: 1-forhold at producere monobiotinylated BDNF monomer. Biotinyleret, modne BDNF med en molekylmasse på ~ 18 kDa blev udvundet og oprenset fra medierne under anvendelse af Ni-harpiks (figur 1C). Biotinylering BDNF var fuldstændig, bedømt ved manglende evne til at detektere modificeret BDNF ved immunoblotting (figur 1D). Streptavidin konjugerede kvantepunkter, QD 655, blev brugt til at mærke mBtBDNF at gøre QD-BDNF. Tilstedeværelsen af AviTag ikke interfererer med aktiviteten af BDNF som mBtBDNF var i stand til at aktivere phosphoryleret TrkB (figur 1E) og stimulere neuritudvækst (figur 1F) i det omfang af rekombinant humant BDNF (rhBDNF). Immunfarvning viser, at QD-BDNF colocalized med TrkB i hippocampus axoner, hvilket indikerer, at QD-BDNF er bioaktivt (figur 1G). At studere BDNF transport blev QD-BDNF tilføjes distale Axon rummikrofluide kulturer indeholdende rotte E18 hippocampale neuroner (figur 2A). QD-BDNF retrograd transport inden axonerne blev erobret af real-time levende billeder af den røde fluorescerende tag (Supporting videoer S1, S2). Ved at analysere kymograph genereret var QD-BDNF observeret at blive transporteret retrogradt på en bevægende hastighed på omkring 1,06 um / sek (figur 3A). GFP eller mCherry-mærkede BDNF er blevet anvendt til at spore axonal bevægelse BDNF. De vigtigste ulemper er, at de ikke er lyse nok til enkelt molekyle studier. Også tilstedeværelsen af ​​både anterograd og retrograd BDNF bevægelser gør det vanskeligt at vurdere, hvorvidt retrogradt transporteres BDNF var i en neurotrophin / receptor-kompleks. I denne video viser vi, de anvendte teknikker til at undersøge levende handel med QD-BDNF i mikrofluide kamre anvender primære neuroner. Den ultrabrightness og fremragende fotostabilitet af quantum dots makes det muligt at udføre langsigtet sporing BDNF transport. Disse teknikker kan udnyttes til at forbedre undersøgelser af axonal funktion i AD, HD og andre neurodegenerative lidelser.

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		<tr height="20">
			<td height="20" style="height:20px;width:261px;">Name</td>
			<td style="width:205px;">Company</td>
			<td style="width:175px;">Catalog Number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platinum pfx DNA polymerase&#160;</td>
			<td>Invitrogen</td>
			<td>11708021</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EcoRI&#160;</td>
			<td>Fermentas</td>
			<td>FD0274</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BamHI&#160;</td>
			<td>Fermentas</td>
			<td>FD0054</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEK293FT cells</td>
			<td>Invitrogen</td>
			<td>R70007</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM-high glucose media</td>
			<td>Mediatech</td>
			<td>10-013-CV</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">d-biotin&#160;</td>
			<td>Sigma</td>
			<td>B4639</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TurboFect&#160;</td>
			<td>Fermentas</td>
			<td>R0531</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PMSF&#160;&#160;</td>
			<td>Sigma</td>
			<td>P7626</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">aprotinin</td>
			<td>Sigma</td>
			<td>A6279</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ni-NTA resins</td>
			<td>Qiagen</td>
			<td>30250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">protease inhibitors cocktail</td>
			<td>Sigma</td>
			<td>&#160;S8820</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">silver staining kit&#160;</td>
			<td>G-Biosciences</td>
			<td>786-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human recombinant BDNF</td>
			<td>Genentech</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microfluidic chambers</td>
			<td>Xona</td>
			<td>SND450</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24x40 mm No. 1 glass coverslips&#160;</td>
			<td>VWR</td>
			<td>48393-060</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">poly-L-Lysine&#160;</td>
			<td>Cultrex</td>
			<td>3438-100-01</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco</td>
			<td>14185-052</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNase I</td>
			<td>Roche</td>
			<td>10104159001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neurobasal&#160;</td>
			<td>Gibco</td>
			<td>21103-049</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS&#160;</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GlutaMax&#160;</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27&#160;&#160;</td>
			<td>Gibco</td>
			<td>17504-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QD655-streptavidin conjugates</td>
			<td>Invitrogen</td>
			<td>&#160;Q10121MP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Avi tag antibody</td>
			<td>GenScript</td>
			<td>A00674</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">streptavidin-agarose beads&#160;</td>
			<td>Life Technology</td>
			<td>&#160;SA100-04</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trichloroacetic acid</td>
			<td>Sigma</td>
			<td>T6399</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HRP-streptavidin&#160;</td>
			<td>Thermo Scientific</td>
			<td>N100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-pTrkB antibody</td>
			<td colspan="2">a generous gift from Dr M. Chao of NYU</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-TrkB antibody</td>
			<td>BD Science</td>
			<td>610101</td>
		</tr>
	</tbody>
</table>

real-time imaging of axonal transport of quantum dot-labeled bdnf in primary neurons

BDNF spiller en vigtig rolle i flere facetter af neuronal overlevelse, differentiering og funktion. Strukturelle og funktionelle underskud i axoner i stigende grad ses som en tidlig træk ved neurodegenerative sygdomme, herunder Alzheimers sygdom (AD) og Huntingtons sygdom (HD). Endnu uklart er den mekanisme (r), hvorved axonal skade induceres. Vi rapporteret udvikling af en ny teknik til at fremstille biologisk aktivt monobiotinylated BDNF (mBtBDNF), der kan anvendes til at spore axonal transport af BDNF. Quantum dot-mærket BDNF (QD-BDNF) blev fremstillet ved konjugering kvantepunkt 655 til mBtBDNF. Mikrofluidisk indretning blev anvendt til at isolere axoner fra neuron cellelegemer. Tilsætning af QD-BDNF til axonale rum tillod levende billeddannelse af BDNF transport i axoner. Vi viste, at QD-BDNF flyttet væsentlige udelukkende retrogradt med meget få pauser på en bevægende hastighed på omkring 1,06 um / sek. Dette system kan bruges til at undersøge migchanisms af forstyrret axonal funktion i AD eller HD, samt andre degenerative sygdomme.

Produktion og oprensning af biologisk aktivt mono-biotinyleret BDNF Ekspressionsvektoren BDNF fusioneret med en AviTag sekvens (GGGLNDIFEAQKIEWHE) blev skabt efter en tidligere offentliggjort protokol 10. Den molekylære masse af fuldlængde fusionsproteinet blev forudsagt til at være ~ 32 kDa ( <a href="http://ca.expasy.org/tools/pi_tool.html" target="_blank">http://ca.expasy.org/tools/pi_tool.html</a> ) Monobiotinylated modne BDNF med en forudsagt molekylmasse på 18 kDa <stron...

Watch this Scientific Journal Video about Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons at JoVE.com

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

Axonal transport af BDNF, en neurotrofisk faktor, er afgørende for overlevelse og funktion af flere neuronale populationer. Visse degenerative sygdomme er kendetegnet ved forstyrrelser af axonal struktur og funktion. Vi demonstrerede de anvendte teknikker til at undersøge levende handel med QD-BDNF i mikrofluide kamre anvender primære neuroner.

Real-time Imaging af axonal Transport af Quantum Dot-mærket BDNF i Primary neuroner

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

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Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

En protokol til Administration af Real-Time fMRI Neurofeedback træning

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Analyse til måling af nukleocytoplasmisk transport i realtid inden for motor neuronlignende NSC-34 celler

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

Visualisering af Axonal Projeksionsmønsteret af Embryoniske Motor Neuroner i<em&gt; Drosophila</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

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 ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Axonal transport af organeller i motorneuronkulturer ved hjælp af microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...