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Abstract
Introduction
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Neuroscience

In vivo Vurdering af Microtubule Dynamics og Orientering i Caenorhabditis elegans Neuroner

Published: November 20, 2021
doi:
10.3791/62744
,
1DBT-National Brain Research Centre

Summary

En protokol til billeddannelse af de dynamiske mikrotubuler in vivo ved hjælp af fluorescerende mærket End bindende protein er blevet præsenteret. Vi beskrev metoderne til at mærke, billede, og analysere de dynamiske mikrotubuler i Posterior lateral microtubule (PLM) neuron af C. elegans.

Abstract

I neuroner, mikrotubule orientering har været en vigtig assessor til at identificere axoner, der har plus-end ud mikrotubuler og dendritter, der generelt har blandet orientering. Her beskriver vi metoder til at mærke, billede, og analysere microtubule dynamik og vækst under udvikling og regenerering af touch neuroner i C. elegans. Ved hjælp af genetisk kodede fluorescerende journalister af microtubule tips, vi afbildet axonal mikrotubuler. De lokale ændringer i microtubule adfærd, der indleder axon regenerering efter axotomi kan kvantificeres ved hjælp af denne protokol. Denne analyse er tilpasningsdygtig til andre neuroner og genetiske baggrunde til at undersøge reguleringen af microtubule dynamik i forskellige cellulære processer.

Introduction

Neuroner har en udførlig arkitektur med specialiserede rum som dendritter, cellelegemer, axoner og synapser. Den neuronale cytoskeleton består af mikrotubuler, mikrofilamenter og neurofilamenter, og deres særskilte organisation understøtter neuronale rum strukturelt og funktionelt1,2,3,4,5,6,7,8,9,10 . I årenes løb er mikrotubule organisation blevet identificeret som en nøgledeterminant for neuronal polaritet og funktion. Som neuroner gennemgå strukturelle remodeling under udvikling eller regenerering, microtubule dynamik og orientering bestemme identiteten, polariseret transport, vækst og udvikling af forskellige neuronale rum7. Det er derfor bydende nødvendigt at vurdere mikrotubule dynamik og orientering in vivo at korrelere med neuronal remodeling proces.

Mikrotubuler består af protofilaments af α og β Tubulin heterodimers med dynamiske plus ender og relativt stabile minus ender11,12. Opdagelsen af plus tip komplekse og tilhørende ende bindende proteiner har gjort det muligt for en platform til at vurdere microtubule organisation13. End bindende proteiner (EBP) forbigående forbundet med de voksende plus ender af microtubule og deres forening dynamik er korreleret med væksten af microtubule protofilaments14,15. På grund af hyppig tilknytning og dissociation af plus tip kompleks med microtubule, punkt spredning funktion af GFP-tagged EBP vises som en “komet” i en timelapse film15,16. Siden den banebrydende observation i pattedyr neuroner16, ende bindende proteiner mærket med fluorescerende proteiner er blevet brugt til at bestemme microtubule dynamik på tværs af forskellige modelsystemer og neuron typer17,18,19,20,21,22,23.

På grund af sit enkle nervesystem og gennemsigtige krop har C. elegans vist sig at være et fremragende modelsystem til at studere neuronal remodeling under udvikling og regenerering in vivo. Her beskriver vi metoder til at mærke, billede, og analysere microtubule dynamik og vækst under udvikling og regenerering af touch neuroner i C. elegans. Ved hjælp af genetisk kodet EBP-2::GFP, vi afbildet mikrotubuler i PLM neuron, som tillod os at bestemme polariteten af mikrotubuler i to forskellige neurites af denne neuron24. Denne metode gør det muligt at observere og kvantificere EBP-kometer som et mål for mikrotubuledynamik i forskellige cellulære sammenhænge, for eksempel kan de lokale ændringer i mikrotubuleadfærd, der indleder axonregenerering efter axotomi, vurderes ved hjælp af vores protokol. Denne analyse kan tilpasses til at undersøge reguleringen af microtubule dynamik i forskellige cellulære processer i forskellige celletyper og genetiske baggrunde.

Protocol

1. Reporter stamme: Kultur og vedligeholdelse BEMÆRK: For at måle mikrotubule dynamik og orientering i PLM neuroner, vi brugte ormen stamme udtrykker EBP-2::GFP under touch neuron specifikke promotor mec-4 (juIs338 allel)18,25,26. Vi bruger standard orm kultur og vedligeholdelse metoder til denne stamme27. …

Representative Results

Som et repræsentativt eksempel har vi beskrevet in vivo observation af EBP kometer i steady-state og regenererende axoner af PLM neuroner. PLM neuroner er placeret i ormens haleregion med en lang forreste proces, der danner en synapse og en kort posteriorproces. PLM neuroner vokser i den forreste-posterior retning tæt på epidermis og er ansvarlige for den blide touch fornemmelse i ormene. På grund af deres forenklede struktur, og amenability til billeddannelse og mikrokirurgi, PLM neuroner er blevet grundigt undersø…

Discussion

Forståelse af microtubule dynamik har været et centralt fokus inden for cytoskeletal forskning gennem årene. Mikrotubuler gennemgår nucleation og katastrofe sammen med en kontinuerlig proces med dynamisk ustabilitet44,45,46,47. Mange af disse oplysninger er opnået gennem in vitro-analyser som lysspredningsudlæsninger af gratis vs. polymeriseret tubulin, mikrot…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi takker Yishi Jin og Andrew Chisholm for den indledende støtte og den belastning, der anvendes i undersøgelsen. Den bakteriestamme OP50 blev kommercielt benyttet fra Caenorhabditis Genetics Center (CGC) finansieret af NIH Office of Research Infrastructure Programs (P40 OD010440). Vi takker også Dharmendra Puri for standardiseringen af de eksperimentelle procedurer. Undersøgelsen er finansieret af kernebevillingen fra National Brain Research Centre (støttet af Department of Biotechnology, Govt. of India), DBT /Wellcome Trust India Alliance Early Career Grant (Grant # IA/E/18/1/504331) til S.D., Wellcome Trust-DBT India Alliance Intermediate Grant (Grant # IA/I/13/1/500874) til A.G.-R og et tilskud fra Science and Engineering Research Board (SERB: CRG/2019/002194) til A.G.-R.

Materials

CZ18975 worm strain Yishi Jin lab CZ18975 Generated by Anindya Ghosh-Roy
Agarose Sigma A9539 Mounting worms
Coverslip (18 mm x 18 mm) Zeiss 474030-9010-000 Mounting worms
Dry bath with heating block Neolab Mounting worms
Glass slides (35 mm x 25 mm) Blue Star Mounting worms
Polystyrene bead solution (4.55 x 10^13 particles/ml in aqueous medium with minimal surfactant) Polysciences Inc. 00876 Mounting worms
Test tubes Mounting worms
OP50 bacterial strain Caenorhabditis Genetics Center (CGC) OP50 Worm handling
60mm petri plates Praveen Scientific 20440 Worm handling
Aspirator/Capillary VWR 53432-921 Worm handling
Incubator Panasonic MIR554E Worm handling
Platinum wire Worm handling
Stereomicroscope with fluorescence attachment Leica M165FC Worm handling
0.3% Sodium Chloride Sigma 71376 Nematode Growth Medium
0.25% Peptone T M Media 1506 Nematode Growth Medium
10mg/mL Cholesterol Sigma C8667 Nematode Growth Medium
1mM Calcium chloride dihydrate Sigma 223506 Nematode Growth Medium
1mM Magnesium sulphate heptahydrate Sigma M2773 Nematode Growth Medium
2% Agar T M Media 1202 Nematode Growth Medium
25mM Monobasic Potassium dihydrogen phosphate Sigma P9791 Nematode Growth Medium
0.1M Monobasic Potassium dihydrogen phosphate Sigma P9791 1X M9 buffer
0.04M Sodium chloride Sigma 71376 1X M9 buffer
0.1M Ammonium chloride Fisher Scientific 21405 1X M9 buffer
0.2M Dibasic Disodium hydrogen phosphate heptahydrate Sigma S9390 1X M9 buffer
Glass bottles Borosil Buffer storage
488 nm laser Zeiss Imaging
5X objective Zeiss Imaging
63X objective Zeiss Imaging
Camera Photometrics Evolve 512 Delta Imaging
Computer system for Spinning Disk unit HP Intel ® Xeon CPU E5-2623 3.00GHz Imaging
Epifluorescence microscope Zeiss Observer.Z1 Imaging
Halogen lamp Zeiss Imaging
Mercury Arc Lamp Zeiss Imaging
Spinning Disk Unit Yokogawa CSU-X1 Imaging
ZEN2 software Zeiss Imaging
Image J (Fiji Version) Image analysis and processing
Adobe Creative Cloud Adobe Image analysis and processing
Computer system for Image Analysis Dell Intel ® Core ™ i7-9700 CPU 3.00GHz Image processing/Representation
DOWNLOAD MATERIALS LIST

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Keywords: Microtubule DynamicsMicrotubule OrientationCaenorhabditis Elegans NeuronsIn Vivo AssessmentCytoskeletonAlpha And Beta TubulinEnd-binding ProteinsEBP-2GFPPLM NeuronsMake-3 PromoterMountingPolystyrene BeadsFluorescence MicroscopySpinning Disk Unit
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Dey, S., Ghosh-Roy, A. In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons. J. Vis. Exp. (177), e62744, doi:10.3791/62744 (2021).
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