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.

Representative 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 and pathogenic repeat with 97 glutamines (HTTQ97-D2, Figure 1B). Initially the fusion protein was tested to make sure it changed from its green spectrum to its red spectrum upon UV irradiation. HTT-D2 successfully switched from green to red exclusively within the illuminated region. According to the power and iterations of the UV pulse, and depending on the z-plane and penetrance of the laser beam through the cuticle of the nematode, a defined portion of HTT-D2 was converted, but not all. A red signal appeared within the photoconverted neurons, colocalizing with the green nonconverted HTT-D2 (Figure 1C,D). Due to the accuracy of the laser scanning photon beams it was possible to convert a precise ROI corresponding to a single neuron. Before conversion, no red signal was visible when the sample was excited in the red channel (Figure 2A). Upon UV irradiation, the green signal diminished as HTT-D2 was converted and a red signal finally appeared (Figure 2B). HTT-D2 was then degraded over time, resulting in a reduction in the levels of red HTT-D2 (Figure 2C) and a possible increase of the green HTT-D2 signal, as more fusion protein was newly synthesized. Because a significant decrease was already prominent at 2 h after conversion, this interval for detecting and quantifying the degradation of HTT-D2 was maintained. It is important to note that degradation is not the only process that can occur after conversion of HTT-D2. Converted HTT-D2 could be trafficked and transported along axons, resulting in a decrease in the red signal not due to clearance. However, with the settings employed here and over the short time span of 2 h, no spreading of the red signal was observed, possibly due to the fact that little HTT-D2 was moved from the soma to the axon. Furthermore, converting and imaging a single whole neuronal soma helped exclude the effects of HTT-D2 diffusion within the same neuron, because all cellular compartments were being analyzed simultaneously. To study both diffusion or transport/trafficking it is advisable to obtain fast and higher magnification images and track a smaller and possibly more motile fraction of the protein of interest. It is also important to note that within a set of experiments, each animal represented one biological repeat. Only one neuron per nematode was imaged, each animal was imaged once per session, and imaging occurred over three sessions, which constituted technical repeats. The three sessions required that the animals be synchronized on fresh plates before each experiment either on day 4 or day 10, allowing for whatever environmental variability is imposed on the nematodes. Three sessions also account for any variability arising from the imaging set up (e.g., the laser power varying due to a different temperature between experiments). All biological replicates obtained during the three sessions (a minimum of 20 animals) were considered individual samples and utilized to establish statistical significance.

After confirming that both C. elegans HTT-D2 strains were effective and establishing the optimal conversion parameters, the differences between the turnover of a disease-causing HTT-D2 protein (i.e., HTTQ97-D2) compared to its physiologically relevant control (HTTQ25-D2) were investigated. First, the degradation of HTT-D2 in different neurons was observed (Figure 3). It is known that subtypes of neurons within the nematode's nervous system vary in their metabolic activity and morphology²¹, possibly making a difference in the degradation and rebalancing of the proteome. The neurons of the tail region were compared with those of the head and found to be significantly more active (Figure 3A). This finding was only valid for HTTQ25-D2 and not for pathogenic HTTQ97-D2, suggesting that the PN was unable to remove HTT-D2 containing longer glutamine stretches throughout the nervous system (Figure 3B).

To further confirm that the model system behaved as expected, conversion experiments were performed over a longer period, allowing the cells' degradation pathways to eliminate the protein of interest almost completely (Figure 4). Indeed, there was significantly more degradation of control HTTQ25-D2 24 h after conversion than after 2 h in the head neurons, and extensively more in the tail neurons (Figure 4A). Again, the posterior tail neurons more actively removed red HTT-D2, even over an increased timespan. A similar trend was detected for the disease-causing HTTQ97-D2, with a very slight reduction of the red HTT-D2 signal over 24 h. However, HTTQ97-D2 was not removed as readily as HTTQ25-D2, especially after 24 h. It may be that only the soluble HTTQ97-D2 fraction is efficiently degraded, accounting for the initial diminishing of the red signal and its further mild decrease over time. Importantly, there were two populations of neurons after 24 h: one with a higher degradation rate and one where the red HTT-D2 signal was not degraded at all, or potentially even increased (Figure 4B). Increased and stable signal possibly represent already aggregated or continuously aggregating species which were substantially harder to clear from the cells due to their tightly packed amyloid nature. These inclusions, present exclusively when glutamine stretches exceeded the 40 repeat threshold, and which appeared microscopically as foci, have been hypothesized to accumulate as deposits that cannot be easily removed²². It is also possible that an increase in the red fluorescent signal was an artifact resulting from technical issues (e.g., use of wrong acquisition parameters, sub-par performance of the microscopy setup, or an erroneous recovery/mounting process). When acquiring images over longer periods of time these variables must be taken into account.

Finally, because neurodegenerative disorders such as HD manifest in adult life, the effect of aging on the rate of HTT-D2 degradation was observed. Conversion experiments were performed in the head and the tail regions of young (day 4) versus old (day 10) nematodes in both HTT-D2 strains (Figure 5). For the head neurons, there was no significant change in the rate of degradation due to aging within the lifetime of both HTTQ25-D2 and HTTQ97-D2, possibly because HTT-D2 was removed equally throughout the life of each nematode. However, a very significant change was recorded when comparing old nematodes containing either a pathological or a physiological glutamine stretch. HTTQ97-D2 was not degraded as efficiently as HTTQ25-D2, highlighting the PN's inability to remove aggregated and possibly toxic species of huntingtin in older nematodes (Figure 5A). Again, a more active and significant turnover in the tail neurons was observed. As in the head neurons, a more robust turnover of HTTQ25-D2 was not observed in the tail neurons of young nematodes compared to the older cohort, and degradation was equal at day 4 and day 10. Conversely, significant changes in degradation rates between the control and the pathogenic HTT-D2 strains appeared in young day 4 nematodes, becoming even more significant at day 10. Importantly, tail neurons were able to cope with toxic HTTQ97-D2 in young nematodes, illustrating that various concomitant PN mechanisms might be at work to remove HTT-D2 (Figure. 5B). Overall, PN activity diminished over time, possibly due to detrimental effects caused by both aging and the presence of aggregates.

Figure 1: C. elegans expressed huntingtin exon-1 fused to Dendra2 in the nervous system. (A) Young, day 4 C. elegans pan-neuronally expressing huntingtin exon-1 containing 25 glutamines, fused to Dendra2 in its unconverted green excitation/emission state. Scale bar = 100 µm. Insets show a magnified image of the head (top) and the tail (bottom) neurons. Inset scale bar = 10 µm. (B) Young, day 4 C. elegans pan-neuronally expressing huntingtin exon-1 containing 97 glutamines, fused to Dendra2 in its unconverted green excitation/emission state. Scale bar = 100 µm. Insets show a magnified image of the tail (top) and the head (bottom) neurons, and the head of a day 7 nematode (far right). Inset scale bar = 10 µm. White arrowheads point to HTTQ97-D2 foci, depicting huntingtin aggregates. (C) Channel merge image of HTTQ25-D2 with conversion of the head region. Box represents the portion of the whole C. elegans that has been UV irradiated. Scale bar = 100 µm. Inset shows the Dendra2 emission at 488 nm (top, green) and the at 561 nm (bottom, red). Scale bar = 10 µm. (D) Channel merge image of HTTQ97-D2 with conversion of the head region. Scale bar = 100 µm. Box represents the portion of the whole C. elegans that has been UV irradiated. Inset shows the Dendra2 emission at 488 nm (top, green) and the at 561 nm (bottom, red). Scale bar = 10 µm. Please click here to view a larger version of this figure.

Figure 2: Converted red HTT-D2 decreased over time in single neurons. Tail neurons of HTTQ25-D2. Two neurons are shown simultaneously and independently photoconverted. Images sequentially depict three time points: (A) Before irradiation (before), (B) immediately after irradiation (conversion), and (C) 2 h after irradiation (after). Top panel (green channel) represents HTT-D2 signal collected at 486-553 nm excitation/emission. The white region of interest delineates the neurons that have been irradiated. The middle panel (red channel) shows the converted HTT-D2 signal collected at 580-740 nm excitation/emission. The bottom panel is a merge of the two channels. Scale bar = 5 µm for all images. Please click here to view a larger version of this figure.

Figure 3: Head and tail neurons exhibited different degradation rates. Column bar graphs show the percentage of red intensity relative to the initial value at time of conversion (i.e., 100%). Converted red Dendra2 signal decreased overall over the 2 h interval as HTT-D2 was degraded within the neurons of C. elegans. Within the nematode's nervous system neuronal subtypes exhibited different degradation rates, with tail neurons showing a more active turnover, but only in nematodes carrying a nonpathogenic polyglutamine stretch. (A) Quantification of degradation in HTTQ25-D2 head versus tail neurons. Mean ± SD, unpaired two-tailed Student's t-test. N = 23-28 samples/nematodes imaged per region, *P < 0.05. (B) Quantification of degradation in HTTQ97-D2 head versus tail neurons. Mean ± SD, unpaired two-tailed Student's t-test. N = 23-28 samples/nematodes imaged per region, ns = non-significant. Please click here to view a larger version of this figure.

Figure 4: HTTQ97-D2 was not significantly cleared after 24 h. Column bar graphs show the percentage of red intensity relative to the initial value at time of conversion (i.e., 100%). Converted red Dendra2 signal decreased as HTT-D2 was degraded within the neurons of C. elegans. HTT-D2 exhibited different rates of degradation. Even over longer periods of time after conversion, pathogenic HTT-D2 could not be removed compared to its healthy control. (A) Quantification of the rate of degradation in HTTQ25-D2 at two time points after conversion (2 h and 24 h) in both head and tail neurons. Mean ± SD, one-way analysis of variance (ANOVA). N = 21-28 samples/nematodes imaged per time/region, *P < 0.05, ****P < 0.0001. (B) Quantification of the rate of degradation in HTTQ97-D2 at two time points after conversion (2 h and 24 h) in both head and tail neurons. Mean ± SD, one-way analysis of variance (ANOVA) followed by Tukey's Multiple Comparison post hoc test.  N = 21-28 samples/nematodes imaged per time/region, ns = non-significant. Please click here to view a larger version of this figure.

Figure 5: Old and young nematodes expressing the pathogenic HTT-D2 did not degrade it efficiently. Column bar graphs show the percentage of red intensity relative to the initial value at time of conversion (i.e., 100%). Converted red Dendra2 signal decreased as HTT-D2 was degraded within the neurons. As the nematode aged, its ability to degrade pathogenic HTT-D2 was additionally impaired throughout its nervous system. (A) Quantification of the rate of degradation in the head neurons of young (day 4) and old (day 10) HTTQ25-D2 nematodes compared to age-matched HTTQ97-D2 nematodes. Mean ± SD, one-way analysis of variance (ANOVA). N = 23-28 samples/nematodes imaged per strain/day, ****P < 0.0001. (B) Rate of degradation 2 h after conversion in the tail neurons of young (day 4) and old (day 10) HTTQ25-D2 nematodes, compared to age-matched HTTQ97-D2 nematodes. Mean ± SD, one-way analysis of variance (ANOVA) followed by Tukey's Multiple Comparison post hoc test. N = 23-28 samples/nematodes imaged per strain/day, *P < 0.05, ***P < 0.001, ****P < 0.0001. Please click here to view a larger version of this figure.

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 complete shift of spectra from green to red²³. This shift allows for the detection and monitoring of any POI linked to Dendra2. Indeed, these fusion constructs were first used to create a C. elegans reporter strain to study the ubiquitin-proteasome system in vivo¹⁶. Dendra2 was also employed to understand the vulnerability of selective neuronal subtypes and their ability to deal with expanded polyglutamine proteins²⁴, or monitor the induction of autophagy in models of motor neuron disease²⁵.

A protocol is presented here to monitor in vivo the degradation of huntingtin, a disease-related aggregation-prone protein in a noninvasive manner. After successfully generating a neuronal C. elegans model of HD, expressing HTT-D2 pan-neuronally, the rates of degradation of expanded and pathogenic HTT compared to its physiological counterpart were quantified. Striking differences were observed between neuronal subtypes, between young and aged nematodes, and between the capacity of the PN to deal with toxic glutamine loads over time. This technique can also be applied to follow the location and movement of huntingtin as well as its fate when perturbations to the PN are introduced. siRNA knockdown of key chaperones or administration of compounds that inhibit proteasome activity can uncover the function and importance of these components in aggregation-prone proteins: for example, whether the PN activates specific nodes to compensate for deficiencies²⁶. It can also explain the detrimental effects caused by a disease compared to those of normal aging.

Although many different questions can be addressed using this technique, once a desired model has been generated, correct conversion and detection parameters must be established to obtain reliable data. Also crucial is to determine conversion settings that allow for sufficient yield of activated protein without photobleaching or phototoxicity and without undesired conversion. Moreover, for every studied protein within a specific model system, either ex vivo or in vivo, it is necessary to experimentally establish a time period sufficiently large to allow for accurate quantification of the degradation rate.

Dendra2 offers a series of advantages over other PAFPs: 1) it is monomeric and very bright; 2) it has a high contrast photoconversion and a stable photoconverted signal; 3) it can be activated with low phototoxicity by a blue 488 nm laser, which is part of most confocal hardware setups; 4) it efficiently matures at 37 °C for application in mammalian cells; 5) it has no toxic side effects when expressed for extended periods of time^23,27; and 6) the system is not affected by variations in expression levels between or within an organism or cell, as only the ratio of the Dendra2 signal before and after conversion is quantified. All listed properties make Dendra2 an ideal fluorophore for tracking protein dynamics in real time and monitoring cell fate.

Unfortunately, Dendra2 fusion proteins suffer from some common limitations of fluorescent protein labelling. The construct is a chimeric species often experimentally overexpressed in biological systems, although endogenous expression could be established via genomic engineering. The rate of degradation of Dendra2 itself potentially influences the degradation of the target protein, though it has been described as a highly stable, long-lived protein²⁷. Furthermore, Dendra2 is not suitable to track proteins with very fast turnovers as it might not have time for its own proper maturation. Lastly, 405 nm lasers, which are uncommon, are preferred for efficient photoswitching, although they are more toxic to the sample. Indeed, less phototoxic blue light can be utilized to both visualize green Dendra2 and convert it when laser power is at high intensity. This particular feature should always be kept in mind, as prolonged exposure will produce unwanted conversion and potentially wrong measurements. Finally, it may be problematic to use Dendra2 in combination with green or red fluorophores. However, many different PAFPs are available to investigate the dynamics of several proteins simultaneously.

Experiments utilizing Dendra2 and other PAFPs have been linked to fluorescence recovery after photobleaching (FRAP) and radioactive pulse-chase labelling techniques. In a FRAP setting it is impossible to distinguish proteins re-entering an ROI from newly formed fluorescent protein, and constant monitoring and visualization of the sample is necessary. With Dendra2, two clearly distinguishable populations are generated that can be independently observed over time so the replaced and newly synthetized "inactive" form of green Dendra2 can be tracked and quantified¹⁶. Dendra2 is also a useful probe in super resolution microscopy such as total internal reflection fluorescent microscopy (TIRF)²⁸ and photoactivation localization microscopy (PALM)²⁹. In the near future such advancements will allow for better localization and potentially single molecule tracking of any POI, allowing to uncover more subtle differences within and between samples and ultimately yielding new information on the life and fate of any POI within a biological system.

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