We acknowledge the DFG (KI-1988/5-1 to JK, NeuroCure PhD fellowship by the NeuroCure Cluster of Excellence to MLP) for funding. We also acknowledge the Imaging Core Facility of the Leibniz Research Institute for Molecular Pharmacology Berlin (FMP) for providing the imaging set up. In addition, we would like to thank Diogo Feleciano who established the Dendra2 system in the lab and provided instructions.

1. Generation of&#160;&#160;C. elegans expressing neuronal Huntingtin-Dendra2 fusion protein
<ol>
	<li>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.</li>...

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The authors have nothing to disclose.

To comprehend a protein&#39;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 ...

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&#39;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&#39;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-fold5,6. Huntingtin is the protein responsible for causing Huntington&#39;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&#160;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&#39;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.

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			<td>Carl Roth GmbH + Co. KG</td>
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			<td>Agarose, Universal Grade</td>
			<td>Bio &#38; Sell GmbH</td>
			<td>BS20.46.500</td>
			<td>Mounting slide component</td>
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			<td>BD Bacto Peptone</td>
			<td>BD-Bionsciences</td>
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			<td>NGM component</td>
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			<td>Deckgl&#228;ser-18x18mm</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>0657.2</td>
			<td>Cover slips</td>
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			<td>EC Plan-Neufluar 20x/0.50 Ph2 M27</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td>Objective</td>
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			<td>Fiji/ImageJ 1.52p</td>
			<td>NIH</td>
			<td></td>
			<td>Analysis Software</td>
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			<td>Levamisole Hydrochloride</td>
			<td>AppliChem GmbH</td>
			<td>A4341</td>
			<td>Anesthetic</td>
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			<td>LSM710-ConfoCor3</td>
			<td>Carl Zeiss AG</td>
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			<td>Laser Scanning Confocal Micoscope</td>
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			<td>Mounting stereomicroscope</td>
			<td>Leica Camera AG</td>
			<td></td>
			<td>Mounting microscope</td>
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			<td>neuronal-HTTQ25-Dendra2</td>
			<td>this paper</td>
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			<td>C. elegans strain</td>
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			<td>neuronal-HTTQ97-Dendra2</td>
			<td>this paper</td>
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			<td>C. elegans strain</td>
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			<td>OP50 Escherichia coli</td>
			<td>CAENORHABDITIS GENETICS CENTER (CGC)</td>
			<td>OP50</td>
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			<td>Sodium Chloride</td>
			<td>Carl Roth GmbH + Co. KG</td>
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			<td>Standard-Objekttr&#228;ger</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>0656.1</td>
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			<td>ZEN2010 B SP1</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td>Confocal acquisition software</td>
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in vivo quantification of protein turnover in aging c. elegans using photoconvertible dendra2

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.

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...

Watch this Scientific Journal Video about In Vivo Quantification of Protein Turnover in Aging C. Elegans using Photoconvertible Dendra2 at JoVE.com

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

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

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

Proteins are synthesized and degraded constantly within a cell to maintain homeostasis. Being able to monitor the degradation of a protein of interest is ...

This protocol allows for tracking and quantification of the degradation of a protein of interest in live and aging C.elegans. This in vivo and noninvasive technique requires only little microscopy set up to reliably convert Dendra2 into a bright and stable fluorophore. The method is adaptable to mammalian systems, as well as zebrafish.To study, put in turnover within the proteostatis network as well as transport and trafficking. Take care not to provoke unwanted conversion of Dendra2 while scanning with the blue light laser, and to test and optimize the acquisition settings and conversion settings for each system and fusion protein. Begin by growing age-matched nematodes at 20 degrees Celsius to the desired age for the experiment.On the day of the imaging experiment, melt general grade agarose at a 3%concentration in double distilled water. While the agarose is cooling, cut the tip of a one milliliter pipette tip to facilitate aspiration of approximately 400 microliters of the melted agarose. Carefully add a few drops of agarose onto a clean glass slide and immediately place another slide on top of the drops to create a thin pad of agarose between the two glass pieces.When the agarose has dried, carefully remove the top slide. When enough slides have been prepared, open the confocal microscope software and set the light path for excitation and emission. For green Dendra2 add 486 to 553 nanometers, and for red Dendra2 add 580 to 740 nanometers.Adjust the power and gain of both of the channels according to the intensity of the fluorophore and set the pinhole to fully open. Select a sequential channel mode and set the track to switch every frame. Set the scan mode as frame and the frame size as 1024 by 1024 with a line step of one.Set the averaging to two and to average by the mean method and mode of unidirectional line. Set the bit depth to eight bits. Define the multidimensional acquisition setting for the conversion of Dendra2.For conversion and bleaching, use the 405 nanometer diode laser set to 60%energy power. Select a time series of two cycles with a 0.0 millisecond interval between the cycles and a normal start and stop. Set the bleaching to start after scan one of two, to repeat for 30 iterations and to stop when the intensity drops to 50%Then, define the speed of the acquisition pixel dwell to fast for conversion and to medium for capturing a snap image.To mount the nematodes onto the slides, use a permanent marker to draw and number a window with four squares on the opposite of the agarose pad on each slide. Add 15 microliters of levamisole to the middle of the agarose pad and use a stereo microscope and a wire pick to transfer for age-matched nematodes into the droplet. Use an eyelash pick to gently move each nematode into one window of the square.When all of the nematodes have been repositioned, wait until the worms have stopped moving before placing a cover slip over the liquid to immobilize the nematodes during imaging. Then, place the slide onto the confocal microscope stage. For green Dendra2 conversion, use the eyepiece of the 20 X objective under green fluorescence to locate the first nematode and focus on the head or tail.Switch to confocal mode and increase or decrease the gain and laser power to obtain a saturated, but not overexposed image. Draw a region of interest around the selected neuron and draw a second larger region of interest around the tail that includes the first region of interest. Set the first region to be acquired, bleached, and analyzed.Set the second region of interest to be acquired and analyzed, but not bleached. Set the speed of scanning to maximum and start the scan to convert the selected Dendra2 neurons. Immediately after conversion, begin live scanning with the green 561 nanometer laser to visualize Dendra2 in the red channel and find the focus and respective maximum projection of the converted neuron.Quickly set the scan rate to a lower pixel dwell speed and acquire a snapshot image of both channels at a higher resolution. This image is considered at time zero after conversion. Then, save the scan with an identifiable name and or number, followed by the time zero label.To track Dendra2 degradation over time, open the times zero image of the respective nematode neuron. Using the same image setting, begin scanning live in the red channel and bring the converted red neuron into focus. Then, without changing any acquisition parameters, obtain a snapshot at the same speed as the first image.Be sure to define your conversion setting carefully to convert Dendra2 efficiently and sufficiently and to maintain the same acquisition parameters throughout different time points. To analyze the converted Dendra2 images, open the time zero and second time point images in Fiji and open Analyze and Set Measurements. Select the Area and Integrated Density functions and select the image obtained with the red channel.Using the Polygon Selection Tool, draw a region of interest around the time zero converted neuron. To properly identify the contours of the neuron, select Image, Adjust, and Threshold to highlight the intensity thresholds. Once the selection has been defined, click Analyze and Measure.A pop up results window will appear, including the area, integrated density, and raw integrated density values for the selected region of interest. Perform the same process of selection and measurement for the image from the second time point. Then, copy the values into the spreadsheet.In this representative analysis before conversion, no red signal was visible when the sample was excited in the red channel. Upon irradiation, the green signal diminished as HTT-D2 was converted and a red signal subsequently appeared. HTT-D2 expression degraded over time, resulting in a reduction in the levels of red HTT-D2 signal and a possible increase of the green HTT-D2 signal as more fusion protein was newly synthesized.Notably, the neurons of the tail region were determined to be significantly more active compared with those of the head. Further, there was significantly more degradation of control HTTQ25-D2 24 hours after conversion, then after two hours in both the head and tail neurons. This difference was not observed in pathogenic HTTQ97-D2, however, suggesting that the proteostasis network was unable to remove HTT-D2 containing longer glutamine stretches throughout the nervous system.HTTQ97-D2 was not degraded as efficiently as HTTQ25-D2 in very old nematodes, highlighting the inability of the proteostasis network to remove aggregated and possibly toxic species of Huntingtin in older animals. Importantly, tail neurons were able to cope with toxic HTTQ97-D2 in young nematodes, suggesting that various concomitant proteostasis network mechanisms might be at work to remove HTT-D2. For each sample acquire and non-overexposed and non-saturated image immediately after conversion and reuse the same setting for that sample throughout time.With this protocol, it is possible to test changes in a proteostatis network, and also, with the help of super-resolution microscopy, to track the spreading of disease associated proteins.

in-vivo-quantification-protein-turnover-aging-c-elegans-using

Research

JoVE Journal

Biology

6.0K visualizações.  Leibniz Research Institute for Molecular Pharmacology im Forschungsverbund. Este protocolo permite o rastreamento e quantificação da degradação de uma proteína de interesse em C.elegans vivos e envelhecidos. Esta técnica in vivo e não invasiva requer apenas pouca microscopia configurada para converter dendra2 de forma confiável em um fluoróforo brilhante e estável. O método é adaptável aos sistemas de mamíferos, bem como aos zebrafish.Para estudar, colocar em rotatividade dentro da rede de proteostatis, bem como transporte e tráfico. Tome cuidad...