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Abstract
Introduction
Protocol
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Bioengineering

Dextran Mærkning og optagelse i levende og funktionelle Murine Cochlear hårceller

Published: February 08, 2020
doi:
10.3791/60769
,
1Molecular Physiology and Biophysics Section,National Institute of Neurological Disorders and Stroke, National Institutes of Health

Summary

Her præsenterer vi en metode til visualisering af udbredelsen af 3 kDa Texas Rød-mærket dextran i auditive hårceller med funktionelle mechanotransduction kanaler. Desuden kan dextrans af 3-10 kDa bruges til at studere endocytose i hår og støtte celler i orglet corti.

Abstract

Hårcellemechanotransduction (MET) kanal spiller en vigtig rolle i hørelsen. Den molekylære identitet og de strukturelle oplysninger om markedsøkonomisk behandling er dog fortsat ukendt. Elektrofysiologiske undersøgelser af hårceller viste, at MET-kanalen har en stor ledningsevne og er gennemtrængelig for relativt store fluorescerende kationiske molekyler, herunder nogle styrylfarvestoffer og Texas Rød-mærket aminoglycoside antibiotika. I denne protokol beskriver vi en metode til at visualisere og evaluere udbredelsen af fluorescerende dextrans i hårceller i orglet i Corti explants, der kan bruges til at assay for funktionelle MET kanaler. Vi fandt, at 3 kDa Texas Rød-mærket dextran specifikt etiketter funktionelle auditive hårceller efter 1-2 h inkubation. Især 3 kDa dextran etiketter de to kortere stereocilia rækker og ophobes i cellekroppen i et diffust mønster, når funktionelle MET kanaler er til stede. En yderligere vesicle-lignende mønster af mærkning blev observeret i cellekroppen af hårceller og omkringliggende støtteceller. Vores data tyder på, at 3 kDa Texas-Red dextran kan bruges til at visualisere og studere to veje til cellulære farvestof optagelse; en hårcellespecifik indgangsrute gennem funktionelle MET-kanaler og endocytose, et mønster, der også er tilgængeligt for større dextran.

Introduction

Hårcellerne i det indre øre er de sensoriske celler, der registrerer lyd og skjulte de mekanisk stimuli i elektriske signaler, som i sidste ende fortolkes af vores hjerne. Disse celler har en trappe-formet bundt af tre rækker af actin-baserede filamenter, kendt som stereocilia, som stikker ud fra deres apical region1,2. De mekaniske stimuli afleder stereocilia-glødetrådene mod den længste række og udløser åbningen af mechanotransduction (MET)kanaler3. Åbningen af MET-kanalerne fører til en tilstrømning af kationer, der depolariserer cellen og dermed signalerer frigivelsen af synapse vesikler på de basale region af hårcelle.

De biofysiske egenskaber af MET kanal afgørende for hørelse naster. Disse kanaler er bl.a. Bemærkelsesværdigt, store fluorescerende molekyler såsom FM1-43 og Texas Rød-mærket aminoglycosides er permeant blokkere af MET kanal, hvilket resulterer i deres ophobning i hårcellekroppen, der kan visualiseres ved hjælp af fluorescens mikroskopi11,12,13,14. Omvendt er den molekylære identitet og strukturen af MET-kanalen og dens gennemtrængelsesvej forblevet undvigende. Stigende eksperimentelle beviser viser , at transmembrane-lignende kanal protein 1 (TMC1) er en del af MET kanal i modne hårceller15,16,17,18,19. Mutationer i transmembrane-lignende kanal 1 (TMC1) ændre MET kanal egenskaber19,20,21,22 og forårsage døvhed. Desuden lokaliserer TMC1 til det sted, hvor MET kanal18,23 og interagerer med tip-link ansvarlig for at overføre den mekaniske kraft til MET kanal24,25. Desuden har de seneste bioinformatik analyse identificeret TMC proteiner som evolutionære relateret til mechanosensitive kanaler TMEM63/OSCA proteiner og TMEM16 proteiner, en familie af calcium-aktiverede chlorid kanaler og lipid scramblases26,27,28. En strukturel model af TMC1 baseret på forholdet mellem disse proteiner afslørede tilstedeværelsen af et stort hulrum på protein-lipid interface27. Dette hulrum huser de to TMC1 mutationer, der forårsager autosomal dominerende høretab (DFNA36)27,29,30,31,32, og selektiv ændring af cystein mutanter for rester i hulrummet ændre MET kanal egenskaber28, hvilket indikerer, at det kunne fungere som gennemfarvning vej met kanal. Den store størrelse af denne forudsagte hulrum i TMC proteiner kunne forklare evnen af store molekyler til at gennemsyre MET kanal. For at teste forudsigelsen om, at MET-kanalen indeholder en usædvanlig stor gennemtrængelsesvej og for at skubbe grænserne for størrelsen af hulrummet observeret i TMC1, udviklede vi en protokol til at udføre optagelseeksperimenter i Corti-explants organ med et større molekyle, 3 kDa dextran fluorescerende mærket med Texas Red.

Dextran er en kompleks forgrenet polysaccharid består af mange D-glukose molekyler bundet af alfa-1,6 glycosidiske forbindelser. Dens høje opløselighed i vand, lav celle toksicitet, og bioinertity gør det til et alsidigt værktøj til at studere flere cellulære processer. Desuden er dextran fås i en bred vifte af størrelser og fluorescerende mærket med fluorophores i flere farver. Fluorescerende mærket dextrans er almindeligt anvendt i celle og væv permeabilitet forskning33,34, at studere endocytose i flere cellulære systemer35,36, og for neurale sporing37,38. På det auditive område er dextranmolekyler også blevet brugt til at vurdere afbrydelsen af cellecellekrydset og tabet af den auditive sensoriske epitelintegritet efter udsættelse for intens støj i chinchillaorgelet i Corti39,40.

I dette arbejde udnyttede vi egenskaberne for nogle af de mindste (3 og 10 kDa) fluorescerende dextrans til at udføre optagelsesforsøg i murine indre ørehårceller og udforske størrelsen af permeationsvejen for den indre ørehårscelle MET-kanal. Derudover brugte vi en laser-scanning konfokale mikroskop (LSM) 880 udstyret med en Airyscan detektor til at visualisere og lokalisere fluorescerende dextran på stereocilia og cellekroppen af auditive hårceller.

Protocol

Dyrepleje- og forsøgsprocedurerne blev udført i følger retningslinjerne for pleje og anvendelse af laboratoriedyr, som blev godkendt af Animal Care and Use Committee fra National Institute of Neurological Disorders and Stroke (Animal protocol #1336 til KJS). 1. Mus Indstil et par ynglende par C57BL/6J vildtype til at yngle i dyreanlægget for at kontrollere datoen for nedløbets fødsel og holde styr på hvalpenes alder. 2. Cochleae Dissektion <…

Representative Results

Vi observerede robust og specifik mærkning af hårceller efter 2 h inkubation af orglet af Corti explants fra vilde-type postnatal-dag-6 (P6) mus med 3 kDa dextran fluorescerende mærket med Texas Red (dextran-TR)(Figur 2A-B). Dextran mærkning blev observeret i både indre og ydre hårceller (IHC og OHC) på basale, midterste og apiske regioner af orglet Corti(Figur 2B). Fluorescerende mærket phall…

Discussion

Denne protokol beskriver, hvordan man udfører optagelse eksperimenter i murine organ Corti explants med 3 kDa dextran Texas Red. Formålet med denne metode er at teste, om molekyler større end andre tidligere testede var også i stand til specifikt at mærke auditive hårceller og gennemsyre gennem MET kanal. Lignende eksperimentelle protokoller er tidligere blevet brugt til at vurdere gennemtrængeligheden af hårceller til andre fluorescerende farvestoffer såsom FM1-43 (0,56 kDa)12,</sup…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi takker Vincent Schram fra NICHD mikroskopi og billeddannelse kerne for at bistå i konfokale billede erhvervelse, og Tsg-Hui Chang for uvurderlig hjælp med koloniforvaltning og mus pleje. Denne forskning blev støttet af Intramural Research Program af NINDS, NIH, Bethesda, MD, til KJA A.B. blev støttet af Intramural Research Program af NINDS, NIH, og af en Robert Wenthold Postdoctoral Fellowship fra intramural forskningsprogram af NIDCD.

Materials

#1.5 glass coverslips 18mm Warner Instruments 64-0714
Alexa Fluor 488 Phalloidin ThermoFisher A12379
Alexa Fluor 594 Phalloidin ThermoFisher A12381
alpha Plan-Apochromat 63X/1.4 Oil Corr M27 objective Carl Zeiss 420780-9970-000
Amiloride hydrochloride EMD MILLIPORE 129876
Benchwaver 3-dimensional Rocker Benchmarks scientific B3D5000
C57BL/6J wild-type mice strain 000664 The Jackson Laboratory
Cell impermeant BAPTA tetrapotassium salt ThermoFisher B1204
Dextran, Fluorescein, 10,000 MW, Anionic, Lysine Fixable ThermoFisher D1820
Dextran, Texas Red, 10,000 MW, Lysine Fixable ThermoFisher D1863
Dextran, Texas Red, 3000 MW, Lysine Fixable ThermoFisher D3328
Formaldehyde Aqueous Solution EM Grade Electron microscopy science 15710
HBSS, calcium, magnesium, no phenol red ThermoFisher 14025
HBSS, no calcium, no magnesium, no phenol red ThermoFisher 14170
Image J or FIJI NIH http://fiji.sc/
Immersol 518F oil immersion media Carl Zeiss 444970-9000-000
Leibovitz's L-15 Medium, GlutaMAX Supplement ThermoFisher 31415029
neomycin trisulfate salt hydrate Sigma N6386
PBS (10X), pH 7.4 ThermoFisher 70011069
Phalloidin-CF405M Biotium 00034
ProLong Diamond antifade mounting media ThermoFisher P36970
superfrost plus microscope slide Fisherbrand 22-037-246
Triton X-100 Sigma T8787
Zen Black 2.3 SP1 software Carl Zeiss https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html
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Dextran LabelingLive Hair CellsMechanotransduction ChannelsCochlear DissectionFluorescent Dextran
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Ballesteros, A., Swartz, K. J. Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells. J. Vis. Exp. (156), e60769, doi:10.3791/60769 (2020).
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