JoVE Journal > Bioengineering
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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Bioengineering

In vitro Rekonstitution af Actin Cytoskelettet Inde i Giant Unilamellar Vesikler

Published: August 25, 2022
doi:
10.3791/64026
, ,
1Department of Biomedical Engineering,Yale University, 2Systems Biology Institute,Yale University, 3Department of Physics,Yale University

Summary

I dette manuskript demonstrerer vi de eksperimentelle teknikker til at indkapsle F-actincytoskelettet i gigantiske unilamellare lipidvesikler (også kaldet liposomer) og metoden til at danne et cortex-biomimicking F-actinlag ved den indre folder af liposommembranen.

Abstract

Actincytoskelettet, det vigtigste mekaniske maskineri i cellen, formidler adskillige væsentlige fysiske cellulære aktiviteter, herunder celledeformation, opdeling, migration og vedhæftning. Imidlertid kompliceres studiet af dynamikken og strukturen i actinnetværket in vivo af den biokemiske og genetiske regulering i levende celler. For at opbygge en minimal model uden intracellulær biokemisk regulering er actin indkapslet inde i gigantiske unilamellare vesikler (GUV’er, også kaldet liposomer). De biomimetiske liposomer er cellestørrelse og letter en kvantitativ indsigt i cytoskeletnetværkets mekaniske og dynamiske egenskaber, hvilket åbner en levedygtig rute for bottom-up syntetisk biologi. For at generere liposomer til indkapsling anvendes den omvendte emulsionsmetode (også kaldet emulsionsoverførselsmetoden), hvilket er en af de mest succesrige teknikker til indkapsling af komplekse opløsninger i liposomer til fremstilling af forskellige celleefterlignende systemer. Med denne metode tilsættes en blanding af proteiner af interesse til den indre buffer, som senere emulgeres i en phospholipidholdig mineralolieopløsning til dannelse af monolags lipiddråber. De ønskede liposomer genereres fra monolags lipiddråber, der krydser en lipid / olie-vand-grænseflade. Denne metode muliggør indkapsling af koncentrerede actinpolymerer i liposomerne med ønskede lipidkomponenter, hvilket baner vejen for in vitro-rekonstitution af et biomimicking cytoskeletnetværk.

Introduction

Actincytoskelettet spiller en grundlæggende rolle i konstruktionen af cellens intracellulære arkitektur ved at koordinere kontraktilitet på molekylært niveau og kraftgenerering 1,2,3. Som et resultat formidler det adskillige væsentlige cellulære aktiviteter, herunder celledeformation 4,5, division6, migration 7,8 og adhæsion9. In vitro-rekonstitutionen af actin-netværk har fået enorm opmærksomhed i de senere år 10,11,12,13,14,15,16,17. Målet med rekonstitution er at opbygge en minimal model af cellen blottet for den komplekse biokemiske regulering, der findes i levende celler. Dette giver et kontrollerbart miljø til at undersøge specifikke intracellulære aktiviteter og letter identifikation og analyse af forskellige komponenter i actincytoskelettet18,19. Endvidere giver indkapslingen af in vitro actinnetværk inde i phospholipid gigantiske unilamellare vesikler (GUV’er, liposomer) et begrænset, men deformerbart rum med en semipermeabel grænse. Det efterligner det fysiologiske og mekaniske mikromiljø af actinmaskineriet i cellen 9,20,21,22.

Blandt forskellige metoder til fremstilling af liposomer er lipidfilmhydreringsmetoden (også kendt som hævelsesmetoden) en af de tidligste teknikker23. Den tørre lipidfilm hydrerer med tilsætning af buffere og danner membranøse bobler, der til sidst bliver vesikler24. For at producere større vesikler med et højere udbytte anvender en forbedret metode, der går videre fra filmhydreringsmetoden, kendt som elektroformationsmetoden25, et AC-elektrisk felt til effektivt at fremme hydreringsprocessen26. De største begrænsninger ved disse hydreringsbaserede metoder til actinindkapsling er, at den har lav indkapslingseffektivitet af stærkt koncentrerede proteiner, og den er kun kompatibel med specifikke lipidsammensætninger24. Den omvendte emulsionsteknik har til sammenligning færre begrænsninger for lipidkomponenter og proteinkoncentrationer 20,27,28,29. I denne metode tilsættes en blanding af proteiner til indkapsling til den indre vandige buffer, som senere emulgeres i en lipidholdig mineralolieopløsning, der danner lipid-monolagsdråber. Monolags lipiddråber krydser derefter gennem en anden lipid / olie-vand-grænseflade gennem centrifugering for at danne dobbeltlags lipidvesikler (liposomer). Denne teknik har vist sig at være en af de mest succesrige strategier til actinindkapsling24,30. Separat er der nogle mikrofluidiske enhedsmetoder, herunder pulserende jetting31,32, forbigående membranudstødning33 og cDICE-metoden34. Lighederne mellem den omvendte emulsionsmetode og den mikrofluidiske metode er lipidopløsningsmiddelet (olie), der anvendes, og indførelsen af lipid / olie-vand-grænseflade til dannelse af den ydre folder af liposomer. I modsætning hertil kræver dannelsen af liposomer ved den mikrofluidiske metode en opsætning af mikrofluidiske anordninger og ledsages af olie fanget mellem dobbeltlagets to foldere, hvilket kræver et ekstra trin til fjernelse af olie35.

I dette manuskript brugte vi den omvendte emulsionsteknik til at fremstille liposomer, der indkapslede et polymeriseret F-actin-netværk som tidligere anvendt22. Proteinblandingen til indkapsling blev først anbragt i en buffer med ikke-polymeriserende betingelser for at opretholde actin i sin kugleformede (G) form. Hele processen blev udført ved 4 °C for at forhindre tidlig actinpolymerisation, som senere blev udløst ved at lade prøven varme op til stuetemperatur. En gang ved stuetemperatur polymeriserer actinet i sin filamentøse (F) form. En række actinbindende proteiner kan tilsættes til den indre vandige bufferopløsning for at studere proteinfunktionaliteter og egenskaber og dermed yderligere give indsigt i dets interaktion med actinnetværket og membranoverfladen. Denne metode kan også anvendes til indkapsling af forskellige proteiner af interesse36 og store genstande (mikropartikler, selvkørende mikrosvømmere osv.) tæt på størrelsen af de endelige liposomer28,37.

Protocol

1. Fremstilling af buffere og proteinopløsninger Den vandige indre ikke-polymerisationsbuffer (INP) fremstilles i et samlet volumen på 5 ml ved at blande 0,1 mM CaCl2, 10 mM HEPES (pH 7,5), 1 mM DTT, 0,5 mM Dabco, 320 mM saccharose og 0,2 mM ATP. Forbered proteinblandingen (PM) ved at tilsætte proteiner til INP-bufferen ved 4 °C med følgende koncentrationer: 11,2 μM ikke-fluorescerende G-actin, 2,8 μM fluorescerende mærket actin og 0,24 μM Arp2/3 (materiale…

Representative Results

Fremstillingen af liposomer baseret på den omvendte emulsionsteknik er illustreret grafisk og skematisk i figur 1. Først blev tomme (bare) liposomer (~ 5-50 μm i diameter), der var sammensat af phospholipid (EPC) og fluorescerende lipid (DHPE) fremstillet. Et lyst, langt rødt fluorescerende farvestof blev indkapslet i bare liposomer som et kontroleksperiment. Hvorvidt et lipidmonolag med succes er dannet i dråbens perifere enhed, kunne bestemmes ved at observ…

Discussion

Flere nøgletrin bestemmer succesen med et højt udbytte af liposomer under forberedelsesprocessen. For helt at opløse lipidfilmen i olien skal prøven sonikeres, indtil lipidfilmen i bunden af hætteglasset forsvinder helt. Efter sonikeringen skal lipid-olieblandingen opbevares natten over ved stuetemperatur under mørke forhold for at lipidmolekylerne kan sprede sig yderligere29. Blandingen kan opbevares ved 4 °C i op til en uge. Ved fremstilling af en FB/olieemulsion med monolags lipiddråber…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi anerkender finansiering af ARO MURI W911NF-14-1-0403 til MPM, National Institutes of Health (NIH) R01 1R01GM126256 til MPM, National Institutes of Health (NIH) U54 CA209992, NIH RO1 GM126256, NIH U54 CA209992, University of Michigan / Genentech, SUBK00016255 og Human Frontiers Science Program (HFSP) tilskudsnummer RGY0073/2018 til M.P.M. Enhver mening, resultater, konklusioner eller anbefalinger, der udtrykkes i dette materiale, er forfatternes og afspejler ikke nødvendigvis synspunkterne fra ARO, NIH eller HFSP. S.C. anerkender frugtbare diskussioner med V. Yadav, C. Muresan og S. Amiri.

Materials

1,2-dioleoyl-sn-glycero-3-{[n(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DOGS-NTA-Ni)  Avanti Polar Lipids Inc. 231615773 Nickel Lipid
1,4-Diazabicyclo[2.2.2]octane Sigma D27802-25G DABCO
Actin protein (>99% pure): rabbit skeletal muscle Cytoskeleton, Inc AKL99-D non-fluorescent G-actin
Actin protein (rhodamine): rabbit skeletal muscle Cytoskeleton, Inc AR05 fluorescently labeled actin
Adenosine 5′-triphosphate disodium salt hydrate Sigma A2383-10G ATP
Alexa Fluor 647 dye ThermoFisher fluorescent dye
Andor iQ3 Andor Technologies control and acquisition software for confocal microscope
Arp2/3 Protein Complex: Porcine Brain Cytoskeleton, Inc RP01P-A Arp 2/3
Calcium chloride dihydrate Sigma 10035048 CaCl2
Chamlide Chambers (4-well for 12 mm round coverslip) Quorum Technologies incubation chamber
Cofilin protein: human recombinant Cytoskeleton, Inc CF01-C cofilin
Confocal Microscope (63× oil-immersion objective) Andor Technologies LEICA DMi8
D-(+)-GLUCOSE BIOXTRA Sigma G7528 glucose
Dithiothreitol DOT Scientific  DSD11000-10 DTT
Gelsolin Protein: Homo Sapiens Recombinant Cytoskeleton, Inc HPG6 gelsolin
Hamilton 1750 Gastight Syringe, 500 µL, cemented needle, 22 G, 2" conical tip Cole-Parmer UX-07940-53 glass syringe
HEPES AmericanBio 7365-45-9
ImageJ/Fiji https://imagej.net/tutorials/
L-alpha-Phosphatidylcholine Avanti Polar Lipids Inc. 97281442 EPC
Magnesium chloride Sigma 7786303 MgCl2
Mineral oil, BioReagent, for molecular biology, light oil Sigma 8042475 mineral oil
N-WASP fragment WWA (aa400–501, VCA-His) VCA-His is purified using lab protocol. The protocol can be provided upon reasonable requests
Oregon Green 488 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (Oregon Green 488 DHPE) Thermo Fisher O12650 DHPE
Potassium chloride Sigma 7447407 KCl
Sucrose Sigma 57-50-1 sucrose
β-Casein from bovine milk Sigma C6905-250MG
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Tags

Keywords: Actin CytoskeletonIn Vitro ReconstitutionGiant Unilamellar VesiclesLiposome YieldEncapsulation EfficiencyProtein EncapsulationMicroparticle EncapsulationSelf-propelling MicroswimmersNon-polymerization BufferPolymerization BufferEGGPCNickel Lipid
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Chen, S., Sun, Z. G., Murrell, M. P. In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles. J. Vis. Exp. (186), e64026, doi:10.3791/64026 (2022).
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