Method Article

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

DOI:

10.3791/61004

⸱

March 27th, 2020

 ,  ,  ,  , 
1Leibniz Research Institute for Molecular Pharmacology im Forschungsverbund Berlin, 2NeuroCure Cluster of Excellence, Charité - Universitätsmedizin Berlin, 3Molecular Neuroscience Group, Department of Chemical Engineering and Biotechnology, University of Cambridge, 4Cell Biology, University of Bremen

In This Article

Summary

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Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

Abstract

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Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington's, Parkinson's, or Alzheimer's disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

Introduction

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Protein aggregation occurs both in aging and disease. The pathways that lead to the formation and deposition of large amyloids or amorphous inclusions are difficult to follow and their kinetics are similarly challenging to unravel. Proteins can misfold due to intrinsic mutations within their coding sequences, as in the case of genetic diseases. Proteins also misfold because the proteostasis network (PN) that keeps them soluble and properly folded is impaired, as happens during aging. The PN includes molecular chaperones and degradation machineries and is responsible for the biogenesis, folding, trafficking, and degradation of proteins1.

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Protocol

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1. Synchronization of C. elegans

  1. Synchronize C. elegans either via alkaline hypochlorite solution treatment or via simple egg laying for 4 h at 20 °C8.
  2. Grow and maintain nematodes at 20 °C on nematode growth medium (NGM) plates seeded with OP50 E. coli according to standard procedures9. Age the nematodes until the desired developmental stage or day.
    NOTE: In this protocol, young adults are imaged on day 4 and old nematodes are imaged on day 8 of life.

2. RNAi-mediated knockdown of chaperone machinery via feeding

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Results

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The protocol shows how to accurately monitor the formation of aggregated species in living C. elegans, both during its natural aging and when subjected to stress. We selected four different strains of transgenic nematodes expressing polyglutamine proteins of either 40Q, 44Q, or 85Q repeats. These proteins are synthesized in different tissues and were fused to different fluorophores. The C. elegans strains either expressed Q40-mRFP in the body wall muscles (mQ40-RFP), Q40.......

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Discussion

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The protocol presented here describes a microscopy-based technique to identify aggregated species in the C. elegans model system. FLIM can accurately characterize the presence of both aggregated and soluble species fused to a fluorophore via measurement of their fluorescence lifetime decays. When a fusion protein starts to aggregate its recorded average lifetime will shift from a higher to a lower value16. The propensity of aggregation can then be deduced by the drop in lifetime: the lowe.......

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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The muscle-Q40-mRFP strain provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The neuronal-Q40-CFP was a kind gift of the Morimoto Lab. We acknowledge the DFG (KI-1988/5-1 to JK, NeuroCure PhD fellowship by the NeuroCure Cluster of Excellence to MLP), EMBO (Short term fellowship to MLP) and the Company of Biologists (travel grants to CG and MLP) for funding. We also acknowledge the Advanced Light Microscopy imaging facility at the Max Delbrück Centre for Molecular Medicine, Berlin, for providing the setup to image the YFP constructs.

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Agar-Agar Kobe ICarl Roth GmbH + Co. KG5210.2NGM component
Ahringer Library hsp-1 siRNASource BioScience UK LimitedF26D10.3
AmpicillinCarl Roth GmbH + Co. KGK029.3Antibiotic
B&H DCS-120 SPC-150Becker & Hickl GmbHFLIM Aquisition software
B&H SPC830-SPC ImageBecker & Hickl GmbHFLIM Aquisition software
BD Bacto PeptoneBD-Bionsciences211677NGM component
C. elegans iQ44-YFPCAENORHABDITIS GENETICS CENTER (CGC)OG412
C. elegans iQ85-YFPKind gift from Morimoto Lab
C. elegans mQ40-RFPKind gift from Morimoto Lab
C. elegans nQ40-CFPKind gift from Morimoto Lab
Deckgläser-18x18mmCarl Roth GmbH + Co. KG0657.2Cover slips
Isopropyl-β-D-thiogalactopyranosid (IPTG)Carl Roth GmbH + Co. KG2316.4
Leica M165 FCLeica Camera AGMounting Stereomicroscope
Leica TCS SP5Leica Camera AGConfocal Microscope
Levamisole HydrochlorideAppliChem GmbHA4341Anesthetic
OP50 Escherichia coliCAENORHABDITIS GENETICS CENTER (CGC)OP50
PicoQuant PicoHarp300PicoQuant GmbHFLIM Aquisition software
Sodium AzideCarl Roth GmbH + Co. KGK305.1Anesthetic
Sodium ChlorideCarl Roth GmbH + Co. KG3957.2NGM component
Standard-ObjektträgerCarl Roth GmbH + Co. KG0656.1Glass slides
Universal AgaroseBio & Sell GmbHBS20.46.500
Zeiss AxioObserver.Z1Carl Zeiss AGConfocal Microscope
Zeiss LSM510-Meta NLOCarl Zeiss AGConfocal Microscope

References

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  1. Klaips, C. L., Jayaraj, G. G., Hartl, F. U. Pathways of cellular proteostasis in aging and disease. Journal of Cell Biology. 217 (1), 51-63 (2018).
  2. Kikis, E. A. The struggle by Caenorhabditis elegans to maintain proteostasis during aging and disease. Bio....

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Tags

Fluorescence Lifetime ImagingAmyloid FibrilizationC Elegans ModelProtein Aggregation AnalysisNoninvasive MonitoringFLIM AcquisitionLifetime MeasurementPhoton DetectionAgarose Pad PreparationNeurodegenerative Disease Study

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