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Presented here is a protocol to monitor degradation of the protein huntingtin fused to the photoconvertible fluorophore 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.
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 wavelen....
1. Generation of C. elegans expressing neuronal Huntingtin-Dendra2 fusion protein
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.......
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 .......
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.
....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 |
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