Name: in vitro polymerization of f-actin on early endosomes
Uploaded: 2017-08-28T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Polymerization of F-actin on Early Endosomes at JoVE.com

Støtte ble mottatt fra Swiss National Science Foundation; Swiss Sinergia programmet; den polsk-sveitsiske forskning program (PSPB-094/2010); NCCR i kjemisk biologi; og LipidX fra det sveitsiske SystemsX.ch initiativet, evaluert av Swiss National Science Foundation (å J. G.). O. M. ble støttet av en EMBO langsiktige fellesskap (ALTF-516-2012).

1. løsninger og forberedelser Merk: alle buffere og løsninger bør være forberedt i dobbel-destillert (dd) H 2 O. Fordi hydration delstaten sukrose varierer, siste konsentrasjonen av alle sukrose løsninger må bestemmes ved hjelp av en refractometer.
<ol><li>Forberede fosfat-bufret saline uten divalent kasjoner (PBS-): 137 mM Born, 2.7 mM KCl, 1,5 mM KH 2 PO 4 og 6.5 mM Na 2 HPO 4. Justere pH 7,4. Sterilisere av autoklavering (1 sy...

<ol>
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C., Gruenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moesin+and+cortactin+control+actin-dependent+multivesicular+endosome+biogenesis.">Moesin and cortactin control actin-dependent multivesicular endosome biogenesis.</a> Mol Biol Cell. 27 (21), 3305-3316 (2016).</li><li>Sonnichsen, B., De Renzis, S., Nielsen, E., Rietdorf, J., Zerial, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+membrane+domains+on+endosomes+in+the+recycling+pathway+visualized+by+multicolor+imaging+of+Rab4,+Rab5,+and+Rab11.">Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11.</a> J Cell Biol. 149 (4), 901-914 (2000).</li><li>Morel, E., Parton, R. 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Utgangen spiller en avgjørende rolle i endosome membran dynamics4,14. Tidligere rapporterte vi at utgangen nucleation og polymerisasjon forekommer på tidlig endosomes, danner små F-utgangen flekker eller nettverk. Disse F-utgangen nettverkene er absolutt nødvendig for membran transport utover tidlig endosomes langs veien degradering. Virker på alle trinn i denne nucleation og polymerisasjon prosessen hindrer endosome modning og således nedstrøms transport ...

I høyere eukaryote celler, er proteiner og lipider internalisert i tidlig endosomes der sortering oppstår. Noen proteiner og lipider, som er forutbestemt til å være reutilized, er innlemmet i rørformede regioner av tidlig endosomes og deretter transportert til plasma membranen eller trans-Golgi nettverk (TGN)1,2. Andre proteiner og lipider pakkes derimot selektivt i områder av tidlig endosomes som viser en multivesicular utseende. Disse regionene utvide og på løsgjøring fra tidlig endosomal membraner, til slutt moden til gratis endosomal bærer blemmer eller multivesicular organer (ECV/MVBs), som er ansvarlig som mot slutten endosomes1, 2.
Utgangen spiller en avgjørende rolle i membranen remodeling prosessen knyttet endosomal sortering kapasitet og endosome biogenesis. Protein sortering langs resirkulering veiene plasma membranen eller TGN avhenger retromer komplekse og tilhørende proteiner. Dette sortering maskiner synes å være knyttet til dannelsen av resirkulering tubuli via samhandling retromer komplekset, med VEPS og Scar homologue (vask) komplekse og forgrenede begrepsordbok3,4,5 . Derimot molekyler skjebnebestemt til fornedrelse, spesielt aktivert signalnettverk reseptorer, sorteres i intraluminal blemmer (ILVs) av endosomal sortering komplekser kreves for transport (ESCRT)2,6, 7. Mens mulige rolle begrepsordbok i ESCRT-avhengige sortering prosessen ikke er kjent, spiller F-utgangen en viktig rolle i biogenesis av ECV/MVBs og transport utover tidlig endosomes. Spesielt fant vi at annexin A2 binder kolesterol-beriket regioner av tidlig endosome, og spire1, nucleates F-utgangen polymerisasjon. Dannelsen av forgrenede begrepsordbok nettverket observert på endosomes krever forgrening aktiviteten til utgangen-relaterte protein (ARP) 2/3 kompleks, samt ERM protein moesin og utgangen-bindende protein cortactin8,9.
Her beskriver vi en mikroskopi-basert i vitro analysen som rekonstruerer nucleation og polymerisering av F-utgangen på tidlig endosomal membraner i test-rør. Denne analysen er brukt tidligere å undersøke rollen til annexin A2 i F-utgangen nucleation og moesin og cortactin i dannelsen av endosomal utgangen nettverk8,9. Med denne i vitro protokollen blir de komplekse reaksjoner som oppstår på endosomes under begrepsordbok polymerisasjon mottakelig for biokjemiske og molekylære analyse av de sekvensielle trinnene av prosessen, inkludert begrepsordbok nucleation, lineær polymerisasjon forgrening og crosslinking.

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			<td height="20" style="height:20px;width:477px;">NaCl</td>
			<td style="width:223px;">Sigma-Aldrich</td>
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			<td style="width:443px;"></td>
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			<td height="20" style="height:20px;">KCl</td>
			<td>Acros Organics</td>
			<td>196770010</td>
			<td></td>
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			<td height="20" style="height:20px;">KH2PO4</td>
			<td>AppliChem</td>
			<td>A1042</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Na2HPO4</td>
			<td>Acros Organics</td>
			<td>424370025</td>
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			<td height="20" style="height:20px;">Hepes</td>
			<td>AppliChem</td>
			<td>A3724</td>
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			<td height="20" style="height:20px;">Magnesiun acetate tetrahydrate</td>
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			<td>63047</td>
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			<td height="20" style="height:20px;">Dithiothreitol (DTT)</td>
			<td>AppliChem</td>
			<td>A2948</td>
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			<td height="20" style="height:20px;">Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>10125</td>
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			<td height="20" style="height:20px;">NaOH</td>
			<td>Fluka</td>
			<td>71690</td>
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			<td>Merck Millipore</td>
			<td>107687</td>
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			<td height="20" style="height:20px;">Leupeptin</td>
			<td>Roche</td>
			<td>11017101001</td>
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			<td height="20" style="height:20px;">Pepstatin</td>
			<td>Roche</td>
			<td>10253286001</td>
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			<td height="20" style="height:20px;">Aprotinin</td>
			<td>Roche</td>
			<td>10236624001</td>
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			<td height="20" style="height:20px;">Paraformaldehide</td>
			<td>Polysciences. Inc</td>
			<td>380</td>
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			<td height="20" style="height:20px;">Alexa Fluor 555 phalloidin</td>
			<td>Molecular Probes</td>
			<td>A34055</td>
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			<td height="20" style="height:20px;">Actin rhodamine</td>
			<td>Cytoskeleton. Inc</td>
			<td>APHR-A</td>
			<td></td>
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			<td height="20" style="height:20px;">Mowiol 4-88</td>
			<td>Sigma-Aldrich</td>
			<td>81381</td>
			<td>poly(vinyl alcohol), Mw ~31 000&#160;</td>
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			<td height="20" style="height:20px;">DABCO</td>
			<td>Sigma-Aldrich</td>
			<td>D-2522</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl</td>
			<td>AppliChem</td>
			<td>A1086</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">&#946;-casein</td>
			<td>Sigma-Aldrich</td>
			<td>C6905</td>
			<td></td>
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			<td height="20" style="height:20px;">Filter 0.22um</td>
			<td>Millex&#160;</td>
			<td>SL6V033RS</td>
			<td></td>
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			<td height="20" style="height:20px;">Round 10cm dishes for cell culture</td>
			<td>Thermo Fisher Scientific</td>
			<td>150350</td>
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			<td height="20" style="height:20px;">Plastic Pasteur pipette</td>
			<td>Assistent</td>
			<td>569/3 40569003</td>
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		<tr height="20">
			<td height="20" style="height:20px;">15-ml polypropylen tube&#160;</td>
			<td>TPP</td>
			<td>91015</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Hypodermic Needle 22G Black 30mm&#160;</td>
			<td>BD Microlance</td>
			<td>300900</td>
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			<td>BD Plastipak</td>
			<td>300013</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro glass slides&#160;</td>
			<td>Assistent</td>
			<td>2406</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">18X18-mm glass coverslip</td>
			<td>Assistent</td>
			<td>1000/1818</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SW60 centrifuge tube</td>
			<td>Beckman coulter</td>
			<td>344062</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">TLS-55 centrifuge tube</td>
			<td>Beckman coulter</td>
			<td>343778</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">200-&#956;l yellow tip</td>
			<td>Starlab</td>
			<td>S1111-0706</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">1000-&#956;l Blue Graduated Tip</td>
			<td>Starlab</td>
			<td>S1111-6801</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5-ml test tube</td>
			<td>Axygen</td>
			<td>MCT-175-C 311-04-051</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">18-mm diameter round coverslip&#160;</td>
			<td>Assistent</td>
			<td>1001/18</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">35-mm diameter round dish with a 20-mm glass bottom (0.16-0.19&#160;mm)&#160;</td>
			<td>In vitro Scientific</td>
			<td>D35-20-1.5-N</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Refractometer</td>
			<td>Carl Zeiss</td>
			<td>79729</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sorvall WX80 Ultracentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>46900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tabletop ultracentrifuge</td>
			<td>Beckman coulter</td>
			<td>TL-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SW60 rotor</td>
			<td>Beckman coulter</td>
			<td>335649</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TLS-55 rotor</td>
			<td>Beckman coulter</td>
			<td>346936</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal microscopy</td>
			<td>Carl Zeiss</td>
			<td>LSM-780</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fugene HD transfection reagent</td>
			<td>Promega</td>
			<td style="width:145px;">E2311</td>
			<td style="width:443px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein assay reagent A</td>
			<td>Bio-Rad</td>
			<td>500-0113</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein assay reagent B</td>
			<td>Bio-Rad</td>
			<td>500-0114</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein assay reagent S</td>
			<td>Bio-Rad</td>
			<td>500-0115</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell scraper</td>
			<td>Homemade</td>
			<td></td>
			<td>Silicone rubber piece of about 2 cm, cut at a very sharp angle and attached to a metal bar or held with forceps</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Refractometer</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Minimum Essential Medium Eagle (MEM)</td>
			<td>Sigma-Aldrich</td>
			<td>M0643</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FCS</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM Non-Essential Amino Acids (NEAA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pH Meter 691</td>
			<td>Metrohm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ImageJ software</td>
			<td></td>
			<td></td>
			<td>NIH, Bethesda MD</td>
		</tr>
	</tbody>
</table>

in vitro polymerization of f-actin on early endosomes

Mange tidlige endosome funksjoner, spesielt Last protein sortering og membran deformasjon, avhengig av flekker av kort F-utgangen filamenter nucleated på endosomal membranen. Vi har etablert en mikroskopi-basert i vitro analysen som rekonstruerer nucleation og polymerisering av F-utgangen på tidlig endosomal membraner i test-rør, dermed gjengi dette komplekse rekke reaksjoner mottakelig for genetiske og biokjemiske manipulasjoner. Endosomal fraksjoner tilberedes av floatation i sukrose forløpninger fra celler uttrykke tidlig endosomal protein GFP-RAB5. Cytosolic fraksjoner tilberedes fra separate grupper av celler. Både endosomal og cytosolic fraksjoner kan lagres frosset i flytende nitrogen, hvis nødvendig. I analysen, endosomal og cytosolic fraksjoner blandes og blandingen er ruges på 37 ° C under riktige forhold (f.eks ioniske styrke, redusere miljø). Samtidig ønsket reaksjonsblandingen er fast, og F-utgangen er avslørt med phalloidin. Utgangen nucleation og polymerisasjon analyseres deretter av fluorescens mikroskopi. Her rapporterer vi at denne analysen kan brukes til å undersøke hvilken rolle faktorer som er involvert i utgangen nucleation membranen eller i påfølgende forlengelse, forgrening eller crosslinking av F-utgangen filamenter.

For å få innsikt i dannelsen av F-utgangen flekker på tidlig endosome membraner, fulgte vi protokollen i figur 2. Kort, celler var transfekterte med GFP-RAB5 og deretter tidlig endosomes ble utarbeidet av subcellular fraksjoneres. Disse renset tidlig endosomes ble inkubert med stoffer for å gi begrepsordbok selv, samt andre faktorer muligens involvert i reaksjonen. På slutten av inkubasjonstiden, ble reaksjonen stoppet av fiksering av blandingen. Et ekse...

Watch this Scientific Journal Video about In Vitro Polymerization of F-actin on Early Endosomes at JoVE.com

In Vitro Polymerization of F-actin on Early Endosomes

Tidlig endosome funksjoner avhenger av F-utgangen polymerisasjon. Her beskriver vi en mikroskopi-basert i vitro analysen som rekonstruerer nucleation og polymerisering av F-utgangen på tidlig endosomal membraner i test-rør, dermed gjengi dette komplekse rekke reaksjoner mottakelig for biokjemiske og genetiske manipulasjoner.

In Vitro Av F-utgangen på tidlig Endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

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In Vitro Polymerization of F-actin on Early Endosomes

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The Cytoskeleton I: Actin and Microfilaments

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