In Vivo Quantification of Protein Turnover in Aging C. Elegans using Photoconvertible Dendra2

Summary

Presented here is a protocol to monitor degradation of the protein huntingtin fused to the photoconvertible fluorophore Dendra2.

Abstract

Proteins are synthesized and degraded constantly within a cell to maintain homeostasis. Being able to monitor the degradation of a protein of interest is key to understanding not only its life cycle, but also to uncover imbalances in the proteostasis network. This method shows how to track the degradation of the disease-causing protein huntingtin. Two versions of huntingtin fused to Dendra2 are expressed in the C. elegans nervous system: a physiological version or one with an expanded and pathogenic stretch of glutamines. Dendra2 is a photoconvertible fluorescent protein; upon a short ultraviolet (UV) irradiation pulse, Dendra2 switches its excitation/emission spectra from green to red. Similar to a pulse-chase experiment, the turnover of the converted red-Dendra2 can be monitored and quantified, regardless of the interference from newly synthesized green-Dendra2. Using confocal-based microscopy and due to the optical transparency of C. elegans, it is possible to monitor and quantify the degradation of huntingtin-Dendra2 in a living, aging organism. Neuronal huntingtin-Dendra2 is partially degraded soon after conversion and cleared further over time. The systems controlling degradation are deficient in the presence of mutant huntingtin and are further impaired with aging. Neuronal subtypes within the same nervous system exhibit different turnover capacities for huntingtin-Dendra2. Overall, monitoring any protein of interest fused to Dendra2 can provide important information not only on its degradation and the players of the proteostasis network involved, but also on its location, trafficking, and transport.

Introduction

The proteome of a living organism is constantly renewing itself. Proteins are continuously degraded and synthesized according to the physiological demand of a cell. Some proteins are quickly eliminated, whereas others are longer lived. Monitoring protein dynamics is a simpler, more accurate, and less invasive task when using genetically encoded fluorescent proteins (FPs). FPs form autocatalytically and can be fused to any protein of interest (POI), but do not require enzymes to fold or need cofactors save for oxygen¹. A newer generation of FPs has recently been engineered to switch color upon irradiation with a light pulse of determined wavelength. These photoactivatable FPs (PAFPs) allow for labeling and tracking of POIs, or the organelles or cells they reside in, and to examine their quantitative and/or qualitative parameters². FPs make it possible to track any POI's movement, directionality, rate of locomotion, coefficient of diffusion, mobile versus immobile fractions, the time it resides in one cellular compartment, as well as its turnover rate. For specific organelles, locomotion and transport, or fission and fusion events can be determined. For a particular cell type, a cell's position, rate of division, volume, and shape can be established. Crucially, the use of PAFPs allows tracking without continuous visualization and without interference from any newly synthetized probe. Studies both in cells and in whole organisms have successfully employed PAFPs to address biological questions in vivo, such as the development of cancer and metastasis, assembly or disassembly of the cytoskeleton, and RNA-DNA/protein interactions³. In this manuscript, light microscopy and PAFPs are used to uncover the turnover rates of the aggregation-prone protein huntingtin (HTT) in vivo in a C. elegans model of neurodegenerative disease.

The protocol described here quantifies the stability and degradation of the fusion protein huntingtin-Dendra2 (HTT-D2). Dendra2 is a second-generation monomeric PAFP⁴ that irreversibly switches its emission/excitation spectra from green to red in response to either UV or visible blue light, with an increase in its intensity of up to 4,000-fold^5,6. Huntingtin is the protein responsible for causing Huntington's disease (HD), a fatal hereditary neurodegenerative disorder. Huntingtin exon-1 contains a stretch of glutamines (CAG, Q). When the protein is expressed with over 39Q, it misfolds into a mutant, toxic, and pathogenic protein. Mutant HTT is prone to aggregation and leads to neuronal cell death and degeneration, either as short oligomeric species or as larger highly structured amyloids⁷.

The nematode is a model system for studying aging and neurodegeneration thanks to its ease of manipulation, isogenic nature, short lifespan, and its optical transparency⁸. To study the stability of HTT in vivo, a fusion construct was expressed in the nervous system of C. elegans. A HTT-D2 transgene containing either a physiological stretch of 25Qs (HTTQ25-D2) or a pathological stretch of 97Qs (HTTQ97-D2) is overexpressed pan-neuronally throughout the nematode's lifetime⁹. By subjecting live C. elegans to a brief and focused point of light, a single neuron is photoswitched and the converted HTT-D2 is tracked over time. To establish the amount of HTT-D2 degraded, the difference between the red signal of the freshly converted HTT-D2 is compared to the remaining red signal of HTT-D2 after a determined period of time. Therefore, it becomes possible to investigate how huntingtin is degraded when found in its expanded and toxic form compared to its physiological form; how anterior or posterior neurons respond differently to the presence of Q97 versus Q25, especially over prolonged time periods; and how the collapse of the proteostasis network (PN) during aging contribute to the differences in degradation rates. These results only describe a small set of observations on the turnover of HTT-D2. However, many more biological questions relevant to both the field of protein aggregation and proteostasis can be addressed with this in vivo application.

Protocol

1. Generation of  C. elegans expressing neuronal Huntingtin-Dendra2 fusion protein

1. Clone the gene encoding the POI in a nematode expression vector (i.e., pPD95_75, Addgene #1494), by traditional restriction enzyme digest¹⁰, Gibson assembly¹¹, or any method of choice. Insert a promoter to drive expression in a desired tissue or at a desired developmental stage. Insert the Dendra2 fluorophore either N- or C-terminally in frame with the POI.
2. Generate transgenic C. elegans expressing the fusion construct (e.g., via microinjection)¹².
   NOTE: The plasmid carrying the transgene will remain as an extrachromosomal array. Integration of the construct is not necessary but can be performed if desired¹³. In this protocol, C. elegans were microinjected with a plasmid carrying the fusion construct huntingtin exon 1-Dendra2 (HTT-D2) under the control of the pan-neuronal promoter Prgef-1. The C. elegans expression backbone was obtained from Kreis et al.¹⁴, the huntingtin exon 1 with either Q25 or Q97 was obtained from Juenemann et al.¹⁵, and Dendra2 was obtained from Hamer et al.¹⁶.

2. Age matching and maintenance of  C. elegans

1. Age match all nematodes by synchronizing either with alkaline hypochlorite solution treatment¹⁷ or via egg laying for 4 h at 20 °C. For egg laying, place 10 gravid adults on a freshly seeded nematode growth media (NGM) plate and leave for 4 h before removing. The eggs laid in this timespan will give rise to synchronized nematodes.
2. Keep experimental C. elegans on nematode growth media (NGM) plates seeded with the bacterial food source E. coli OP50, following standard nematode husbandry¹⁸.
3. Grow nematodes at 20 °C to the desired stage. For this protocol, the required ages are days 4 and 10.
   NOTE: Young adults at day 4 can be identified by the presence of eggs in their gonads and their high mobility. Aged day 10 nematodes are post-fertile, and undergo tissue deterioration and locomotive decline¹⁹.
4. For day 10 nematodes, passage daily after the L4 stage at day 3, once nematodes are fertile, to avoid a mixed population.

3. Preparation of microscopy slides for imaging

1. Prepare the microscopy slides on the day of imaging. In a microwave, melt general grade agarose at a concentration of 3% (w/v) in ddH₂O. Leave to cool slightly.
2. Cut the tip of a 1 mL pipette tip and aspirate roughly 400 µL of melted agarose. Gently place a few drops of agarose onto a clean glass slide and immediately place another slide on top, making sure that a thin pad of agarose is created between the two. Let dry before gently sliding or lifting the top slide off.
3. Place the agarose pad slides in a humidified container to prevent them from drying out. These can be used within 2-3 h.
   NOTE: Avoid formation of small bubbles in the agarose, as the nematodes can be trapped within.

4. Definition of confocal microscope parameters

NOTE: Before mounting the nematodes and data acquisition, define all settings on the confocal acquisition software. The settings can be adapted to the imaging hardware and software of choice.

1. Open the confocal software and define the laser imaging settings. Set the light path for excitation/emission for green Dendra2 at 486-553 nm and for red Dendra2 at 580-740 nm. Adjust the power and gain of both channels/lasers according to the intensity of the fluorophore. Do not change the digital gain or offset and set the pinhole as fully open.
2. Define the acquisition setup: select a sequential channel mode and switch track every frame. Set the scan mode as frame, and the frame size as 1,024 x 1,024, with a line step of 1. Set the averaging to 2, and average by mean method and mode of unidirectional line. Set the bit depth to 8 bits.
3. Define the multidimensional acquisitions settings for the conversion of Dendra2. For conversion and bleaching use the 405 nm diode laser set at 60% energy power. If available, activate the safe bleaching GaAsP to protect the detectors.
4. Select a time series of two cycles, with a 0.0 ms interval in between, and normal start and stop. Start bleaching after scan 1 of 2 and repeat for 30 iterations. Stop bleaching when the intensity drops to 50%.
5. Define the speed of acquisition/pixel dwell as fast (e.g., maximum = 12) for conversion, and medium speed (e.g., medium = 5) for capturing a snap image.
   NOTE: The bleaching parameters defined here are guidelines. For other Dendra2 tagged POIs the laser power settings and bleaching iterations and values must be established empirically.

5. Mounting of C. elegans onto microscopy slides

NOTE: If possible, place a mounting stereomicroscope close to the confocal microscope setup and mount the nematodes just before imaging.

1. On the glass cover slide on the opposite side of the agarose pad, draw a window with four squares with a permanent marker and number them.
2. Pipette 15 µL of levamisole in the middle of the agarose pad. The concentration of levamisole will vary according to the nematode's age: When imaging day 4 nematodes use 2 mM levamisole; when imaging day 10 nematodes use 0.5 mM levamisole.
3. Transfer four nematodes into the liquid using a wire pick. With the help of an eyelash pick, gently move each individual nematode to a window square. Swivel the eyelash so that any trace of E. coli OP50 is diluted and its fluorescent background does not interfere with signal acquisition.
4. Wait for the nematodes to almost stop moving and gently place a cover slip on top of the liquid to immobilize the nematodes in the layer of levamisole between the agarose pad and the cover slip.
5. Place the inverted slide on the confocal stage to image the nematodes.

6. Conversion of green Dendra2: Data acquisition at time zero

NOTE: The pulse-chase experiments start by irreversibly converting the Dendra2 fusion protein from a green emitting fluorophore to a red one.

1. Using the microscope's eyepiece, locate the first nematode with a 20x objective under green fluorescence. Focus on the head or tail and switch to confocal mode.
2. Start live laser scanning with the 488 nm blue laser to visualize the green Dendra2 in the EGFP green channel (ex/em = 486-553 nm). Select a single neuron and bring it into focus. Zoom in 3x and increase the target of the laser beam⁴.
   NOTE: Select one neuron per nematode. Each neuron will constitute one sample or data point.
3. Find the maximum projection plane and, according to the brightness of the fluorophore, increase or decrease the gain or laser power to obtain a saturated but not overexposed image identifiable by the color range indicator. Once this is defined, stop the scanning.
   NOTE: Do not irradiate the sample for too long or with too much power, as excitation with visible blue 488 nm light can also convert Dendra2, albeit slowly and less efficiently⁴.
4. In the software, open the tab to select the regions of interest (ROIs) and draw a first ROI around the selected neuron. Define a larger second region of interest encompassing the nematode's head and including the first ROI.
5. In the bleaching settings, select for the first ROI to be acquired, bleached, and analyzed. Select for the second ROI to be acquired and analyzed but not bleached.
6. Set the speed of scanning to maximum (i.e., fast pixel dwell) and start the experiment to convert the selected Dendra2 neurons.
   NOTE: Once the experiment is done the acquired picture will result in two images: one before and one after conversion. For the green channel, the first image should have a higher green signal that diminishes in the second image due to the conversion of the green Dendra2. For the red channel, the first image should be negative and show no signal, with a red signal appearing in the after conversion image. If the green signal does not diminish, the conversion did not occur, and the settings, such as the 405 laser power or the number of iterations, should be modified. If there is a red signal in the first image, then the 488 nm laser power used was too high and a portion of the green Dendra2 was already converted to red. In this case, a new neuron/nematode should be selected.
7. Immediately after conversion, start live scanning with the green 561 nm laser to visualize Dendra2 in the red channel (ex/em = 580-740 nm). Find the focus and respective maximum projection of the converted neuron using the range color indicator to avoid overexposure.
8. Quickly set the scan rate to a lower pixel dwell speed (e.g., 5x) and acquire a snapshot image of both channels at a higher resolution. This image is defined as timepoint zero (T0) after conversion.
   NOTE: The speed of acquisition for the converted image can be varied. However, once chosen, this speed needs to stay constant throughout the data collection.
9. Save the scan with an identifiable name and/or number, followed by the time zero label (T0).
   NOTE: It is advisable to also save the image of the conversion experiment (step 6.6) to illustrate the lack of red signal before conversion and its appearance afterwards.

7. Imaging of converted red Dendra2 for data acquisition at a selected second time point

1. To track Dendra2 degradation over time, define a second timepoint to reimage the same nematode/neuron. Select the second timepoint experimentally to address any relevant biological question. For the protocol described here, Dendra2 is imaged both at 2 h (T2) and 24 h (T24) post conversion.
   1. At the selected timepoint, find the same nematode/neuron with the use of the eyepiece and red fluorescence.
   2. Open the T0 image of the respective nematode/neuron and reload/reuse the image settings. Ensure that the acquisition settings of the snapshot are precisely the same when acquiring the T0, T2, and T24 h images.
   3. Scanning live in the red channel, bring the converted red neuron into focus. Because the red Dendra2 degrades over time the range indicator will show a less intense maximum projection. Do not change any acquisition parameters and obtain a snapshot at the same speed (e.g., 5x) as the first image.
2. To track the degradation of Dendra2 after 4 h or longer, rescue the nematodes after conversion.
   1. Remove the slide from the microscope immediately after converting and imaging the four nematodes. Gently remove the coverslip and with the use of a wire pick, lift each nematode from the agarose pad.
   2. Place each nematode individually on an appropriately labelled and identifiable NGM plate.
   3. For the second time point, mount the nematode again onto a fresh agarose pad and proceed with the imaging of the converted red Dendra2 following the instructions in section 6.

8. Image analysis of converted Dendra2

NOTE: Analysis of the degradation of Dendra2 is performed with Fiji/ImageJ software²⁰.

1. Open Fiji and drag and drop the .lsm file into the Fiji bar. Open the T0 image taken just after conversion and the image of the same nematode taken at the selected time point after conversion (T2 or T24 h).
   NOTE: To track the degradation of the protein of interest fused to Dendra2 only the red channel needs to be analyzed.
2. Establish the measurement parameters from the menu: Analyze | Set Measurements. Select the Area and Integrated Density functions.
3. Select the image obtained with the red channel. Select the Polygon Selection Tool from the Fiji bar.
4. Identify the converted neuron on the T0 image and draw an ROI around it using the selection tool.
   1. To properly identify the contours of the neuron, highlight the intensity thresholds by selecting from the bar Image | Adjust | Threshold. Drag the bar cursor to delineate the threshold and track around this area with the polygon tool. To generate an accurate ROI, it is also possible to use the contour of the selected neuron from the green channel.
5. Once the selection has been made in the red channel window, press Analyze | Measure. A pop-up window named Results will appear and include the ROI values for Area, IntDen, and RawIntDen.
6. Perform the same process of selection and measurement for the image of the second time point (T2 or T24 h).
7. Copy the obtained values into a spreadsheet software, taking care to appropriately record the values at T0 after conversion, and at T2 or T24 h after conversion.

9. Calculating the ratio of Dendra2 degradation

1. To calculate the ratio of degradation, first assign a value of 1 (or 100%) to time the degradation from time point zero (i.e., just after conversion, when all of the red Dendra2 converted is still present). This results from dividing the value of IntRawDen of T0 by itself.
2. To calculate the reduction of the red Dendra2 intensity signal over time, and the degradation, divide the value of RawIntDen of the second time point (e.g., T2 or T24 h) by the value of the RawIntDen of T0. The resulting number should be less than 1. These values can also be expressed as percentages, defining T0 as 100%.
3. Repeat section 7 for each nematode converted. For a graphical representation of the degradation of Dendra2, chart a scatter plot or bar graph with the percentage or ratio values of fluorescence decrease obtained in the y axis. Apply any desired statistical analysis and illustrate it on the graph.

Results

Two nematode strains expressing the huntingtin exon-1 protein fragment in frame with the photoconvertible protein Dendra2 were obtained via microinjection and the plasmids were kept as an extrachromosomal array. The fusion construct was expressed in the whole C. elegans nervous system from development throughout aging. Here, HTT-D2 contained either the physiological 25 polyglutamine stretch (HTTQ25-D2, Figure 1A) or a fully penetrant...

Discussion

To comprehend a protein's function it is important to understand its synthesis, location, and degradation. With the development of novel, stable, and bright FPs, visualizing and monitoring POIs has become easier and more efficient. Genetically expressed fusion PAFPs such as Dendra2 are uniquely positioned to study the stability of a POI. Upon exposure to purple-blue light, Dendra2 breaks at a precise location within a triad of conserved amino acids. The fluorophore undergoes a small structural change, resulting in a ...

Materials

Name	Company	Catalog Number	Comments
Agar-Agar Kobe I	Carl Roth GmbH + Co. KG	5210.2	NGM component
Agarose, Universal Grade	Bio & Sell GmbH	BS20.46.500	Mounting slide component
BD Bacto Peptone	BD-Bionsciences	211677	NGM component
Deckgläser-18x18mm	Carl Roth GmbH + Co. KG	0657.2	Cover slips
EC Plan-Neufluar 20x/0.50 Ph2 M27	Carl Zeiss AG		Objective
Fiji/ImageJ 1.52p	NIH		Analysis Software
Levamisole Hydrochloride	AppliChem GmbH	A4341	Anesthetic
LSM710-ConfoCor3	Carl Zeiss AG		Laser Scanning Confocal Micoscope
Mounting stereomicroscope	Leica Camera AG		Mounting microscope
neuronal-HTTQ25-Dendra2	this paper		C. elegans strain
neuronal-HTTQ97-Dendra2	this paper		C. elegans strain
OP50 Escherichia coli	CAENORHABDITIS GENETICS CENTER (CGC)	OP50	Nematode food source
Sodium Chloride	Carl Roth GmbH + Co. KG	3957.2	NGM component
Standard-Objektträger	Carl Roth GmbH + Co. KG	0656.1	Glass slides
ZEN2010 B SP1	Carl Zeiss AG		Confocal acquisition software