Description

​Induced protein dimerization is an invaluable tool in cellular biology to study protein function. Induced dimerization has been widely used to modulate enzymatic activity as well as expression and localization of proteins (Voß et al., 2015; DeRose et al., 2013). A popular method employs the chemical dimerizer rapamycin to induce binding between the FRB domain of the mTOR kinase and the FKBP12 protein (FK506 binding protein 12 kDa) (Putyrski and Schultz, 2012). Rapamycin-induced protein dimerization has been extensively used in cell culture systems and yeast to control RhoA GTPases signaling (Inoue et al., 2005), protein stability (Janse et al., 2004) or phosphoinosite composition of the plasma membrane (Ueno et al., 2011). The small nematode C. elegans is a popular model system for cell biology, however to our knowledge, rapamycin-induced protein dimerization has not been successfully used in this organism. To establish the rapamycin-induced protein dimerization technique in C. elegans we codon-optimized the human FRB and FKBP12 domains and introduced one intron to ensure high expression of the transgenes. The FRB domain was fused to GFP and the plekstrin homology domain (PH) (Audhya et al., 2005) at the C-terminus to anchor it at the plasma membrane and the FKBP12 domain was fused to mCherry (Figure 1A, B). Both transgenes are controlled by the mex-5 promoter and tbb-2 3’UTR to ensure high and ubiquitous expression of the fusion proteins. After we generated single-copy integrations using MosSCI (Frøkjær-Jensen et al., 2008) both strains were crossed together. As expected FRB::GFP::PH localizes to the plasma membrane in the germ line and early embryos and mCherry::FKBP12 is present in the cytoplasm and the nucleus (Figure 1C, D). To induce binding of the FRB and FKBP12 domains and thereby translocation of the mCherry::FKBP12 to the plasma membrane, we injected 1 mM rapamycin into the pachytene region of the germ line. Upon rapamycin injection, mCherry::FKBP12 translocated from the cytoplasm to the plasma membrane in all germ lines (Figure 1C) and early embryos analyzed (Figure 1D). In control worms injected with DMSO, translocation of mCherry::FKBP12 to the plasma membrane was not observed (Figure 1C, D). As expected in rapamycin-injected worms expressing only the mCherry::FKBP12 transgene, no translocation of mCherry::FKBP12 to the plasma membrane was visible (n = 7 gonads). Importantly after rapamycin injection we did not observe alterations in gonad morphology (n = 29 gonads) and early embryonic divisions (n = 17 embryos) or embryonic lethality (+DMSO 281/0; +rapamycin 302/0; number of viable/dead progeny) validating applicability of our system for future cell biological studies. mCherry::FKBP12 localizes to the nucleus and the cytoplasm and therefore dimerization can be induced in both compartments. In case dimerization will be used to selectively target proteins to the nucleus or cytoplasm additional modifications of the presented system will be required. In summary, we establish a rapamycin-inducible dimerization system in C. elegans and demonstrate that it can be used to target a protein of interest to a specific subcellular region in the germ line and in early embryos. Our system can be directly used to target any protein to the plasma membrane of germ cells or early embryos by fusing it to the FKBP12 domain. Moreover, it can be easily modified to target proteins to different subcellular locations and to control protein activity or stability.

Table 1: Used C. elegans strains