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 blev modtaget fra den schweiziske National Science Foundation; den schweiziske Sinergia program; Polsk-schweizisk forskning program (2010/PSPB-094); NCCR i kemisk biologi; og LipidX fra den schweiziske SystemsX.ch initiativ, evalueres af den schweiziske National Science Foundation (til J. G.). O. M. blev støttet af en EMBO langsigtede fellowship (ALTF-516-2012).

1. løsninger og præparater Bemærk: alle buffere og løsninger bør forberedes i dobbeltdestilleret (dd) H 2 O. Fordi tilstanden hydrering af saccharose varierer, den endelige koncentrationen af alle saccharose løsninger skal bestemmes ved hjælp af et refraktometer.
<ol><li>Forbered fosfatbufferet saltopløsning uden divalent kationer (PBS-): 137 mM NaCl, 2,7 mM KCl, 1,5 mM KH 2 PO 4 og 6.5 mM Na 2 HPO 4. PH indstilles til 7,4. Ste...

<ol>
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Aktin spiller en afgørende rolle i endosome membranen dynamics4,14. Vi har tidligere rapporteret at actin Nukleering og polymerisation forekomme på tidlig endosomes, danner små F-actin patches eller netværk. Disse F-actin netværk er absolut nødvendige for membran transport ud over tidlige endosomes langs Nedbrydningsvejen. At gribe ind på alle trin i denne Nukleering og polymerisation proces forhindrer endosome modning og dermed downstream transport mod sl...

I højere eukaryote celler, er proteiner og lipider internaliseret i tidlige endosomes hvor sortering opstår. Nogle proteiner og lipider, som er bestemt til at være reutilized, er indarbejdet i rørformede regioner i de tidlige endosomes og derefter transporteres til plasma membran eller til trans-Golgi network (TGN)1,2. Derimod pakket andre proteiner og lipider selektivt ind i regioner i den tidlige endosomes, der udviser et multivesicular udseende. Disse regioner udvide og efter udstationering fra tidlig endosomal membraner, tiden moden til gratis endosomal carrier blærer eller multivesicular organer (ECV/MVBs), som er ansvarlig for fragt transport mod slutningen endosomes1, 2.
Aktin spiller en afgørende rolle i den membran remodeling proces forbundet med endosomal sortering kapacitet og endosome Biogenese. Protein sortering langs den genanvendelse veje til plasma membran eller TGN afhænger af den komplekse retromer og de tilknyttede proteiner. Denne sortering maskiner synes at være koblet til dannelsen af genanvendelse tubuli via interaktioner retromer komplekse, med HVEPS og ar homolog (vask) komplekse og forgrenede actin3,4,5 . Derimod molekyler bestemt til nedbrydning, især aktiveret signaling receptorer, er sorteret i intraluminal vesikler (ILVs) af endosomal sortering komplekser kræves for transport (ESCRT)2,6, 7. Mens den mulige rolle af actin i ESCRT-afhængige sorterings processen ikke er kendt, spiller F-actin en vigtig rolle i ECV/MVBs Biogenese og transport ud over tidlige endosomes. Vi fandt navnlig, at annexin A2 binder kolesterol-beriget regioner af tidlige endosome, og sammen med spire1, nucleates F-actin polymerisering. Dannelsen af forgrenede actin netværket observeret på endosomes kræver det forgrenede aktivitet af actin-relaterede protein (ARP) 2/3 kompleks, samt ERM protein moesin og actin-bindende protein cortactin8,9.
Her, beskriver vi en mikroskopi-baseret i vitro assay, der genopretter Nukleering og polymerisering af F-actin på tidlig endosomal membraner i reagensglas. Denne analyse er blevet brugt tidligere til at undersøge rollen af annexin A2 i F-actin Nukleering og moesin og cortactin i dannelsen af endosomal actin net8,9. Med dette in vitro- protokollen blive den komplekse række af reaktioner, der opstår på endosomes under actin polymerisering modtagelig den biokemiske og molekylær analyse af de sekventielle trin i processen, herunder actin Nukleering, lineær polymerisering, forgrening, og crosslinking.

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			<td>S1111-0706</td>
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			<td>MCT-175-C 311-04-051</td>
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		<tr height="20">
			<td height="20" style="height:20px;">18-mm diameter round coverslip&#160;</td>
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			<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>
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			<td height="20" style="height:20px;">Refractometer</td>
			<td>Carl Zeiss</td>
			<td>79729</td>
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			<td>Beckman coulter</td>
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		<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>
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		</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>
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in vitro polymerization of f-actin on early endosomes

Mange tidlige endosome funktioner, især cargo protein sortering og membran deformation, afhænger af pletter af kort F-actin filamenter nukleeret på endosomal membran. Vi har etableret en mikroskopi-baseret i vitro assay, der genopretter Nukleering og polymerisering af F-actin på tidlig endosomal membraner i reagensglas, hvilket gør dette kompleks række reaktioner imødekommenhed over for genetiske og biokemiske manipulationer. Endosomal brøker er udarbejdet af opdrift i saccharose forløb fra celler, der udtrykker den tidlige endosomal protein normal god landbrugspraksis-RAB5. Cytosole fraktioner er fremstillet af særskilte partier af celler. Både endosomal og cytosole fraktioner kan opbevares frosset i flydende kvælstof, hvis det er nødvendigt. I analysen, endosomal og cytosole fraktioner blandes, og blandingen er inkuberes ved 37 ° C under de rette betingelser (fx ionisk styrke, at reducere miljø). Dengang ønskede reaktionsblandingen er fast, og F-actin er afsløret med phalloidin. Aktin Nukleering og polymerisation er derefter analyseret af Fluorescens mikroskopi. Vi rapporterer her, at denne analyse kan bruges til at undersøge rollen af faktorer, der er involveret i actin Nukleering på membranen, eller i den efterfølgende strækforlængelse, forgrener sig eller crosslinking af F-actin filamenter.

For at få indsigt i dannelsen af F-actin pletter på tidlig endosome membraner, fulgte vi den protokol, der er skitseret i figur 2. Kort, celler blev transfekteret med normal god landbrugspraksis-RAB5 og derefter tidlige endosomes blev udarbejdet af subcellulært fraktionering. Disse renset tidlige endosomes var inkuberes med cytosol for at give actin selv samt andre faktorer, muligvis involveret i reaktionen. For enden af inkubationstiden blev reaktion stop...

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

Tidlige endosome funktioner afhænger af F-actin polymerisering. Her, beskriver vi en mikroskopi-baseret i vitro assay, der genopretter Nukleering og polymerisering af F-actin på tidlig endosomal membraner i reagensglas, hvilket gør dette kompleks række reaktioner medgørlige biokemiske og genetiske manipulationer.

In Vitro Polymerisering af F-actin 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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