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

Summary

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

Abstract

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

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.

Protocol

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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Agar-Agar Kobe I	Carl Roth GmbH + Co. KG	5210.2	NGM component
Ahringer Library hsp-1 siRNA	Source BioScience UK Limited	F26D10.3
Ampicillin	Carl Roth GmbH + Co. KG	K029.3	Antibiotic
B&H DCS-120 SPC-150	Becker & Hickl GmbH		FLIM Aquisition software
B&H SPC830-SPC Image	Becker & Hickl GmbH		FLIM Aquisition software
BD Bacto Peptone	BD-Bionsciences	211677	NGM component
C. elegans iQ44-YFP	CAENORHABDITIS GENETICS CENTER (CGC)	OG412
C. elegans iQ85-YFP			Kind gift from Morimoto Lab
C. elegans mQ40-RFP			Kind gift from Morimoto Lab
C. elegans nQ40-CFP			Kind gift from Morimoto Lab
Deckgläser-18x18mm	Carl Roth GmbH + Co. KG	0657.2	Cover slips
Isopropyl-β-D-thiogalactopyranosid (IPTG)	Carl Roth GmbH + Co. KG	2316.4
Leica M165 FC	Leica Camera AG		Mounting Stereomicroscope
Leica TCS SP5	Leica Camera AG		Confocal Microscope
Levamisole Hydrochloride	AppliChem GmbH	A4341	Anesthetic
OP50 Escherichia coli	CAENORHABDITIS GENETICS CENTER (CGC)	OP50
PicoQuant PicoHarp300	PicoQuant GmbH		FLIM Aquisition software
Sodium Azide	Carl Roth GmbH + Co. KG	K305.1	Anesthetic
Sodium Chloride	Carl Roth GmbH + Co. KG	3957.2	NGM component
Standard-Objektträger	Carl Roth GmbH + Co. KG	0656.1	Glass slides
Universal Agarose	Bio & Sell GmbH	BS20.46.500
Zeiss AxioObserver.Z1	Carl Zeiss AG		Confocal Microscope
Zeiss LSM510-Meta NLO	Carl Zeiss AG		Confocal Microscope