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.

C. elegans has emerged as a model to study aging and disease due to its short lifespan, isogenic nature, and ease of genetic manipulation. Several C. elegans transgenic strains that express human disease-causing proteins in vulnerable tissues have been created. Importantly, many of the strains containing aggregation-prone proteins recapitulate the hallmark of amyloid disorders, the formation of large inclusions. Thanks to C. elegans' transparent body, these aggregates can be visualized in vivo, noninvasively and nondestructively². Generating any protein of interest (POI) in fusion with a fluorophore allows to investigate its locations, trafficking, interaction network, and general fate.

We present a protocol to monitor the aggregation of disease-causing proteins in living and aging C. elegans via fluorescence lifetime imaging microscopy (FLIM). FLIM is a powerful technique based on the lifetime of a fluorophore, rather than its emission spectra. The lifetime (tau, τ) is defined as the average time required by a photon to decay from its excited state back to its ground state. The lifetime of a given molecule is calculated with the time-domain technique of time-correlated single photon counting (TCSPC). In TCSPC-FLIM, the fluorescent decay function is obtained by exciting the fluorophore with short, high-frequency laser pulses and measuring the emitted photon's arrival times to a detector in respect to the pulses. When scanning a sample, a three-dimensional data array is created for each pixel: the array includes information on the distribution of the photons in their x,y spatial coordinates and their temporal decay curve. A given sample therefore becomes a map of lifetimes revealing information on the protein's structure, binding, and environment^3,4. Each fluorescent protein possesses an intrinsic and precisely defined lifetime, usually of a few nanoseconds (ns), dependent on its physiochemical properties. Importantly, the lifetime of a fluorophore is independent of its concentration, fluorescent intensity, and of the imaging methodology. However, within a biological system, it can be affected by environmental factors such as pH, temperature, ion concentrations, oxygen saturation, and its interaction partners. Lifetimes are also sensitive to internal structural changes and orientation. Fusing a fluorophore to a POI results in a change in its lifetime and consequently information on the behavior of the fused protein. When a fluorophore is surrounded or encapsulated in a tightly bound environment, such as the antiparallel beta sheets of an amyloid structure, it loses energy non-radiatively, a process known as quenching⁵. Quenching of the fluorophore results in a shortening of its apparent lifetime. When soluble, a protein's lifetime will stay closer to its original, higher value. In contrast, when a protein starts to aggregate, its lifetime will inevitably shift to a lower value^6,7. Therefore, it becomes possible to monitor the aggregation propensity of any amyloid-forming protein at different ages in living C. elegans.

Here we describe a protocol to analyze the aggregation of a fusion protein comprising different polyglutamine (CAG, Q) stretches (Q40, Q44, and Q85). We illustrate how the technique can be applied equally to different fluorophores, such as cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and monomeric red fluorescent protein (mRFP); and in all tissues of C. elegans, including the neurons, muscles, and the intestine. Moreover, in the context of proteostasis, FLIM is a very useful tool to observe changes upon depletion of molecular chaperones. Knocking down one of the key molecular chaperones, heat shock protein 1 (hsp-1), via RNA interference produces premature misfolding of proteins. The increase in aggregation load as a result of aging, disease, or deficient chaperones, is then measured as a decrease in fluorescence lifetime.

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 old nematodes are imaged on day 8 of life.

2. RNAi-mediated knockdown of chaperone machinery via feeding

NOTE: Perform knockdown of heat shock protein 1 (hsp-1) chaperone by feeding the corresponding RNAi vector to the nematodes¹⁰. The hsp-1 RNAi plasmid was obtained from the Ahringer library (clone ID: F26D10.3).

1. Grow the HT115 (DE3) E. coli expressing the hsp-1 RNAi plasmid for 6 h to overnight in Luria Bertani (LB) medium containing 50 µg/mL ampicillin.
2. Prepare fresh NGM agar plates containing isopropyl β-D-1-thiogalactopyranoside (IPTG; 1 mM) and ampicillin (25 µg/mL) and seed with the hsp-1 RNAi bacteria. Leave plates to dry and induce at room temperature for 1-3 days.
3. Place synchronized eggs on an siRNA plate and leave to hatch, or place gravid nematodes and allow to lay eggs for 4 h at 20 °C before removing. Grow the nematodes until the desired age or stage.
   NOTE: The second generation of nematodes might present a stronger phenotype of the knockdown. The siRNA protocol and conditions described here are general and adapted to hsp1. RNA interference via siRNA of a specific clone/gene needs to be established and optimized by the end user. It is important to note that not all siRNA have the same efficiency, and it is therefore recommended to test the efficacy of the knockdown by quantification by either quantitative reverse transcription polymerase chain reaction (RT-qPCR) or Western blot.

3. Preparation of microscopy slides

1. On the day of imaging, start by preparing the imaging slides. Melt agarose in ddH₂O at a concentration of 3% (w/v) and let cool slightly.
2. Cut the tip of a 1 mL pipette tip and take roughly 200 µL of melted agarose. Pipette the agarose onto a clean glass slide and immediately place a second one on top, avoiding the formation of any bubbles. Leave to dry and gently remove the top glass slide. The result is a glass slide with an even agarose surface where the nematodes will be positioned.
   NOTE: Each slide will be used to image between 5-10 nematodes. Slides can be prepared and stored for a few hours in a humified box to prevent the agarose from drying out.

4. Mounting nematodes onto microscopy slides

NOTE: FLIM requires the nematodes to be immobilized. Perform this step once the imaging setup (e.g., microscopes, lasers, detectors) is ready to use.

1. Using a platinum wire pick, place nematodes of the desired age onto a fresh unseeded plate and let them crawl to remove the excess OP50 bacteria from their bodies.
2. Prepare the anesthetic compound (sodium azide or levamisole) to immobilize the nematode. Keep a 500 mM NaN₃ stock in the dark at 4 °C and dilute in fresh ddH₂O to a final concentration of 250 mM. If using levamisole, dilute a 20 mM stock to 2 mM working solution in ddH₂O.
   CAUTION: Sodium azide (NaN₃) is highly toxic. Use gloves and protective eyewear and work under a ventilated hood.
3. Working under a stereomicroscope, place a 10 µL drop of anesthetic compound onto an agarose pad and gently transfer 5-10 nematodes into it. Use an eyelash tip to separate the nematodes. Keep them close together but not touching to allow for easier localization of the nematodes during image acquisition.
4. Carefully overlay the nematodes with a coverslip. Take measurements within 1 h after mounting.
   NOTE: Both anesthetics will eventually kill C. elegans. The nematodes must be completely immobile during imaging, because the map of the lifetime is recorded from each pixel. Any movement of the x,y parameters prevents the reading of the lifetime in the same excited pixel.

5. Acquisition of FLIM data

NOTE: In this protocol, the lifetime of the fluorophore is acquired via the time-domain TCSPC method. FLIM requires a pulse of light to be generated by the laser at a set and constant repetition rate. The repetition rate varies according to the laser type and needs to be known by the user. Lifetime measurements are achieved by detectors and electronic equipment installed alongside a conventional microscope. In this protocol, measurements are performed on three different laser scanning confocal microscopes with detectors and software provided by two different companies (Table of Materials) for acquisition of mRFP, CFP, and YFP lifetimes, respectively. Check that the correct filters of emission/excitation are in place and minimize any background or monitor backlight before starting. Before starting any experiment, establish the photostability of the chosen fluorophore. If the fluorophore bleaches within a short time within the nematode tissues, it is not suitable for FLIM measurements in C. elegans.

1. Open the FLIM acquisition software. The FLIM software also allows control of the confocal microscope. Locate the tab/button to allow for the detector's outputs to be enabled and press Enable Outputs.
2. Acquire the instrument response function (IRF), which describes the timing precision of the instrumental setup.
   NOTE: This step should be performed preferably before mounting the nematodes.
   1. If available, remove the excitation/emission filters.
   2. Place an empty coverslip above the objective and find its surface. Record the scatter signal obtained from the coverslip for a minimum of 30 s.
      NOTE: For lifetimes of several nanoseconds, the acquisition software can automatically estimate the IRF shift. Acquiring an IRF is always recommended.
3. Place the slide with the mounted C. elegans on the stage. Using a 10x magnification lens in transmission mode and localize the position of the nematodes on the slide.
4. Remove the slide, switch the objective to a 63x magnification lens, and apply the required immersion medium (e.g., oil). Replace the slide on the stage and localize the nematodes.
5. Locate the Pinhole Manager on the acquisition software and open it to the Maximum. Start scanning the sample, select a region of interest (e.g., head, upper body), and focus on its maximum projection plane.
6. Monitor the laser pulse rate and the three other values present on the interface of the software: The Constant Fraction Discriminator (CFD), the Time-to-Amplitude (TAC), and the Analogue-to-Digital Converter (ADC).
   NOTE: The laser should have a maximum gate of 1 x 10⁸ single photon counts. This number represents the maximum number of photons supplied by the laser. The CFD provides information on the receipt of the single photon pulse in reference to the laser pulse by the detector. This value should be roughly 1 x 10⁵. The TAC discriminates between the time one photon was detected and the next laser pulse. Finally, the ADC converts the TAC voltage into a storable memory signal¹¹. The CFD, TAC, and ADC should all have similar values to ensure that photons emitted by the fluorophore are not lost. Correct evaluation of these parameters ensures that enough photons are being collected to create an accurate lifetime map.
7. On the interface of the FLIM software, preview the number of photons detected: the ADC value should be between 1 x 10⁴ and 1 x 10⁵. If necessary, shift the focus on a different plane or increase the laser power to collect more photons.
   NOTE: In general, the number of recorded photons per second should not exceed 1% of the laser's repetition rate.
8. In the menu bar, select the tab to set the acquisition parameters. Select scan sync in to allow for single photon detection.
9. Set the acquisition to a fixed amount of time or a fixed number of photons. For example, acquire a lifetime decay curve for 2 min or until a single pixel reaches a photon count of 2,000 single events. Press Start to begin acquisition.
   NOTE: Different fluorophores will require different excitation and emission lasers and filters. According to the brightness of the sample, the laser power can also be adjusted, which will not interfere with the lifetime. These protocols use the following excitation/emission settings: YFP ex500/em520-50 nm, mRFP ex561/em580-620 nm. A pulsed two photon laser was employed for CFP measurements using ex800/em440 nm. The amount of time and photon count required for acquisition of a FLIM map will need to be empirically established for each setup and each experimental purpose.

6. Analysis of FLIM data using FLIMfit software

NOTE: Perform data analysis using the FLIMfit software tool developed at Imperial College London¹² (see Figure 1).

1. Open the software and import FLIM data files via File | Load FLIM Data. Load all samples from one condition, even if obtained in different sessions and from different biological repeats.
2. If necessary, segment a single nematode from any FLIM picture via Segmentation | Segmentation Manager. Drag the cropping tool around the area of interest until it is highlighted. Once completed, press OK.
   NOTE: Segmentation must be done for all images.
3. Select a small region where the intensity-based image of a C. elegans appears (Figure 1, Arrows 1). The decay curve of that region will appear in the large decay window on the right side of the interface (Figure 1, Arrow 2).
   NOTE: The decay can be displayed linearly or logarithmically.
4. Set the correct parameters to extrapolate the lifetime via the software's algorithm as described in steps 6.5-6.8.
5. On the Data tab (Figure 1, Arrow 3):
   1. Set an arbitrary Integrated Minimum value to exclude any pixels that are too dim to produce a good fit. Depending on the C. elegans sample this value varies from 40-300. Input different values until a satisfactory preview is achieved.
   2. Select a Time Min and a Time Max number to limit the FLIM signal to these values. All events that appear before and after this threshold will be excluded.
      NOTE: For example, for the analysis of mRFP, the events prior to 800 ps and after 4,000 ps were excluded. These values depend on the lifetime of the fluorophore and need to be determined by the end user.
   3. Do not change the preset Counts/Photon of 1.
   4. Input the Repetition Rate, in MHz, of the laser utilized during acquisition.
      NOTE: For the current protocol, different lasers were utilized with various repetition rates. The two photon laser used for acquisition of CFP lifetimes possesses a repetition rate of 80 MHz, for YFP the laser repeats at 40 MHz, and for mRFP the value is 78.01 MHz. These values were inputted into FLIMfit according to the sample analyzed.
   5. Input a Gate Max value to exclude all saturated pixels.
      NOTE: For lifetime measurements in C. elegans, this value is set to any large number (e.g., 1 x 10⁸).
6. On the Lifetime tab, select a global fitting to be used (e.g., a pixel-wise fitting). See Figure 1, Arrow 4.
   NOTE: A Pixel-wise fitting will produce a decay fitted to each individual pixel. An Image-wise fitting will produce a global fitting of each individual image and display a single lifetime value per image. A Global-wise fitting will produce a single fitting across the whole dataset. A single lifetime value is provided for all images.
7. Do not change any other parameter except for the No. Exp selection if it is known that the chosen fluorescence decay is multiexponential and exhibits more than a single lifetime.
   NOTE: In the present protocol, this function was utilized to calculate the lifetimes of the biexponential CFP fluorophore.
8. Upload the IRF via the IRF menu: IRF | Load IRF. To estimate the IRF shift, select IRF | Estimate IRF Shift. A set of values will automatically appear on the IRF tab. Once this is established, do not change any other parameters of this tab.
9. Once all parameters are set, press Fit Dataset (Figure 1, Arrow 5). The algorithm will produce a fit for the decay curve and establish a lifetime value for each image.
   NOTE: The resulting fit, highlighted in a blue line, should overlap with all the events. A good fit is obtained when all events are aligned along the fit.
10. Click the Parameters tab (Figure 1, Arrow 6), located within the top right menus of the software's interface, and select Statistic: w_mean (weighted mean) and check that the chi2 value is as close as possible to 1.
    NOTE: A chi2 close to one ensures the accuracy of the fit. The lifetime value of the selected image is thus revealed as tau_1.
11. Export any information of interest: File | Export Intensity Images/Fit Result Table/Images/Histograms. Save the data settings used to calculate the lifetime: File | Save Data Settings.
    NOTE: The parameters employed will be saved for future analysis of the selected samples.

7. Graphical representations of FLIM data

NOTE: The lifetimes collected from different samples can be visually represented in various ways. Select to denote the lifetime values either in nanoseconds or picoseconds.

1. Show the quality of the fit and the accuracy of the curve by exporting the decay curve directly from FLIMfit.
2. Represent the distribution of the photons by plotting the frequency of the photon count versus the lifetime value in a histogram.
3. Finally, for statistical comparison, if comparing two or more samples, place lifetime values plus standard deviation of the mean in a scatter plot bar graph. Perform any desired statistical analysis.

Results

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...

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