Summary

Direkte måling af kræfter i rekonstituerede aktive mikrotubulusbundter

Published: May 10, 2022
doi:

Summary

Her præsenterer vi en protokol til rekonstituering af mikrotubulusbundter in vitro og direkte kvantificering af de kræfter, der udøves i dem ved hjælp af samtidig optisk fangst og total intern refleksionsfluorescensmikroskopi. Dette assay muliggør måling på nanoskala-niveau af de kræfter og forskydninger, der genereres af proteinensembler inden for aktive mikrotubulinetværk.

Abstract

Mikrotubulusnetværk anvendes i celler til at udføre en bred vifte af opgaver, lige fra at fungere som spor til vesikeltransport til at arbejde som specialiserede arrays under mitose for at regulere kromosomadskillelse. Proteiner, der interagerer med mikrotubuli, omfatter motorer som kinesiner og dynein, som kan generere aktive kræfter og retningsbestemt bevægelse samt ikke-motoriske proteiner, der tværbinder filamenter i højere ordens netværk eller regulerer filamentdynamik. Hidtil har biofysiske undersøgelser af mikrotubuli-associerede proteiner overvældende fokuseret på rollen som enkeltmotorproteiner, der er nødvendige for vesikeltransport, og der er gjort betydelige fremskridt med at belyse de kraftgenererende egenskaber og mekanokemisk regulering af kinesiner og dyneiner. For processer, hvor mikrotubuli fungerer både som last og spor, såsom under filamentglidning i den mitotiske spindel, forstås der imidlertid meget mindre om den biofysiske regulering af ensembler af de involverede tværbindingsproteiner. Her beskriver vi vores metode til direkte sondering af kraftgenerering og respons inden for tværbundne mikrotubulus minimale netværk rekonstitueret fra oprensede mikrotubuli og mitotiske proteiner. Mikrotubulipar er tværbundet af proteiner af interesse, en mikrotubuli er immobiliseret til et mikroskop dækslip, og den anden mikrotubuli manipuleres af en optisk fælde. Samtidig total intern refleksionsfluorescensmikroskopi muliggør multikanalvisualisering af alle komponenterne i dette mikrotubulinetværk, når filamenterne glider fra hinanden for at generere kraft. Vi demonstrerer også, hvordan disse teknikker kan bruges til at undersøge skubbekræfter, der udøves af kinesin-5-ensembler, og hvordan viskøse bremsekræfter opstår mellem glidende mikrotubuluspar tværbundet af den mitotiske MAP PRC1. Disse assays giver indsigt i mekanismerne for spindelsamling og funktion og kan tilpasses mere bredt til at studere tæt mikrotubulusnetværksmekanik i forskellige sammenhænge, såsom axon- og dendritter af neuroner og polære epitelceller.

Introduction

Celler anvender mikrotubulusnetværk til at udføre en lang række mekaniske opgaver, lige fra vesikeltransport 1,2,3 til kromosomadskillelse under mitose 4,5,6. Mange af de proteiner, der interagerer med mikrotubuli, såsom de molekylære motorproteiner kinesin og dynein, genererer kræfter og reguleres af mekaniske belastninger. For bedre at forstå, hvordan disse kritiske molekyler fungerer, har forskere anvendt enkeltmolekyle biofysiske metoder, såsom optisk fangst og TIRF-mikroskopi, til direkte at overvåge kritiske parametre såsom ubelastede trinhastigheder, processivitet og krafthastighedsforhold for individuelle proteiner. Den mest almindeligt anvendte eksperimentelle geometri har været at fastgøre motorproteiner direkte til fangstperler, hvis sfæriske geometri og størrelse efterligner vesikler, der gennemgår motordrevet transport. Talrige kinesiner, herunder kinesin-1 7,8,9, kinesin-2 10,11,12, kinesin-3 13,14,15,16 kinesin-517,18, kinesin-8 19,20 samt dynein- og dyneinkomplekser21,22, 23,24,25, er blevet undersøgt med disse metoder.

I mange cellulære processer bruger motoriske og ikke-motoriske proteiner imidlertid mikrotubuli både som spor og last26,27. Desuden fungerer disse proteiner i disse scenarier, hvor mikrotubulifilamenter er tværbundet i højere ordens bundter, som ensembler snarere end enkeltenheder. For eksempel inden for delende somatiske celler organiserer tætte filamentnetværk sig selv for at opbygge det mitotiske spindelapparat28,29,30. Det interpolære spindelmikrotubulinetværk er meget dynamisk og er stort set arrangeret med minusender, der peger mod spindelpolerne og plus-ender overlapper nær spindelækvator. Filamenter i spindlen er tværbundet af motorproteiner såsom kinesin-5 31,32,33, kinesin-12 34,35,36 og kinesin-1437,38,39 eller af ikke-motoriske proteiner såsom PRC1 40,41,42,43 eller NuMA 44,45, 46. De bevæger sig ofte eller oplever mekanisk belastning under processer som polflux eller mens de koordinerer kromosomcentrering under metafase eller kromosomadskillelse under anafase 47,48,49,50,51,52. Integriteten af spindelapparatet i mikronskala gennem mitose er derfor afhængig af en omhyggeligt reguleret balance mellem skubbe- og trækkræfter, der genereres og opretholdes af dette netværk af interagerende filamenter. Imidlertid er de nødvendige værktøjer til at undersøge denne mekaniske regulering og forklare, hvordan proteinensembler arbejder sammen for at koordinere mikrotubulusbevægelser og producere de kræfter, der er nødvendige for korrekt at samle spindlen, først for nylig blevet udviklet, og vi er lige begyndt at forstå de biofysiske regler, der definerer dynamiske mikrotubulusnetværk.

Målet med dette manuskript er at demonstrere de trin, der kræves for at rekonstituere tværbundne mikrotubulipar in vitro, immobilisere disse bundter i et mikroskopikammer, der muliggør samtidig fluorescensvisualisering af både mikrotubuli og tværbindingsproteiner og nanoskala kraftmåling og behandle disse data robust. Vi beskriver de trin, der er nødvendige for stabilt at polymerisere fluorescensmærkede mikrotubuli, forberede mikroskopdæksler til fastgørelse, forberede polystyrenperler til optiske fangsteksperimenter og samle tværbundne filamentnetværk, der bevarer deres in vivo-funktionalitet , samtidig med at de giver mulighed for direkte biofysisk manipulation.

Protocol

1. Fremstilling af mikrotubuli BEMÆRK: Ved anvendelse af GFP-mærkede tværbindingsproteiner, rød (f.eks. Rhodamin) og far-rød (f.eks. Biotinyleret HiLyte647, kaldet biotinyleret langt rød i resten af teksten) fungerer organisk fluoroformærkning af mikrotubuli godt. Minimal krydstale mellem alle tre kanaler kan opnås under billeddannelse ved hjælp af et højkvalitets quad band total intern refleksionsfluorescens (TIRF) filter. Forbered GMPCPP mikrotubulusfrøl…

Representative Results

Fremstillingen af mikrotubulusbundter, der er egnede til biofysisk analyse, betragtes som vellykket, hvis flere af nøglekriterierne er opfyldt. For det første skal billeddannelse i tre farver afsløre to justerede mikrotubuli med en koncentration af tværbindingsprotein, der fortrinsvis dekorerer overlapningsområdet (figur 5B, C og figur 6B). Ideelt set bør afstanden mellem overlapningskanten og den frie ende af rhodaminmikr…

Discussion

Mikrotubulusnetværk anvendes af utallige celletyper til at udføre en bred vifte af opgaver, der grundlæggende er mekaniske. For at beskrive, hvordan celler fungerer i både sunde og sygdomstilstande, er det afgørende at forstå, hvordan disse mikronskalanetværk er organiseret og reguleret af de nanometerstore proteiner, der kollektivt bygger dem. Biofysiske værktøjer såsom optisk pincet er velegnede til at undersøge mekanokemien af nøgleproteiner i denne skala. Afspejler mangfoldigheden af mikrotubulusnetværks…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Forfatterne ønsker at anerkende støtte fra R21 AG067436 (til JP og SF), T32 AG057464 (til ET) og Rensselaer Polytechnic Institute School of Science Startup Funds (til SF).

Materials

10W Ytterbium Fiber Laser, 1064nm IPG Photonics YLR-10-1064-LP
405/488/561/640nm Laser Quad Band Set for TIRF applications Chroma TRF89901v2
6x His Tag Antibody, Biotin Conjugate Invitrogen #MA1-21315-BTIN
Acetone, HPLC grade Fisher Scientific 18-608-395
Alpha casein from bovine milk Sigma 1002484390
ATP Fisher Scientific BP413-25
Benzonase Novagen 70746-3
Biotin-PEG-SVA-5000 Laysan Bio, Inc. NC0479433
BL21 (DE3) Rosetta Cells Millipore Sigma 71-400-3
Catalase MP Biomedicals LLC 190311
CFI Apo 100X/1.49NA oil immersion TIRF objective Nikon N/A
Chloramphenicol ACROS Organics 227920250
Coverslip Mini-Rack, for 8 coverslips Fisher Scientific C14784
Delicate Task Wipers Kimberly-Clark 34120
Dextrose Anhydrous Fisher Scientific BP3501
D-Sucrose Fisher Scientific BP220-1
DTT Fisher Scientific BP172-25
Ecoline Immersion Thermostat E100 with 003 Bath LAUDA-Brinkmann 27709
EDTA Fisher Scientific BP118-500
EGTA Millipore Corporation 32462-25GM
FIJI / Image J https://fiji.sc/ N/A
Frosted Microscope Slides Corning 12-553-10 75mmx25mm, with thickness of 0.9-1.1mm
Glucose Oxidase MP Biomedicals LLC 195196 Type VII, without added oxygen
GMPCPP Jena Biosciences JBS-NU-405S Can be stored for several months at -20 °C and up to a year at -80 °C
Gold Seal-Cover Glass Thermo Scientific 3405
HEPES Fisher Scientific BP310-500
Imidazole Fisher Scientific 03196-500
IPTG Fisher Scientific BP1755-10
Laboratory dessicator Bel-Art 999320237 190mm plate size
Kanamycin Sulfate Fischer Scientific BP906-5
KIF5A K439 (aa:1-439)-6His Gilbert Lab, RPI N/A doi.org/10.1074/jbc.RA118.002182
Kimwipe Kimberley Clark Z188956 lint-free tissue
Immersion Oil, Type B Cargille 16484
Lens Tissue ThorLabs MC-5
LuNA Laser launch (4 channel: 405, 488, 561, 640nm) Nikon N/A
Lysozyme MP Biomedicals LLC 100834
Magnesium Acetate Tetrahydrate Fisher Scientific BP215-500
Microfuge 18 Beckman Coulter 367160
MPEG-SVA MW-5000 Laysan Bio, Inc. NC0107576
Neutravadin Invitrogen PI31000
Nikon Ti-E inverted microscope Nikon N/A Nikon LuN4 Laser
Ni-NTA Resin Thermo Scientific 88221
Oligonucleotide – CACCTATTCTGAGTTTGCGCGA
GAACTTTCAAAGGC
IDT N/A
Oligonucleotide – GCCTTTGAAAGTTCTCGCGCAA
ACTCAGAATAGGTG
IDT N/A
Open-top thickwall polycarbonate tube, 0.2 mL, 7 mm x 22 mm Beckman Coulter 343755
Optima-TLX Ultracentrifuge Beckman Coulter 361544
Paclitaxel (Taxol equivalent) Thermo Fisher Scientific P3456
PIPES ACROS Organics 172615000
PMSF Millipore 7110-5GM
Porcine Tubulin, biotin label Cytoskeleton, Inc. T333P
Porcine Tubulin, HiLyte 647 Fluor Cytoskeleton, Inc. TL670M far red labelled
Porcine Tubulin, Rhodamine Cytoskeleton, Inc. TL590M
Porcine Tubulin, Tubulin Protein Cytoskeleton, Inc. T240
Potassium Acetate Fisher Scientific BP364-500
Prime 95B sCMOS camera Photometric N/A
Quadrant Detector Sensor Head ThorLabs PDQ80A
Quikchange Lightning Kit Agilent Technologies 210518
Sodium Bicarbonate Fisher Scientific S233-500
Sodium Phosphate Dibasic Anhydrous Fisher Scientific BP332-500
Square Cover Glasses Corning 12-553-450 18 mm x 18 mm, with thickness of 0.13-0.17 mm
Streptavidin Microspheres Polysciences Inc. 24162-1
Superose-6 Column GE Healthcare 29-0915–96
TCEP Thermo Scientific 77720
TLA-100 Fixed-Angle Rotor Beckman Coulter 343840
Ultrasonic Cleaner (Sonicator) Vevor JPS-08A(DD) 304 stainless steel, 40 kHz frequency, 60 W power
Vectabond APTES solution Vector Laboratories SP-1800-7
Windex Powerized Glass Cleaner with Ammonia-D S.C. Johnson SJN695237

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Palumbo, J., Tai, E., Forth, S. Directly Measuring Forces Within Reconstituted Active Microtubule Bundles. J. Vis. Exp. (183), e63819, doi:10.3791/63819 (2022).

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