microPublication Biology2578-9430Caltech Library10.17912/W2BH3HNew MethodsMethodsC. elegans
Rapamycin-induced protein dimerization as a tool for
C. elegans
research​
MangalSriyash1ZielichJeffrey1LambieEric J.1ZaninEsther1§
Department of Cell and Developmental Biology, Ludwig-Maximillians-University, Munich, Planegg-Martinsried, Germany.
WehmanAnn
Correspondence to: Esther Zanin (
zanin@biologie.uni-muenchen.de
)
(A)
The mCherry-tagged FKBP12 and GFP-PH-tagged FRB transgenes under control of the
mex-5
promoter and
tbb-2
3′ UTR were integrated into chromosome II and chromosome I, respectively.
(B)
Schematic representation of rapamycin-induced heterodimerization of FRB and FKBP12 fusion proteins in the
C. elegans
gonad. The GFP-tagged FRB domain is tethered to the plasma membrane by the PH domain and the mCherry-tagged FKBP12 is present in the cytoplasm. After injection of 1 mM rapamycin into the gonad, heterodimers of FRB and FKBP12 fusion proteins form and mCherry::FKBP12 translocates to the plasma membrane.
(C)
Confocal images of germ line of an adult worm expressing FRB::GFP::PH and mCherry::FKBP12 2-3 hours after injection with DMSO (
n
= 19) or 1 mM rapamycin (
n
= 29) into the gonad.
n
= number of gonads. Yellow insets highlight the plasma membrane of gonadal germ cells and magenta insets highlight the plasma membrane of oocytes. Scale bar 10 μm.
(D)
Confocal images of two-cell and four-cell embryos imaged approximately 2-3 hours after the gonads were injected with DMSO (2-cell embryo
n
= 10; 4-cell embryo
n =
9) or 1 mM rapamycin (2-cell embryo
n
= 8; 4-cell embryo
n =
9). Insets highlight the plasma membrane between the blastomeres. Scale bar 5μm.
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
Strain Name
Genotype
Reference
EG6699
ttTi5605
II;
unc-119(ed3)
III; oxEx1578
Frøkjær-Jensen
et al.
, 2008
EG8078
oxTi185
I;
unc-119(ed3)
III
Frøkjær-Jensen
et al.
, 2014
ZAN87
estSi50[pEZ156;pmex-5::frb::gfp::ph::tbb2; cb-unc-119(+)]
I;
unc-119(ed3)
III
This study.
ZAN98
estSi54[pEZ159;pmex-5::mCherry::fkbp12::tbb2; cb-unc-119(+)]
II;
unc-119(ed3)
III
This study.
ZAN101
estSi50[pEZ156;pmex-5::frb::gfp::ph::tbb2; cb-unc-119(+)]
I;
estSi54[pEZ159;pmex-5::mCherry::fkbp12::tbb2; cb-unc-119(+)]
II;
unc-119(ed3)
III
This study.
Reagents
Generation of
C. elegans strains C. elegans
strains were grown at 20oC on NGM agar plates according to standard procedures (Stiernagle, 2006). Gibson cloning (E2611; NEB) was used to construct transgenes encoding FRB::GFP::PH and mCherry::FKBP12 in pCFJ350. cDNA sequences of human FRB (NM_004958) and FKBP12 (CR542168) were codon-optimized (Redemann
et al.
, 2011) for expression in
C. elegans
and introns were introduced between amino acids 25(K)-26(G) and amino acids 35(K)-36(K), respectively and DNA was synthesized by Eurofins Genomics. In the FRB domain ‘threonine’ 2098 was mutated to ‘leucine’ which allows the binding to rapamycin derivatives that do not interact with mTOR kinase (Bayle
et al.
, 2006). Single-copy insertions of FRB::GFP::PH and mCherry::FKBP12 were generated on chromosomes I and II, respectively, using the MosSCI method (Frøkjær-Jensen
et al.
, 2008; 2014). Expression of the transgenes was controlled by the
mex-5
promoter and the
tbb-2
3′ UTR (Zeiser
et al.
, 2011). Finally, the two
C. elegans
strains
frb::gfp::ph
and
mCherry::fkbp12
were crossed together to obtain expression of both transgenes in one strain.
Rapamycin injection
10 mM stock of rapamycin (Cayman Chemical, 13346) was prepared in DMSO and stored at -20oC. The two gonad arms of adult worms were injected with 1 mM rapamycin or 10% DMSO (control) diluted to their final concentration in water.
Fluorescence Microscopy
For imaging 
C. elegans
 embryos, adult worms were dissected 1.5 to 2 hours after injection in a 4-μl drop of M9 buffer on an 18 × 18-mm coverslip, and the coverslip was inverted onto a 2% agarose pad. For imaging adult 
C. elegans 
worms, a few animals were mounted on a 10% agarose pad (prepared in 0.6x M9 buffer) with 1 ul of immobilizing 0.10 micron beads (00876, Polysciences) (Kim 
et al.
, 2013). An 18 × 18-mm coverslip was placed on top and the surrounding region of the agarose pad was filled with mineral oil to prevent shrinking of the agarose pad. All images were acquired at 25°C on an eclipse Ti spinning disk confocal (Nikon), which was controlled by NIS Elements 4.51 and equipped with a 100x 1.45-NA Plan-Apo-chromat oil immersion objective, a 488-nm and 561-nm laser line, and an Andor DU-888 X-11056 camera.
Funding
E.Z. was supported by the Emmy-Noether-Program (ZA619/3-1) from the DFG, and this work was also supported by DFG grant LA3380/2 to E.J.L.
AudhyaAHyndmanFMcLeodIXMaddoxASYates JR3rdDesaiAOegemaK20051024A complex containing the Sm protein CAR-1 and the RNA helicase CGH-1 is required for embryonic cytokinesis in Caenorhabditis elegans.17120021-9525267279}10.1083/jcb.20050612416247027BayleJHGrimleyJSStankunasKGestwickiJEWandlessTJCrabtreeGR200611Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity.1311074-552199107}10.1016/j.chembiol.2005.10.01716426976DeRoseRMiyamotoTInoueT201319Manipulating signaling at will: chemically-inducible dimerization (CID) techniques resolve problems in cell biology.46530031-6768409417}10.1007/s00424-012-1208-623299847Frøkjaer-JensenCDavisMWHopkinsCENewmanBJThummelJMOlesenSPGrunnetMJorgensenEM20081026Single-copy insertion of transgenes in Caenorhabditis elegans.40111061-403613751383}10.1038/ng.24818953339Frøkjær-JensenCDavisMWSarovMTaylorJFlibotteSLaBellaMPozniakovskyAMoermanDGJorgensenEM2014316Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon.1151548-7091529534}10.1038/nmeth.288924820376InoueTHeoWDGrimleyJSWandlessTJMeyerT200561An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways.261548-7091415418}10.1038/nmeth76315908919JanseDMCrosasBFinleyDChurchGM2004323Localization to the proteasome is sufficient for degradation.279200021-92582141521420}10.1074/jbc.M40295420015039430KimESunLGabelCVFang-YenC201313Long-term imaging of Caenorhabditis elegans using nanoparticle-mediated immobilization.81e53419e5341910.1371/journal.pone.005341923301069PutyrskiMSchultzC2012511Protein translocation as a tool: The current rapamycin story.586150014-579320972105}10.1016/j.febslet.2012.04.06122584056RedemannSSchloissnigSErnstSPozniakowskyAAylooSHymanAABringmannH2011130Codon adaptation-based control of protein expression in C. elegans.831548-7091250252}10.1038/nmeth.156521278743StiernagleT2006211Maintenance of C. elegans.111}10.1895/wormbook.1.101.118050451UenoTFalkenburgerBHPohlmeyerCInoueT20111213Triggering actin comets versus membrane ruffles: distinctive effects of phosphoinositides on actin reorganization.42031945-0877ra87ra8710.1126/scisignal.200203322169478VoßSKlewerLWuYW2015929Chemically induced dimerization: reversible and spatiotemporal control of protein function in cells.281367-5931194201}10.1016/j.cbpa.2015.09.00326431673ZeiserEFrøkjær-JensenCJorgensenEAhringerJ2011526MosSCI and gateway compatible plasmid toolkit for constitutive and inducible expression of transgenes in the C. elegans germline.65e20082e2008210.1371/journal.pone.002008221637852