Materials and reagents - sources
All standard chemicals were from Sigma Aldrich, ThermoFisher Scientific or Carl Roth GmbH unless stated otherwise. All enzymes were obtained from New England Biolabs. The following reagents, bacterial strains, cell lines and resources were used and respective sources listed:
REAGENT or RESOURCE
|
SOURCE
|
IDENTIFIER
|
---|
Bacterial and Virus Strains
|
E. coli DH5α
|
ThermoFisher Scientific
|
18,258,012
|
E. coli OP50
|
CGC
|
N/A
|
E. coli SW106
|
NCI Frederick
|
N/A
|
Biological Samples
| | |
C. elegans cDNA
|
This study
| |
Chemicals
|
Forskolin
|
Sigma-Aldrich
|
CAS 66575–29-9
|
Sodium azide
|
Roth
|
CAS 26628–22-8
|
Hoechst33342
|
Sigma-Aldrich
|
CAS 23491–52-3
|
DMSO
|
Sigma-Aldrich
|
CAS 67–68-5
|
Lipofectamine 2000
|
ThermoFisher
|
#11668019
|
Trypsin
|
ThermoFisher
|
#25300054
|
HBSS
|
ThermoFisher
|
#15356878
|
HEPES
|
ThermoFisher
|
#15630080
|
MetafectenePro
|
Biontex
|
#T040
|
Coelenterazine H
|
Prolume/Nanolight
|
CAS 50909–86-9
|
LysoTracker Blue DND-22
|
ThermoFisher
|
#L7525
|
Peptides
| | |
Peptides
|
This paper
|
N/A
|
Critical Commercial Assays
| | |
pGL4.29[luc2P/Hygro/CRE] reporter gene plasmid
|
Promega
|
#E8471
|
ONE-Glo Luciferase Assay System
|
Promega
|
#E6120
|
Nano-Glo® Live Cell Assay System
|
Promega
|
#N2011
|
AlphaScreen cAMP Detection Kit
|
PerkinElmer
|
6760635D
|
Experimental Models: Cell Lines
| | |
HEK293
|
German Collection of Microorganisms and Cell Cultures (DSMZ)
|
ACC 305
|
COS-7
|
German Collection of Microorganisms and Cell Cultures (DSMZ)
|
ACC 60
|
Experimental Models: Organisms/Strains
| | |
Caenorhabditis elegans strains, see Table S3
|
This paper
|
N/A
|
Oligonucleotides
| | |
Primers, see Table S2
|
SeqLab
|
N/A
|
Scientific instruments
| | |
Plate reader
|
PerkinElmer/ Tecan
| |
Micro injector
|
Eppendorf
| |
DMi8 confocal microscope
|
Leica
| |
Axiovert Observer Z1 microscope
|
Zeiss
| |
Peptide synthesis
Peptides were synthesized by solid-phase peptide synthesis (SPPS) using a Syro II peptide synthesizer (MultiSynTech; resins and amino acid from Iris Biotech) in 15 μmol scale, following the 9-fluorenylmethoxycarbonyl/tert-butyl (Fmoc/tBu) strategy (reviewed in [59]). Briefly, the peptide sequence is built up from C to N terminus on an immobilized solid-phase as a growing peptide chain by repeated steps of coupling N-terminally Fmoc protected amino acid derivatives, and deprotection of the N-terminal Fmoc group to enable the coupling of the next amino acid. Control peptides were generated by scrambling of the peptide sequence and synthesized in parallel. Peptides were cleaved from the resin by incubation with trifluoroacetic acid (TFA)/H2O/triisopropylsilane (90/5/5, v/v/v), which simultaneously removes the side chain protection groups from reactive side chains. All peptides were purified to ≥95% homogeneity by preparative HPLC (Shimadzu) using a Phenomenex Aeris, 100 Å (C18) column and linear gradients of solvent B (acetonitrile+ 0.08% trifluoroacetic acid) in A (H2O + 0.1% trifluoroacetic acid). The identity of the peptides was confirmed by MALDI-ToF mass spectrometry (Ultraflex III MALDI ToF/ToF, Bruker, Billerica, USA).
To generate the amidated C terminus of FLP-34-1, a TentaGel R RAM resin (Iris Biotech) was used as solid phase. For a fluorescent variant of FLP-34-1, 5(6)-carboxytetramethylrhodamine (TAM) was coupled to the free N-terminus using 2 eq each of the fluorescent dye, diisopropylethylamine (DIEA), and 1.9 eq. 1-[Bis (dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b] pyridinium 3-oxide hexafluorophosphate (HATU) in dimethylformamide (DMF) at room temperature overnight in the dark. Peptides were then further cleaved from the resin and purified as described above.
LAT-1 peptides (wild-type and scrambled sequence) were fluorescently labeled at the C terminus. For that purpose, 15 μmol TentaGel S HMP resin (Iris Biotech) was loaded with Nα-protected 2,3-diaminoproprionic acid with an orthogonally protected side chain (Fmoc-Dap (Dde)-OH). After automated elongation of the peptide sequence up to the N terminus, Dap (Dde) was selectively deprotected on resin by repeated addition of 2% hydrazine (v/v) in DMF (10 × 10 min), and coupled to a TAM-fluorophore as described above. For non-fluorescent peptide variants, the liberated Dap side chain was acetylated on resin using 10 eq of acetic anhydride (Ac2O) and DIEA in dichloromethane (DCM) at room temperature for 15 min. Peptides were further cleaved from the resin and purified as described above.
C. elegans lysis
10 adult wild-type hermaphrodites were transferred into lysis buffer (1x PCR buffer, 2 μg/ml proteinase K), once freeze cracked at − 80 °C for 5 min and lysed at 60 °C for 45 min. Proteinase K was inactivated at 90 °C for 15 min and the mixture was used as DNA template for PCR reactions.
Generation of constructs
NPR-11 constructs fused to a nanoluciferase
Npr-11p::Nluc::npr-11 and npr-11p::Nluc::npr-11::gfp
For expressing a variant of the nanoluciferase (Nluc, Promega, USA), fused to the N terminus of npr-11, 3 kb upstream of npr-11 was amplified from fosmid WRM0616cB05 with primer pnpr-11_SbfI_f and pnpr-11_XbaI_r attaching SbfI and XbaI restriction sites. The fragment was cloned into two modified pPD95.79 (from A. Fire, Addgene plasmid #1496) expression vectors, one with and one without a GFP, using SbfI and XbaI. npr-11 cDNA was purchased from GenScript and amplified with npr-11_nluc_f and npr-11_XmaI_r/ npr-11_XmaI_GFP_r (for sequences see Table S2) generating an overlap to the Nluc at the 5′ end and an XmaI restriction site at the 3′ end. The Nluc with an additional SGGGGS linker at the 3′ end was amplified from pNL1.3_secNluc plasmid (Promega, USA) with primers Nluc_XbaI_f and Nluc_NPR-11_r generating an XbaI restriction site at 5′ and an overlap to npr-11 at the 3′ end. The Nluc and npr-11 fragments were fused together by overlap PCR and inserted into the modified pPD95.79 with pnpr-11 using XbaI and XmaI restriction sides (pSP167 with GFP, pSP168 without GFP). Primer sequences are shown in Table S2.
Npr-11p::npr-11::Nluc
For studying intracellular BRET, the Nanoluciferase (Nluc,Promega, USA) was fused to the C terminus of npr-11. cDNA of npr-11 was amplified with XbaI_npr-11_f/npr-11_Nluc_r from pSP168 inserting an XbaI restriction site at the 5′ end and parts of a SGGGGS linker at the 3′ end. The Nluc was amplified from pNL1.3_secNluc plasmid (Promega, USA) using Nluc_Linker_npr-11_f/Nluc_XmaI_r to add a 5′ SGGGGS Linker and an XmaI restriction site at the 3′ end. Both fragments were fused together via overlap PCR and inserted into a modified pPD95.79 containing 3 kb upstream of npr-11 (generated as described above) using XbaI and XmaI restriction sites resulting in pSP185. Primer sequences are shown in Table S2.
Npr-11 constructs for in vitro analysis
For cAMP reporter gene assays, a plasmid was used containing the cDNA of npr-11 fused C-terminally to the enhanced yellow fluorescent protein (eYFP) in the pVitro2-hygro-mcs vector (InvivoGen) generated previously [23]. An npr-11 fused to eCFP used for imaging was generated from this construct by PCR-overlap extension, using the primers pVitro_prolong_for and NPR11-Linker_rev to amplify the NPR-11 part, while eCFP was amplified from Y1-eCFP_N1 [60] using the primers Linker-eCFP_for and N1_rev. The genetic fusion npr-11:eCFP was then ligated into the pVitro2 vector using the restriction enzymes EcoRV and XbaI (all enyzmes from ThermoFisher). The cDNA of the membrane marker CAAX::mNG was cloned from the expression plasmid containing synthetic introns (see below) using PCR overlap extension with the primers HindIII_Kozak_mNG_part1_for, mNG_part1_rev, mNG_part2_for, mNG_part2_rev, mNG_part3_for, mNG_part3_rev, mNG_part4-Li-CAAX_for, mNG_part4-CAAX_NotI_XhoI_XbaI-rev, and sub-cloned into an empty pcDNA3 vector using HindIII and XbaI.
For NanoBRET binding assays, the nanoluciferase (Nluc, Promega, USA) was genetically fused to the N terminus of npr-11, spaced by a SGGGGS linker as previously described [35]. To facilitate expression and targeting to the plasma membrane, the Nluc sequence was preceded by a secretion signal derived from human IL-6 as previously described (secNluc [5];). The Nluc sequence was amplified from the pNL1.3_secNluc plasmid (Promega, USA).
Rpl-28p::mNeonGreen::CAAX
For a stable mNeonGreen (mNG) localization in all somatic cells on the inner plasma membrane, mNG was fused to the CAAX motif of C. elegans let-60 separated by a DNA linker. The linker (amino acid sequence: GSAGTMASNNTASG) and the CAAX sequence (amino acid sequence: KPQKKKKCQIM*) were added to mNG amplified from vector pDD346 (from D. Dickinson, Addgene plasmid #133311) by three subsequent overlap PCR with following primers: mNG_XmaI_f (1. - 3.), mNG_CAAX_1_r (1.), mNG_CAAX_2_r (2.) and mNG_CAAX_3_EcoRI_r (3.) attaching unique restriction sides for XmaI and EcoRI. The resulting construct was cloned upstream of a 1500 bp sequence 5′ of rpl-28 amplified from pGC185 [61] with primers rpl-28_SbfI_f and rpl-28_XmaI_r into pPD95.79 with SbfI and XmaI yielding plasmid pSP177. For primer sequences see Table S2.
Lat-1p::lat-1(1–249)::Nluc::lat-1(250–650)::GFP::lat-1(651–1015)
The Nluc was inserted into LAT-1 between the hormone-binding and the GPCR autoproteolysis-inducing domain (GAIN) after amino acid position 249 into vector pSP5 [30], which contains the genomic sequence of lat-1 with the 7 kb promoter sequence, using recombineering [62]. A recombineering targeting cassette consisting of three parts – a kanamycin resistance gene, the first seven amino acids of the lat-1 exon 5 and the Nluc – was generated. For this purpose, five different fragments were amplified and fused together using an overlap PCR resulting in construct lat-1(1)::kanR::lat-1(2)::Nluc::lat-1(3). Primer pairs were the following: lat-1_1_f and lat-1_1_r for lat-1(1), kanR_f and kanR_f for the kanamycin resistance cassette, lat-1_2_f and lat-1_2_r for lat-1(2), lat-1_Nluc_f and lat-1_Nluc_r for the Nluc, and lat-1_3_f and lat-1_3_r for lat-1(3). Primer sequences are listed in Table S2. pSP5 was transformed together with the respective fragment into electro-competent E. coli SW106 cells expressing λ Red genes that promote homologous recombination [62]. Positive cells were selected via kanamycin resistance yielding plasmid pSP181.
C. elegans strains
C. elegans strains were maintained as described in [63]. All strains used in this study are listed in Table S3. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).
Generation of transgenic C. elegans strains
All transgenic strains expressed a stable extrachromosomal array. Those strains were obtained by microinjection of plasmid DNA into the syncytial gonad of young adult hermaphrodites according to [64, 65]. The injection mix contained the DNA of interest (10 ng/μl for plasmids encoding npr-11; 1 ng/μl for plasmids encoding lat-1), a plasmid carrying a selection marker (pRF4 (rol-6 (su1006)): 100 ng/μl [65]; IR98 (hygromycin resistance): 30 ng/μl [66]) and was filled up with pBluescript II SK+ vector DNA (Stratagene) as stuffer DNA to achieve a final concentration of 120 ng/μl. After injection, the worms were left to regenerate at 15 °C for three days and positive progeny was selected. For selection for hygromycin B resistance, worms were kept on NGM plates containing 0.3 mg/ml hygromycin B. F2 individuals stably expressing the co-injection marker were established as line.
Preparation of C. elegans for luminescence detection and BRET measurements
All measurements were conducted using synchronized young adult worms (1 day post L4). Individuals were washed twice with HBSS (Hanks’ balanced salt solution with 25 mM HEPES, pH 7.4, 37 °C) and the number of worms/ μl was calculated.
For luminescence measurements of intact worms, animals (1; 10; 50; 100 or 500) were directly transferred into the wells of a white flat-bottom 384-well plate in 50 μl HBSS buffer. Worms to be cut were pre-washed with M9 + 0.1% Tween to avoid sticking of worms to the dish and subsequently transferred in 2 ml of HBSS. Incisions were made in the middle of the body with a scalpel blade and worms were immediately afterwards transferred with a glass pipette into HBSS buffer on ice. For mechanically cracking, a worm suspension was homogenized in tubes with 150 μl HBSS and six glass beads (Ø = 3 mm) using with a Precellys homogenizer (Bertin instruments, France) for 40 s at 5000 rpm. Samples were placed on ice until further usage.
Luminescence detection
50 prepared worms were transferred into a white flat bottom 384-well plate in a total volume of 50 μl HBSS + 25 mM HEPES (pH 7.4; denoted BRET buffer). Immediately after adding coelenterazine H solution (Nanolight/ Prolume, USA) to a final concentration of 4 μM, luminescence was detected using an EnVision plate reader (PerkinElmer, USA) and a NanoBRET Blue 460/80 nm filter with 1000 ms integration time (five repeats every 5 min for long time observation).
In vivo bioluminescence energy transfer (BRET) binding assay
For BRET binding assays in vivo, the nanoluciferase (Nluc) was applied as energy donor and either peptides labeled with the TAM fluorophore or the mNeonGreen fluorescent protein fused to a receptor served as acceptor.
The peptide-receptor binding BRET was conducted with TAM-FLP-34-1/TAM-pLAT-1 and their corresponding scrambled versions dissolved in H2O + 0.5% BSA and 5% DMSO. 30 Nluc-expressing worms with incisions in 50 μl BRET buffer were incubated with dissolving buffer or peptide solutions of final concentrations ranging from 0.05–10 μM in a 384-well plate. During a 25 min incubation with gentle shaking, 10 μl Nano-Glo Live Cell Reagent (N2011, Promega, USA) of a 5x stock were added according to manufacturer’s instructions after 15 min to each well. Competition-binding assays were performed similarly, but additionally, unlabeled peptide (final concentrations 0.016–10 μM) was pipetted into each well and 5 μl of a 10x stock Nano-Glo Live cell reagent was applied after 15 min.
The Ki of FLP-34-1 was calculated defining TAM-FLP-34-1 as ‘hot’ ligand with c = 1.6 μM and Kd = 1.5 μM with a one-site fit Ki with GraphPad Prism version 6 (GraphPad Software).
Luminescence (L) and fluorescence (F) were detected with a microplate plate reader (Tecan Spark) using the following filter set: donor = 430–470 nm, acceptor = 550–700 nm with 1000 ms integration time. BRET was calculated by dividing F values by L values. For each experiment, the background signal of donor-only cells was subtracted, such that the BRET ratio at 0 μM ligand equals 0.00. In Fig. 3B (left) and Fig. 3D, the mean of n = 4 (NPR-11) or n = 3 (LAT-1) independent, individually background-corrected single experiments is presented. The netBRET presented in Fig. 3B (right) is calculated by subtracting the averaged, baseline-corrected values of the scrambled control from the averaged, baseline-corrected BRET values of the active peptide. The Kd value of TAM-FLP-34-1 in vivo was calculated using GraphPad Prism 6 version (GraphPad Software) with a one-site total and nonspecific binding fit.
Luminescence levels depended on the used strain, reaching (without ligand) approximately 2000 AU in Nluc::npr-11 animals and up to 32,000 AU in Nluc::lat-1 expressing worms. For enhanced bystander BRET analyses, 30 cut worms (APR716 and APR718) in 50 μl HBSS buffer were incubated for 10 min with 10 μl of Nano-Glo Live Cell Reagent (5x stock). Luminescence and fluorescence were measured once prior to peptide stimulation. Afterwards, FLP-34-1 (final concentration 5 μM) or H2O with 0.5% BSA and 2.5% DMSO as control were added to each well and luminescence and fluorescence was measured over the course of 80 min. The acceptor filter for this setting was altered to 505–605 nm, while the donor filter remained at 430–470 nm, as before.
In vitro bioluminescence energy transfer (BRET) binding assay
The binding of TAM-FLP-34-1 and its scrambled analog was also tested in vitro using membranes of transfected HEK293 cells as described previously [35]. Briefly, membranes of HEK293 cells transiently transfected with Nluc::NPR-11::eYFP were prepared analogously to a described protocol [67, 68]. TAM-labeled peptide in a concentration range of 10− 12 M to 10− 5 M was incubated with the membranes containing 0.5 μg total protein in 90 μl BRET buffer containing 0.1% bovine serum albumin and Pefabloc SC in solid black 96 well plates for 10 min under gentle agitation at room temperature. Directly before the measurements, coelenterazine H in BRET buffer (10x stock solution) was added to a final concentration of 4 μM, and BRET was measured in a microplate reader (Tecan Spark) with the following filter settings: luminescence (L) 430–470 nm, fluorescence (F) 550–700 nm. The BRET ratio was calculated by the ratio of F/L. The Kd values were obtained from a three-parameter logistic fit in GraphPad Prism version 5.03 (GraphPad Software).
Cell culture
All in vitro experiments were carried out using either HEK293 cells (Homo sapiens, embryonic kidney, DSMZ (German Collection of Microorganisms and Cell Cultures) ACC 305) or COS-7 cells (African green monkey, cercopithecus aethiops, kidney, DSMZ (German Collection of Microorganisms and Cell Cultures) ACC 60). HEK293 cells were kept in Dulbecco’s Modified Eagle’s Medium (DMEM) with Ham’s F-12 (v/v) and 15% (v/v) heat-inactivated fetal calf serum (FCS), while COS-7 cells were cultured in DMEM with 10% (v/v) FCS and 1% (v/v) Penicillin-Streptomycin. All cells were kept at 37 °C under a humidified atmosphere (5% CO2).
cAMP assay
NPR-11 activation was read out in the Gi/o pathway by a cAMP reporter gene assay in transiently transfected HEK293 cells as described previously [23]. Briefly, HEK293 cells were grown to 70% confluency and transiently co-transfected with the receptor plasmid (2 μg) and the reporter gene plasmid pGL4.29 [luc2P/CRE/Hygro] (Promega; 2 μg) with MetafectenePro (Biontex). Cells were then re-seeded into 384-well plates. On the next day, the medium was removed, and the cells were stimulated with peptide solution (20 μl) containing 5 μM forskolin (stimulating intracellular cAMP levels) in DMEM and incubated for 4 h. Luciferase substrate OneGlo in lysis buffer (Promega) was added and incubated for 5 min. Luminescence was measured in a microplate reader (Tecan Spark). Data analysis was performed with GraphPad Prism version 5.03 (GraphPad Software) and is presented as x-fold of forskolin.
Gs-coupling of LAT-1, quantified by detection of intracellular cAMP accumulation, was measured using an AlphaScreen cAMP detection kit (PerkinElmer, USA). Briefly, COS-7 cells (15,000 cells/ well) were transfected in 96-well plates with either 200 ng of LAT-1-encoding plasmid or empty vector using Lipofectamine2000 (ThermoFisher Scientific). 48 h post transfection, the cells were stimulated with 100 μM peptide solution in HBSS containing 1 mM 3-isobutyl-1-methylxanthine (IBMX) and 1% DMSO and control buffer without peptide for 30 min at 37 °C. Subsequently, the medium was displaced with lysis buffer (5 mM HEPES, 0.3% Tween-20, 0.1% BSA, 1 mM IBMX, pH 7.6) and plates were frozen at − 80 °C until further use. The following steps were conducted as stated in the manufactures’ protocol of the AlphaScreen cAMP detection kit (PerkinElmer, USA) and an EnVision plate reader (PerkinElmer, USA) was used to detect the fluorescence. The data were analyzed with GraphPad Prism version 6 (GraphPad Software) are presented in x-fold over the corresponding empty vector control.
Fluorescence microscopy and image processing
To determine the distribution of TAM-labeled peptides in worms in vivo, confocal fluorescence microscopy was performed. Wild-type worms were anesthetized with 30 mM NaN3 and either intact or scratched incubated with 5 μM TAM-FLP-34-1/ TAM-pLAT-1 and their corresponding scrambled versions in H2O + 0.5% BSA for 10 min. After having been washed once in H2O + 0.5% BSA, worms were mounted on a microscopy slide containing a 2% agar pad and a drop of H2O + 0.5% BSA. Images were recorded as stacks with spatial spacing of 2 μm. Fluorescence was tracked with a Leica DMi 8 microscope (model TL LED, 20x/ 0.75 immersions oil objective) and a DPSS 61 Laser (excitation = 561 nm, emission = 566–700 nm) at room temperature. Images were recorded with a Leica DFC9000 GTC camera and corresponding software (Leica Application Suite X). Contrast, brightness and stack overlay were processed using Fiji [69].
Distribution of the CAAX::mNG fusion protein was determined in intact nematodes expressing CAAX::mNG with an argon laser on 488 nm excitation and 493–550 nm emission with the same technique as described above. The same settings were used to image Nluc::NPR-11::GFP and Nluc::LAT-1::GFP.
Localization of NPR-11::eCFP and CAAX::mNG before and after TAM-FLP-34-1 stimulation was determined in HEK293 cells. The cells were grown on 8-well μ-slides (Ibiditreat) to a confluency of 70–80% and transfected with 1 μg vector DNA (4:1 npr11::eCFP over CAAX::mNG) using Lipofectamine2000 (ThermoFisher Scientific) following the manufacturer’s protocol. On the next day, medium was changed to OptiMEM (Invitrogen Life Technologies) for imaging.
Potential co-localization with recycling endosomes was assessed from co-expression of rab11-eCFP (kind gift from R. Schülein, Leibniz-Institute of Molecular Pharmacology, Berlin, Germany), by transfecting plasmids encoding NPR-11::eYFP and rab11::eCFP in a 9:1 ratio (total 1 μg vector DNA) using Lipofectamine2000.
Potential co-localization with lysosomes was investigated in cells expressing only NPR-11:eYFP (1 μg vector DNA transfected with Lipofectamine2000 as described above), and lysosomes were stained for 30 min with Lysotracker blue, (Invitrogen) before peptide stimulation.
Cells were examined before, 30 min and 60 min after peptide stimulation (100 nM TAM-FLP-34-1 in OptiMEM), as well as 30 min and 60 min after agonist wash-out (3 × 5 times with warm OptiMEM). Images were acquired at 37 °C using an Axiovert Observer Z1 microscope (with Apotome, Plan-Apochromat 63x/1.40 Oil DIC objective, filter 47 (436(20)/480(40) for eCFP with acquisition time 1000 ms; filter 46 (500(20)/535(30)) for eYFP and mNG with acquisition time 2000 ms; and filter 31 (565(30)/620(60) for TAM with acquisition time 400 ms; Carl Zeiss). Acquisition time was identical in all experiments and all pictures were processed in the same way.
Processed images were stacked in ImageJ [70] and analyzed as follows: Membrane fluorescence in Fig. 5A was measured from 5 cross-sections per cell, expressing both NPR-11::eCFP and CAAX::mNG, using the plugin “Stack -> measure Stack”, which basically reslices the stack and outputs the maximum fluorescence in each channel. Quantification represents the mean of the maximum membrane fluorescence from three independent experiments with each N > 10 cells, and five cross sections per cell. Linescans in Fig. 6 were generated from a selected line in the image stack using “Stack -> Reslice” and the fluorescence intensity was then normalized for each channel.