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Rapid and tunable control of protein stability in Caenorhabditis elegans using a small molecule.

Cho U, Zimmerman SM, Chen LC, Owen E, Kim JV, Kim SK, Wandless TJ - PLoS ONE (2013)

Bottom Line: To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20-25°C.We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans.These data reinforce that DD can function in virtually any organism and temperature.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Systems Biology, Stanford University, Stanford, California, USA.

ABSTRACT
Destabilizing domains are conditionally unstable protein domains that can be fused to a protein of interest resulting in degradation of the fusion protein in the absence of stabilizing ligand. These engineered protein domains enable rapid, reversible and dose-dependent control of protein expression levels in cultured cells and in vivo. To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20-25°C. This raises the possibility that our technology could be adapted for use at any temperature. We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans. These data reinforce that DD can function in virtually any organism and temperature.

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Related in: MedlinePlus

Trimethoprim rapidly stabilizes DDs expressed in C.elegans in a dose-dependent manner.(A) Representative images of transgenic worms expressing eft-3pro:YFP-DD clone #7 from a single copy MosSci insertion grown in the absence or presence of 1 mM trimethoprim (TMP) for 24 hours at 20°C. (B) Wild type (N2) and YFP-DD transgenic worms were placed on plates containing 0.008, 0.04, 0.2, or 1 mM trimethoprim or control plates (DMSO vehicle only) as synchronized L1 larvae and imaged after 24 hours at 20°C. Graph shows average fold-induction of YFP expression relative to a non-transgenic control at increasing doses of trimethoprim (n>20 worms per dose). Error bars are ± SEM. (C) Wild-type (N2) or YFP-DD transgenic worms were placed on plates containing either 1 mM trimethoprim or DMSO vehicle control and imaged after 2, 6, 12, 24, and 48 h at 20°C. The graph shows the YFP expression of trimethoprim treated worms normalized to age-matched DMSO treated worms at each time point (n>20 worms per dose). Error bars are ± SEM. (D) YFP-DD transgenic worms were placed on plates containing 1 mM trimethoprim or DMSO vehicle control as L1s and grown for 24 hours, at which point one group was maintained on trimethoprim (“+TMP”), one was moved from trimethoprim to control plates (“washout”), and one was maintained on DMSO control plates. The graph shows background subtracted YFP expression of washout worms relative to +TMP worms at each timepoint (n>20 worms per condition). Error bars are ± SEM.
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pone-0072393-g004: Trimethoprim rapidly stabilizes DDs expressed in C.elegans in a dose-dependent manner.(A) Representative images of transgenic worms expressing eft-3pro:YFP-DD clone #7 from a single copy MosSci insertion grown in the absence or presence of 1 mM trimethoprim (TMP) for 24 hours at 20°C. (B) Wild type (N2) and YFP-DD transgenic worms were placed on plates containing 0.008, 0.04, 0.2, or 1 mM trimethoprim or control plates (DMSO vehicle only) as synchronized L1 larvae and imaged after 24 hours at 20°C. Graph shows average fold-induction of YFP expression relative to a non-transgenic control at increasing doses of trimethoprim (n>20 worms per dose). Error bars are ± SEM. (C) Wild-type (N2) or YFP-DD transgenic worms were placed on plates containing either 1 mM trimethoprim or DMSO vehicle control and imaged after 2, 6, 12, 24, and 48 h at 20°C. The graph shows the YFP expression of trimethoprim treated worms normalized to age-matched DMSO treated worms at each time point (n>20 worms per dose). Error bars are ± SEM. (D) YFP-DD transgenic worms were placed on plates containing 1 mM trimethoprim or DMSO vehicle control as L1s and grown for 24 hours, at which point one group was maintained on trimethoprim (“+TMP”), one was moved from trimethoprim to control plates (“washout”), and one was maintained on DMSO control plates. The graph shows background subtracted YFP expression of washout worms relative to +TMP worms at each timepoint (n>20 worms per condition). Error bars are ± SEM.

Mentions: Encouraged, we sought to apply these new DDs in C. elegans, which is a widely used model organism that is maintained in the laboratory at 15–25°C. We chose to focus on clone #7 from the YFP-DD library, because this new DD showed the largest dynamic range between vehicle-treated and trimethoprim-treated cells of the seven mutants shown in Figure 3. We generated worms expressing YFP-DD clone #7 under the control of the ubiquitous eft-3 promoter by single copy MosSci integration into the ttTi4348 locus on chromosome I. Synchronized L1 larvae were grown on NGM plates containing 0.008, 0.04, 0.2, or 1 mM trimethoprim dissolved in DMSO or control plates (DMSO vehicle) for 24 hours at 20°C. In the absence of trimethoprim, there was no detectable YFP expression in DD-YFP worms compared to N2 non-transgenic controls, indicating complete degradation of YFP-DD. With increasing doses of trimethoprim, YFP expression levels increased, with a minimum of 30-fold dynamic range at 8 µM trimethoprim and a maximum of 45-fold at 1 mM trimethoprim (Fig. 4A−B). At the 1 mM dose, trimethoprim had no effect on larval development rate (Fig. S1).


Rapid and tunable control of protein stability in Caenorhabditis elegans using a small molecule.

Cho U, Zimmerman SM, Chen LC, Owen E, Kim JV, Kim SK, Wandless TJ - PLoS ONE (2013)

Trimethoprim rapidly stabilizes DDs expressed in C.elegans in a dose-dependent manner.(A) Representative images of transgenic worms expressing eft-3pro:YFP-DD clone #7 from a single copy MosSci insertion grown in the absence or presence of 1 mM trimethoprim (TMP) for 24 hours at 20°C. (B) Wild type (N2) and YFP-DD transgenic worms were placed on plates containing 0.008, 0.04, 0.2, or 1 mM trimethoprim or control plates (DMSO vehicle only) as synchronized L1 larvae and imaged after 24 hours at 20°C. Graph shows average fold-induction of YFP expression relative to a non-transgenic control at increasing doses of trimethoprim (n>20 worms per dose). Error bars are ± SEM. (C) Wild-type (N2) or YFP-DD transgenic worms were placed on plates containing either 1 mM trimethoprim or DMSO vehicle control and imaged after 2, 6, 12, 24, and 48 h at 20°C. The graph shows the YFP expression of trimethoprim treated worms normalized to age-matched DMSO treated worms at each time point (n>20 worms per dose). Error bars are ± SEM. (D) YFP-DD transgenic worms were placed on plates containing 1 mM trimethoprim or DMSO vehicle control as L1s and grown for 24 hours, at which point one group was maintained on trimethoprim (“+TMP”), one was moved from trimethoprim to control plates (“washout”), and one was maintained on DMSO control plates. The graph shows background subtracted YFP expression of washout worms relative to +TMP worms at each timepoint (n>20 worms per condition). Error bars are ± SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750007&req=5

pone-0072393-g004: Trimethoprim rapidly stabilizes DDs expressed in C.elegans in a dose-dependent manner.(A) Representative images of transgenic worms expressing eft-3pro:YFP-DD clone #7 from a single copy MosSci insertion grown in the absence or presence of 1 mM trimethoprim (TMP) for 24 hours at 20°C. (B) Wild type (N2) and YFP-DD transgenic worms were placed on plates containing 0.008, 0.04, 0.2, or 1 mM trimethoprim or control plates (DMSO vehicle only) as synchronized L1 larvae and imaged after 24 hours at 20°C. Graph shows average fold-induction of YFP expression relative to a non-transgenic control at increasing doses of trimethoprim (n>20 worms per dose). Error bars are ± SEM. (C) Wild-type (N2) or YFP-DD transgenic worms were placed on plates containing either 1 mM trimethoprim or DMSO vehicle control and imaged after 2, 6, 12, 24, and 48 h at 20°C. The graph shows the YFP expression of trimethoprim treated worms normalized to age-matched DMSO treated worms at each time point (n>20 worms per dose). Error bars are ± SEM. (D) YFP-DD transgenic worms were placed on plates containing 1 mM trimethoprim or DMSO vehicle control as L1s and grown for 24 hours, at which point one group was maintained on trimethoprim (“+TMP”), one was moved from trimethoprim to control plates (“washout”), and one was maintained on DMSO control plates. The graph shows background subtracted YFP expression of washout worms relative to +TMP worms at each timepoint (n>20 worms per condition). Error bars are ± SEM.
Mentions: Encouraged, we sought to apply these new DDs in C. elegans, which is a widely used model organism that is maintained in the laboratory at 15–25°C. We chose to focus on clone #7 from the YFP-DD library, because this new DD showed the largest dynamic range between vehicle-treated and trimethoprim-treated cells of the seven mutants shown in Figure 3. We generated worms expressing YFP-DD clone #7 under the control of the ubiquitous eft-3 promoter by single copy MosSci integration into the ttTi4348 locus on chromosome I. Synchronized L1 larvae were grown on NGM plates containing 0.008, 0.04, 0.2, or 1 mM trimethoprim dissolved in DMSO or control plates (DMSO vehicle) for 24 hours at 20°C. In the absence of trimethoprim, there was no detectable YFP expression in DD-YFP worms compared to N2 non-transgenic controls, indicating complete degradation of YFP-DD. With increasing doses of trimethoprim, YFP expression levels increased, with a minimum of 30-fold dynamic range at 8 µM trimethoprim and a maximum of 45-fold at 1 mM trimethoprim (Fig. 4A−B). At the 1 mM dose, trimethoprim had no effect on larval development rate (Fig. S1).

Bottom Line: To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20-25°C.We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans.These data reinforce that DD can function in virtually any organism and temperature.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Systems Biology, Stanford University, Stanford, California, USA.

ABSTRACT
Destabilizing domains are conditionally unstable protein domains that can be fused to a protein of interest resulting in degradation of the fusion protein in the absence of stabilizing ligand. These engineered protein domains enable rapid, reversible and dose-dependent control of protein expression levels in cultured cells and in vivo. To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20-25°C. This raises the possibility that our technology could be adapted for use at any temperature. We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans. These data reinforce that DD can function in virtually any organism and temperature.

Show MeSH
Related in: MedlinePlus