Cloning of GTA tags
To generate the N- and C-terminal GTA tag constructs pMB41 and pMB72, we inserted TEV and Avi sequences obtained as synthetic DNA constructs from GenScript (http://www.genscript.com), and codon-optimized GFP/GalK sequences from vector pBALU1 [26] into vector pUC19 (NEB, http://www.neb.com) by Gibson assembly. Vector maps and sequences are available in Additional file 16. pMB41 and pMB72 are available through Addgene (see “Availability of data and materials” below).
Recombineering
The recombineering approach we used has been described previously [26]. Briefly, the fosmid to be engineered is transformed into E. coli strain SW105, a galK-defective strain carrying a heat shock-inducible phage λ Red recombinase, and an arabinose-inducible Flp recombinase. Bacteria carrying the fosmid are transformed with a PCR product consisting of the tag to be introduced, which also carries the wild-type galK sequence, flanked on both sides by 50 nucleotides identical to the insertion site. Expression of λ Red induces homologous recombination between the fosmid and the PCR product, and successful recombinants are selected on media with galactose as the only carbon source. Finally, galK sequences, which are flanked by FRT sites, are eliminated from the fosmid by expression of the Flp recombinase.
We used the following fosmids and primers to generate the PCR products used as templates for homologous recombination: For tagging dlg-1 we used the C-terminal tag, fosmid WRM067dB05, and primers rec_dlg-1_C-term_F (5′-actccatcatcagccgtgaatcgcagacgccaatttgggtgccacgtcatggaggaggatctggaggaggaggatctggaggagga) and rec_dlg-1_C-term_R (5′-acatatttcttgaagaaacgattatttgtctaaaaaatatccaatttcatctattcatgccattcaatcttctgagcttcg); for let-413 the C-terminal tag, fosmid WRM0640dF02, and primers rec_let-413_C-term_F (5′-ggtccccatcgccagtttcgagaacatctgtgagtaggccatgtgagtatggaggaggatctggaggaggaggatctggaggagga) and rec_let-413_C-term_R (5′-gaatgtcaaaaaaaaaacgtctaatgtctagttttcagccaaaatcggcctcattcatgccattcaatcttctgagcttcg); for lgl-1 the C-terminal tag, fosmid WRM065bB11, and primers rec_lgl-1_C-term_F (5′-gaagtacggtgaatttgaactttcgcggttggagcagtacgcacaagtcaggaggaggatctggaggaggaggatctggaggagga) and rec_lgl-1_C-term_R (5′-aaaattaatatatatcaacaggaaaacgatttttaaaaaaaatgcatctattcatgccattcaatcttctgagcttcg); for par-3 the C-terminal tag, fosmid WRM064bG02, and primers rec_par-3_C-term_F (5′-gccaataccgtcgcagagatcagggaccgcctcatcgttttccccagtacggaggaggatctggaggaggaggatctggaggagga) and rec_par-3_C-term_R (5′-gattccgtatttttcgcggctgcgtaatataactttgagaaaaaactgacctattcatgccattcaatcttctgagcttcg); and for cdc-42 the N-terminal tag, fosmid WRM0612bG08, and primers rec_cdc-42_N-term_F (5′-ctataaagacgtaattttaatacttttattcattttttttttcaggcgaaaaaaatgggacttaatgatattttcgaagctcag) and rec_cdc-42_N-term_R (5′-gttttaccgacagctccatctccaacgacgacgcacttgatcgtctgcatacctcctcctccagatcctcctcct). To check whether the recombineering procedure was successful we performed a PCR reaction on the constructs with primers rec_dlg-1_C-term_check_F (5′-aagctcaagcgcagtattcc) and rec_dlg-1_C-term_check_R (5′-tttcttgaattgagaacttggaaa) for dlg-1, primers rec_let-413_C-term_check_F (5′-cgattggtattccgattggt) and rec_let-413_C-term_check_R (5′-gccgaacagtaacggagatt) for let-413, primers rec_lgl-1_C-term_check_F (5′-gggagttatgtacaggcatctagta) and rec_lgl-1_C-term_check_R (5′-taagccagccgctagcac) for lgl-1, primers rec_par-3_C-term_check_F (5′-tatgccgcgaaggagaagta) and rec_par-3_C-term_check_R (5′-ttcgctcagcggaattatc) for par-3, and primers rec_cdc-42_N-term_check_F (5′-tcgtttattaaggcgtttaccg) and rec_cdc-42_N-term_check_R (5′-cgatcgtaatcttcctgtcc) for cdc-42.
Cloning the BirA constructs
The C. elegans codon-optimized BirA sequence was obtained as a synthetic DNA construct from GenScript (http://www.genscript.com). To generate the BirA expression constructs pMB37 (Prps-27::BirA), pMB71 (Pelt-2::BirA), and pMB73 (Pwrt-2::BirA), the BirA sequence was cloned into vector pPD158.87 (Addgene #1709) using KpnI and EcoRI restriction sites. For vector pBT331 (Prgef-1∷BirA), the BirA sequence was cloned into vector backbone pPD95.77 (Addgene plasmid #1495) using KpnI and EcoRI. Next, promoter regions were amplified from C. elegans genomic DNA and cloned into appropriate restriction sites. For pMB37, the rps-27 promoter was amplified using primers Prps-27F (5′-aaa CTGCAGttcaatcggtttttccttgcttgc) and Prps-27R (5′-aaaGGTACCattccacttgttgagcggggctg), and cloned using PstI/KpnI. For pMB71, the elt-2 promoter was amplified using primers Pelt-2F (5′-aaaCTGCAGtaatttcgaaatgtatgaactccaattc) and Pelt-2R (5′-aaaCCCGGGctataatctattttctagtttc), and cloned using PstI/SmaI. For pMB73, the wrt-2 promoter was amplified using primers Pwrt-2F (5′-aaaCTGCAGcaggtcgactccacgtaatttc) and Pwrt-2R (5′-aaaCCCGGGGATCCccgagaaacaattggcaggttg), and cloned using PstI/SmaI. For pBT331, the rgef-1 promoter was amplified using primers Prgef-1F (5′- aaaCTGCAGcgtttccgatacccccttatatc) and Prgef-1R (5′-aaaCCCGGGgatcctttactgctgatcgtcg), and cloned using PstI/SmaI. Vector maps and sequences are available in Additional file 16. The BirA expression constructs are available through Addgene (see “Availability of data and materials” below).
Cloning the control constructs
To generate expression constructs pMB43 (Prps-27::GTA), pMB76 (Pelt-2::GTA), and pMB77 (Pwrt-2::GTA), we first removed the GalK sequences from the GTA tag vector pMB71 by expression of Flp recombinase. The resulting GFP-2xTEV-Avi sequence was then amplified using primers GTA_F (5′-aaaGGTACCggtagaaaaaatgagtaaaggagaagaacttttc) and GTA_R (5′-aaaGCTAGCttattcatgccattcaatcttctgag), and cloned into vector pPD158.87 (Addgene #1709) using KpnI and NheI. Next, promoter regions were amplified from C. elegans genomic DNA and cloned into appropriate restriction sites. For pMB43, the rps-27 promoter was amplified using primers Prps-27F (5′-aaaCTGCAGttcaatcggtttttccttgcttgc) and Prps-27R (5′-aaaGGTACCattccacttgttgagcggggctg), and cloned using PstI/KpnI. For pMB76, the elt-2 promoter was amplified using primers Pelt-2F (5′-aaaCTGCAGtaatttcgaaatgtatgaactccaattc) and Pelt-2R (5′-aaaCCCGGGctataatctattttctagtttc), and cloned using PstI/SmaI. For pMB77, the wrt-2 promoter was amplified using primers Pwrt-2F (5′-aaaCTGCAGcaggtcgactccacgtaatttc) and Pwrt-2R (5′-aaaCCCGGGGATCCccgagaaacaattggcaggttg), and cloned using PstI/SmaI. For pMB43, the let-858 3′-UTR was replaced with the tbb-2 3′-UTR by PCR amplifying the tbb-2 3′-UTR with primers tbb-2U_F (5′-aaaGCTAGCatgcaagatcctttcaagc) and tbb-2U_R (5′-aaaGGGCCCtgatccacgatctggaagatttc), and cloning using NheI and ApaI restriction sites. Vector maps and sequences are available in Additional file 16.
C. elegans strains and culture conditions
Unless otherwise indicated, strains were maintained at 15 °C as previously described [73]. Transgenic strains were generated by injecting constructs into the gonad of young adult N2 animals using conventional micro-injection procedures. N2 animals were obtained from the Caenorhabditis Genetics Center. The amounts of each construct injected are indicated for each strain, and were supplemented to a final DNA concentration of 80 ng/μl with PstI digested phage λ DNA. The resulting transgenic strains carrying extrachromosomal arrays were subjected to gamma irradiation to integrate the construct into the C. elegans genome. The following strains were generated:
BAT5: barIs3 [Prgef-1::BirA, Pceh-36::mCherry] X
BOX20: mibIs7[Pwrt-2::BirA 10 ng/μl + Pmyo-2::mCherry 2.5 ng/μl]II
BOX27: mibIs14[Pelt-2::BirA 10 ng/μl + Pmyo-2::mCherry 2.5 ng/μl]I
BOX41: mibIs23[lgl-1::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 5 ng/μl]V
BOX43: mibIs25[Avi-2xTEV-GFP::cdc-42 10 ng/μl, Pmyo-3::mCherry 5 ng/μl]X
BOX51: mibIs26[par-3::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 5 ng/μl]V
BOX55: mibIs30[let-413::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 5 ng/μl]X
BOX56: mibIs31[dlg-1::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 5 ng/μl]V
BOX58: mibIs33[Prps-27::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]I
BOX60: mibIs35 [Prgef-1::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl] II
BOX61: mibIs36[Pwrt-2::GFP-2xTEV-Avi 10 ng/μl, Prab-3::mCherry 5 ng/μl]X
BOX62: mibIs37[Pelt-2::GFP-2xTEV-Avi 10 ng/μl, Prab-3::mCherry 5 ng/μl]X
BOX65: mibIs40[Prps-27::GFP-2xTEV-Avi 10 ng/μl, Prab-3::mCherry 5 ng/μl]III
BOX99: mibIs14[Pelt-2::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]I; mibIs37[Pelt-2::GFP-2xTEV-Avi 10 ng/μl, Prab-3::mCherry 5 ng/μl]X
BOX100: mibIs7[Pwrt-2::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]II; mibIs37[Pelt-2::GFP-2xTEV-Avi 10 ng/μl, Prab-3::mCherry 5 ng/μl]X
BOX101: mibIs14[Pelt-2::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]I; mibIs36[Pwrt-2::GFP-2xTEV-Avi 10 ng/μl, Prab-3::mCherry 5 ng/μl]X
BOX102: mibIs7[Pwrt-2::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]II; mibIs36[Pwrt-2::GFP-2xTEV-Avi 10 ng/μl, Prab-3::mCherry 5 ng/μl]X
BOX103: mibIs33[Prps-27::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]I; mibIs26[par-3::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 5 ng/μl]V
BOX104: mibIs7[Pwrt-2::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]II; mibIs26[par-3::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 5 ng/μl]V
BOX105: mibIs14[Pelt-2::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]I; mibIs26[par-3::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 5 ng/μl]V
BOX106: mibIs33[Prps-27::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]I; mibIs31[dlg-1::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 5 ng/μl]V; dlg-1(ok318)X
BOX107: mibIs7[Pwrt-2::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]II; mibIs31[dlg-1::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 5 ng/μl]V; dlg-1(ok318)X
BOX108: mibIs14[Pelt-2::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]I; mibIs31[dlg-1::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 5 ng/μl]V; dlg-1(ok318)X
BOX109: mibIs35 [Prgef-1::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl] II; mibIs31 [dlg-1::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 10 ng/μl] V ; dlg-1(ok318) X
BOX110: mibIs33[Prps-27::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]I; mibIs30[let-413::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 5 ng/μl]X
BOX111: mibIs7[Pwrt-2::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]II; mibIs30[let-413::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 5 ng/μl]X
BOX112: mibIs14[Pelt-2::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]I; mibIs30[let-413::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 5 ng/μl]X
BOX113: mibIs33[Prps-27::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]I; mibIs23[lgl-1::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 5 ng/μl]V; lgl-1(tm2616)X
BOX114: mibIs7[Pwrt-2::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]II; mibIs23[lgl-1::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 5 ng/μl]V; lgl-1(tm2616)X
BOX115: mibIs14[Pelt-2::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]I; mibIs23[lgl-1::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 5 ng/μl]V; lgl-1(tm2616)X
BOX116: mibIs40[Prps-27::GFP-2xTEV-Avi 10 ng/μl, Prab-3::mCherry 5 ng/μl]III; mibIs33[Prps-27::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]I
BOX117: mibIs7[Pwrt-2::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]II; mibIs40[Prps-27::GFP-2xTEV-Avi 10 ng/μl, Prab-3::mCherry 5 ng/μl]III
BOX118: mibIs14[Pelt-2::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl]I; mibIs40[Prps-27::GFP-2xTEV-Avi 10 ng/μl, Prab-3::mCherry 5 ng/μl]III
BOX119: mibIs35 [Prgef-1::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl] II; mibIs40 [Prps-27::GFP-2xTEV-Avi 10 ng/μl, Prab-3::mCherry 5 ng/μl] III
BOX133: mibIs7 [Pwrt-2::BirA 10 ng/μl, Pmyo-2::mCherry] II; mibIs25 [cdc-42::GFP-2TEV-Avi 10 ng/μl, Pmyo-3::mCherry 10ngμl] X
BOX134: mibIs33 [Prps-27::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl] I; mibIs25 [cdc-42::GFP-2TEV-Avi 10 ng/μl, Pmyo-3::mCherry 10 ng/μl] X
BOX135: mibIs14 [Pelt-2::BirA 10 ng/μl, Pmyo-2::mCherry 10 ng/μl] I; mibIs25 [cdc-42::GFP-2TEV-Avi 10 ng/μl, Pmyo-3::mCherry 10 ng/μl] X
BOX188: maph-1.1(mib12[GFP::maph-1.1]) I
BOX209: maph-1.1(mib12[GFP::maph-1.1]) I; mibIs35 [Prgef-1::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl] II; mibIs40 [Prps-27::GFP-2xTEV-Avi 10 ng/μl, Prab-3::mCherry 5 ng/μl] III
BOX212: maph-1.1(mib12[GFP::maph-1.1]) I; mibIs35 [Prgef-1::BirA 10 ng/μl, Pmyo-2::mCherry 2.5 ng/μl] II; mibIs31 [dlg-1::GFP-2xTEV-Avi 10 ng/μl, Pmyo-3::mCherry 10 ng/μl] V
SV1311: atad-3(ok3093) II; hels97[Patad-3::ATAD-3, cb unc-119] IV (referred to as ATAD-3FL)
SV1312: atad-3(ok3093) II; hels98[atad-3::ATAD-3-ETAV, cb unc-119] IV (referred to as ATAD-3ΔETAV).
Western blot analysis
Protein samples were separated on 10 % acrylamide gels, and subjected to western blotting on polyvinylidene difluoride membrane (Immobilon-P; Millipore). Blots were blocked with 5 % skim milk in phosphate-buffered saline with Tween (PBST; 7 mM Na2HPO4, 3 mM NaH2PO4, 140 mM NaCl, 5 mM KCl, 0.05 % Tween-20) for 1 h at room temperature. For detection of GFP, blots were incubated with rabbit polyclonal anti-GFP (Abcam ab6556, 1:1000) or anti-biotin (Abcam ab1227, 1:1000) in PBST + 5 % skim milk for 1 h at room temperature, washed with PBST three times for 10 min each at room temperature, incubated with anti-rabbit IgG antibody conjugated to horseradish peroxidase (Jackson Immuno Research 111035003, 1:10,000) for 45 min at room temperature, washed with PBST three times for 10 min each at room temperature, and finally washed once with PBS at room temperature for 10 min. Blots were developed using enhanced chemiluminescent western blotting substrate (Bio-Rad Laboratories).
C. elegans liquid culture
Liquid cultures were started with semi-synchronized L1 animals obtained by starvation. Depending on the growth rate of the transgenic strain to be cultured, 20–60 9 cm nematode growth media (NGM) plates with OP50 bacteria were seeded with 15–45 L4 animals per plate. After 6 or 7 days at 20 °C, no bacteria were left on the plates and the plates were covered with starved L1 animals. All animals were washed off the plates and transferred to a 2 l Erlenmeyer flask containing 500 ml S-medium supplemented with penicillin-streptomycin (5000 U/ml, Life Technologies 15070-63) diluted 1:100, and nystatin suspension (10,000 U/ml, Sigma N1638) diluted 1:1000 [74]. A pellet of OP50 E. coli bacteria obtained from a 0.5 l overnight culture in lysogeny broth (LB) was added as food source. Animals were allowed to develop until the L3/L4 stage in an incubator at 20 °C shaking at 200 rpm. To harvest the animals, the culture was transferred to 50 ml conical tubes and cooled on ice for 20 min. Animals were then pelleted by centrifugation (all centrifugation steps in this protocol were performed at 400 g for 2 min at 4 °C). After aspirating the supernatant, animals were pooled in a single 50 ml tube, and washed twice in ice-cold M9 lacking MgSO4 [74]. After the second wash step, animals were resuspended in 20 ml of ice-cold M9 lacking MgSO4, followed by the addition of 20 ml of ice-cold 60 % sucrose in H2O. After vigorous mixing of the sucrose/worm mixture, 4 ml of ice-cold M9 lacking MgSO4 was gently layered on top, and the worms were centrifuged at 400 g for 2 min at 4 °C. A layer of animals was now visible on top of the sucrose, while contaminants sedimented at the bottom. The sucrose float steps were performed as quickly as possible, as otherwise the layer of animals failed to form properly. To maximize recovery, 30 ml of supernatant was aspirated from the sucrose float, and distributed into four 50 ml tubes that were subsequently filled by addition of room-temperature M9 lacking MgSO4. The room temperature M9 allowed the animals to digest OP50 bacteria in their intestine. The four tubes were placed on ice to cool down for 30 min, after which the animals were washed twice in lysis buffer (150 mM NaCl, 20 mM Tris pH 7.8, 5 mM EDTA). During the first wash the animals were again pooled into one tube. After a final wash in lysis buffer supplemented with 1 % Triton X-100, as much lysis buffer as possible was removed, and the C. elegans pellet was frozen in liquid nitrogen and stored at −80 °C.
Lysis
C. elegans pellets were lysed by sonication using a Diagenode Bioruptor (http://www.diagenode.com) fitted with a 15 ml tube holder. To each frozen pellet was added 5 ml of lysis buffer (150 mM NaCl, 20 mM Tris pH 7.8, 5 mM EDTA) supplemented with 1 % Triton X-100, 0.5 tablet of protease inhibitor (Roche, 05892791001), and 7 μl of β-mercaptoethanol. Pellets were gently swirled until thawed. Lysates were transferred to 15 ml TPX hard plastic tubes and placed in the Bioruptor filled with ice-water. Samples were lysed nine times for 30 s, with 30 s intervals. After the third and sixth lysis period, lysates were mixed by gently swirling the tube. To remove cellular debris, lysates were distributed to 2 ml Eppendorf tubes, centrifuged at 16,000 g for 15 min at 4 °C, and collected in a fresh conical-bottom 15 ml polypropylene tube. The concentration of protein in the lysates was determined by Bradford assay (Bio-Rad Laboratories) and the lysates were diluted to 1 mg protein/1 ml lysate.
Affinity purification
Affinity purifications were performed with streptavidin-coated beads (Chromotek, HP57.1). Prior to use, beads were washed twice in lysis buffer (150 mM NaCl, 20 mM Tris pH 7.8, 5 mM EDTA) supplemented with 1 % Triton X-100 in a 1.5 ml Eppendorf tube, pelleting the beads by centrifugation at 7500 g for 30 s. After the final wash, beads were resuspended in lysis buffer, in the original volume. Next, 25 μl of beads were added to 15 ml of lysate (15 mg protein) in a 15 ml conical-bottom polypropylene tube, after which the tubes were rotated at 4 °C for 1.5 h. Following the incubation, beads were pelleted by centrifugation at 3220 g for 5 min at 4 °C, resuspended in 0.5 ml TEV buffer (20 mM Tris pH 8.0, 150 mM NaCl, and 0.3 % NP40), and transferred to an Eppendorf tube. Beads were then washed three times with 500 μl TEV buffer, pelleting the beads by centrifugation at 7500 g for 30 s at 4 °C. Finally, the beads were resuspended in 15 μl TEV buffer and 2 μl of TEV protease (Promega, V6101) was added to the samples. TEV cleavage was performed overnight at 4 °C in a shaking block for Eppendorf tubes, shaking at 800 rpm.
Mass spectrometry
Protein reduction and alkylation was performed with 10 mM dithiothreitol (56 °C for 1 h) and 50 mM 2-chloro-iodoacetamide (30 min at room temperature in the dark), respectively, after which in-gel digestion was performed with trypsin overnight at 37 °C. Peptides were extracted with 100 % acetonitrile. The samples were analyzed on an LTQ Orbitrap Elite (Thermo Scientific, Bremen) connected to a Proxeon UHPLC system (Thermo Scientific, Odense). The nanoLC was equipped with a 20 mm × 100 μm internal diameter Reprosil C18 trap column and a 400 mm × 50 μm internal diameter Poroshell C18 analytical column (Zorbax, Agilent), all packed in-house. Solvent A consisted of 0.1 M acetic acid (Merck) in deionized water (Milli-Q, Millipore), and solvent B consisted of 0.1 M acetic acid in 80 % acetonitrile (Biosolve). Trapping was performed at a flow of 5 μl/min for 10 min and the fractions were eluted using a flow rate of 150 nl/min (120 min LC method). The mass spectrometer was operated in positive ion mode and in data-dependent mode to automatically switch between MS and MS/MS. The three most intense ions in the survey scan (350–1500 m/z, resolution 60,000, AGC target 1e6) were fragmented with higher energy collisional dissociation (HCD; AGC target 6e4), with the normalized collision energy set to 32 %. The signal threshold for triggering an MS/MS event was set to 500 counts. Charge state screening was enabled, and precursors with unknown charge state or a charge state of 1 were excluded. Dynamic exclusion was enabled (exclusion size list 500, exclusion duration 40 s).
Mass spectrometry data analysis
Peak lists were generated from the raw data files using Proteome Discoverer version 1.4.1.14 (Thermo Scientific, Bremen). For each affinity purification, one peak list was generated per gel lane. Peak lists were searched against a C. elegans database (UniProt, Jan 2014, 25,863 entries) supplemented with frequently observed contaminants using Mascot software version 2.4.01 (Matrix Science, UK). Trypsin was chosen with two missed cleavages allowed. Carbamidomethylation (C) was set as a fixed modification and oxidation (M) was set as variable modification. The searches were performed using a peptide mass tolerance of 50 ppm and a product ion tolerance of 0.05 Da (HCD), followed by data filtering using percolator, resulting in 1 % false discovery rate (FDR). Only ranked 1 peptide spectrum matches with Mascot scores >20 were accepted. The spectral counts for each triplicate of controls and the four tissues were uploaded to the CRAPome online interface version 1.1 [42] for statistical validation.
Lysis and immunoprecipitations for DLG-1/ATAD-3
Strains were grown in S-medium, either containing HB101 bacteria or bacterial feeding strains targeting dlg-1, atad-3, or gfp (control) to induce RNAi. Embryo pellets were obtained by hypochlorite treatment of adult worms. Embryo pellets were ground two times for 30 s at a frequency of 1500 beats/min using a Mikro-Dismembrator (Sartorius). Ground embryo pellets were lysed in lysis buffer [20 mM Tris-HCl pH 7.8, 250 mM NaCl, 15 % glycerol, 1 % Triton X-100, 0.5 mM EDTA, 1 mM β-mercaptoethanol, 10 mM 1-naphthyl phosphate monosodium salt monohydrate, 50 mM sodium fluoride, 10 mM sodium pyrophosphate decahydrate, 100 μM sodium orthovanadate, and protease inhibitors (Roche complete, Mini, EDTA-free)] for 15 min at 4 °C. The lysate was cleared at 13,000 rpm for 15 min at 4 °C. For immunoprecipitations, 1 mg of total protein was used with either 1 μl mouse anti-PSD95 antibody (Abcam ab2723, RRID AB_303248) non-covalently bound to 5 μl protein G Sepharose beads, 1 μl rabbit anti-ATAD-3 antibody [44] non-covalently bound to 7.5 μl protein A Sepharose beads, 2 μl rabbit anti-GFP antibody (ThermoFisher A-11122, RRID AB_10073917) non-covalently bound to 7.5 μl protein A Sepharose beads (negative control), or 2 μl rabbit anti-eIF4E antibodies [75] non-covalently bound to 7.5 μl protein A Sepharose beads (negative control). Immunoprecipitations were performed for 1 h at 4 °C. Input lysates (1/25) and immunoprecipitations were loaded on gel. Standard procedures were used for SDS-PAGE and western blotting. Mouse anti-PSD95 (1:1000) and rabbit anti-ATAD-3 (1:500) were used for detection. Horseradish peroxidase-conjugated protein A (VWR International) was used at 1:5000 for ATAD-3 probed blots. The signal was revealed with chemiluminesence (Bio-Rad Laboratories). To examine protein levels in ATAD-3FL, and ATAD-3ΔETAV strains, 40 L4 staged larvae grown at indicated temperatures were collected and boiled for 5 min in 1 × Laemmli sample buffer. Samples were run on an SDS-PAGE gel, and blotted according to standard procedures. Immunoblots were probed with rabbit anti-ATAD-3 (1:500) and mouse anti-actin (1:1000) (MP Biomedicals).
Immunoprecipitations for DLG-1/MAPH-1.1
Animals were grown and lysed, and GTA-tagged proteins were purified as described above for the MS experiments. Input lysates (1/100) and immunoprecipitations were loaded on gel. Standard procedures were used for SDS-PAGE and western blotting. For detection of GFP, blots were incubated with rabbit polyclonal anti-GFP (Abcam ab6556, 1:1000) in PBST + 5 % skim milk for 1 h at room temperature, washed with PBST three times for 10 min at room temperature, incubated with anti-rabbit IgG antibody conjugated to horseradish peroxidase (Jackson Immuno Research 111035003, 1:10,000) for 45 min at room temperature, washed with PBST three times for 10 min at room temperature, and finally washed once with PBS at room temperature for 10 min.
Yeast two-hybrid assays
To generate DB::DLG-1 and DB::BAZ fusions, relative fragments were PCR amplified with primers containing EcoRI and BamHI restriction sites in their tails, and cloned into vector pGBTK7 (Clontech, Palo Alto, CA, USA). The following primers were used: PDZ1_F (5′-agGAATTCgtcttggagaaaggtcac), PDZ1_R (5′-tggtggGGATCCcggagccgatgg), PDZ2_F (5′-tccatcgGAATTCattcatccacc), PDZ2_R (5′-tcccatGGATCCgcggttgtag), PDZ3_F (5′-gactacGAATTCtctcaaatgg), PDZ3_R (5′-atGGATCCctcttgtggtctgtactg), BAZ_PDZ1-3_F (5′-tgGAATTCgagagcaagcgaaaggagccc), and BAZ_PDZ1-3_R (5′-gcGGATCCcaagatcttgcggcctaccagc).
To generate AD∷ATAD-3 aa388-595 and AD::ATAD-3 aa388-591 (ΔETAV) fusions, relative fragments were PCR amplified with primers containing BamHI and XhoI restriction sites in their tails, and cloned into vector pACT2 (Clontech). The following primers were used: ATAD-3_F (5′-tcggcGGATCCcaattcataaag), ATAD-3_R595 (5′-taaacCTCGAGttaaacagcagtttctctcttc), and ATAD-3_R591 (5′-taaacCTCGAGttatctcttcaacgta). DB and AD plasmids were co-transformed into yeast strain Y190 (Clontech) using the LiAc transformation method [76]. For X-gal assays, yeast were grown overnight at 30 °C on a nitrocellulose filter on top of a yeast extract peptone dextrose (YEPD) agar plate. A Whatman filter paper was placed in a petri dish containing 2 ml of Z-buffer (60 mM Na2HPO4, 40 M NaH2PO4, 10 M KCl, 1 M MgSO4, pH 7), 5.4 μl β-mercaptoethanol, and 33.4 μl X-Gal (stock solution 20 mg/ml in N,N-dimethyl formamide). The nitrocellulose filter with yeast was then fixed in liquid nitrogen for 10 s, thawed at room temperature, and placed on the Whatman filter paper for 30 min at 30 °C.
Generation of atad-3 transgenic strains
For MosSCI integration of atad-3 constructs on chromosome IV, we generated Patad-3::atad-3::atad-3 3′UTR and Patad-3::atad-3ΔETAV::atad-3 3′UTR constructs, and cloned these into the pCFJ1178 vector (a gift from E. Jorgensen, HHMI, University of Utah, USA). For Patad-3 we used a 1050 bp 5′ region of atad-3. As a 3′ UTR we used a 300 bp flanking region of atad-3. Patad-3::atad-3-ETAV::atad-3 3′UTR lacked 12 bp at the 3′-end of the coding region. MosSCI integration was performed as previously described [46, 77]. Briefly: the following injection mixture was injected into unc-119 animals: 10 ng/μl targeting vector, 10 ng/μl pCFJ601 (Peft-3:Mos1 transposase), 2.5 ng/μl pCFJ90 (Pmyo-2:mCherry:unc-54 UTR), and 10 ng/μl pGH8 (Prab-3:mCherry:unc-54 UTR), and wild-type non-fluorescent animals were selected from the F2 progeny.
Progeny counting and scoring of embryonic lethality
Starting at the L4 stage, individual animals were cultured at 15 °C or 25 °C and transferred to a fresh plate every 24 h. Hatched and unhatched progeny were counted 24 h after removal of the P0. Bars in Fig. 6 represent mean values, and error bars the standard deviation. The progeny of four animals was counted for each genotype and temperature.
Phylogenetic analysis and protein alignments
To identify proteins related to MAPH-1.1, MAPH-1.2, and MAPH-1.3, we performed an iterative JackHMMER search with each of the three proteins against the Reference Proteomes dataset (three iterations). To generate the phylogenetic tree, we selected MAP1 homologs from a subset of species. These sequences were aligned using the online version of MAFFT with the settings E-INS-i iterative refinement method (http://mafft.cbrc.jp/alignment/server/) [78]. From the aligned sequences, a phylogenetic tree was produced using FastTree 2 with the default settings [79]. The online Interactive Tree of Life tool was used to visualize the phylogenetic tree (http://itol.embl.de/) [80]. To calculate similarity of MAPH-1.1 to human MAP1S and Drosophila Futsch, we performed pairwise alignments with MAFFT as described above, and calculated protein similarity using the online Sequence Manipulation Suite (http://www.bioinformatics.org/sms2/ident_sim.html) [81].
Genome engineering of GFP::maph-1.1
To engineer the GFP::maph-1 locus, we used homology directed repair of a CRISPR/Cas9 induced double strand break (DSB). To increase the efficiency of DSB generation we generated a new subgenomic RNA (sgRNA) expression vector (pJJR50) that contains the A-U flipped and hairpin extended sgRNA sequence described in Chen et al. [82], under control of the R07E5.16 U6 promoter [83]. Target sequences are cloned into BbsI digested vector as pairs of annealed oligonucleotides with a 5′-TCTT overhang added to the forward oligo, and a 5′-AAAC overhang added to the reverse oligo. The maph-1.1 sgRNA target sequence was GCGTGCATCTACGTCTTGGG, and oligos used to insert the sequence into pJJR50 were 5′- tcttGCGTGCATCTACGTCTTGGG and 5′- aaacCCCAAGACGTAGATGCACGC. To generate the repair template, we inserted 450–650 bp sequences flanking the start codon of maph-1.1 into the self-excising selection cassette vector pDD282 as described [84]. Several mutations were introduced into the sgRNA target site to prevent cutting by Cas9 after repair. The following mixture was injected into 20 N2 adults: 50 ng/ml Peft-3::Cas9 (Addgene #46168) [85], 100 ng/ml sgRNA in pJJR50, 20 ng/ml maph-1.1 repair template, and 2.5 ng/ml pCFJ90 [46]. Injected animals were placed on individual NGM plates. After 2–3 days at 25 °C, 500 μl of 5 mg/ml hygromycin in water was added to each plate, and healthy non-red-fluorescent Rol animals were selected after 3–5 days. To eliminate the marker cassette, 8–16 L2 animal were heat shocked for 4 h at 34 °C, and non-Rol progeny were selected. We obtained two independent lines, both of which displayed the same expression pattern. Vector maps and sequences are available in Additional file 16.
Expression in hippocampal neurons and fixation
To express MAPH-1.1 and DLG-1 in hippocampal neurons, we generated constructs consisting of MAPH-1.1 N-terminally tagged with GFP, and DLG-1 C-terminally tagged with mCherry, driven by the βactin promoter. MAPH-1.1 and DLG-1 were PCR amplified from a mixed-stage C. elegans cDNA library, and inserted into a vector containing the pβactin promoter and GFP or mCherry [86]. MAPH-1.1 was amplified using primers 5′-aaaGGCGCGCCAatgccggaggaatatatcatg and 5′-tttGCGGCCGCttagagcaaatcgactctggcc, and cloned using AscI and NotI. DLG-1 was amplified using primers 5′-aaaAAGCTTatgtcccacgagtcatcgg and 5′-tttGGTCTCGTCGACttatgacgtggcacccaaattggcg, digested with HindIII and BsaI, and ligated into vector digested with HindIII and SalI. Vector maps and sequences are available in Additional file 16. Primary hippocampal neurons prepared from embryonic day 18 rat brains [86] were transfected at 4 days in vitro (DIV4) for MAPH-1 and microtubule imaging, or DIV22 for DLG-1 imaging, using Lipofectamine 2000 (Life Technologies) and cultured for 2 additional days. For microtubule imaging, cells were first extracted with 0.3 % glutaraldehyde (GA) in PEM80-buffer (80 mM PIPES, 1 mM EGTA, 4 mM MgCl2, pH 6.9) for 1 min at 37 °C. Next, fixation was performed in 4 % paraformaldehyde (PFA) for 10 min. Subsequently, cells were washed twice for 5 min in PBS (Lonza BE17-517Q, w/o Ca and Mg) and cells were further permeabilized for 10 min in PBS + 0.2 % Triton-X100. Cells were then washed three times for 5 min in PBS and incubated for 45 min in blocking solution (2 % w/v bovine serum albumin [BSA], 0.2 % w/v gelatin, 10 mM glycine, 50 mM NH4Cl in PBS, pH 7.4). Primary antibodies to α-tubulin (Sigma, mouse clone B-5-1-2, 1:800) and MAP2 (Abcam ab5392, chicken, 1:1000) were incubated overnight at 4 °C in blocking solution. Cells were washed three times in PBS and incubated with secondary antibodies anti-Mouse Alexa Fluor568 (Life Technologies, goat, 1:800) and anti-Chicken Alexa Fluor647 (Life Technologies, goat, 1:800) for 1.5 h at room temperature. Finally, cells were washed three times in PBS and mounted in Mowiol 4-88 (Sigma-Aldrich). For imaging of postsynaptic densities, fixation (without prior extraction) was performed in 4 % PFA for 10 min. Subsequently, cells were washed three times for 10 min in PBS (Lonza BE17-517Q, w/o Ca and Mg), and mounted in Mowiol 4-88.
Microscopy and image processing
Still images of live animals were captured on a spinning disc platform consisting of a Nikon Ti-U inverted microscope with a motorized XY stage and a Piezo Z stage, ×60 and × 100 PLAN APO 1.4 numerical aperture (NA) oil objectives, a Yokogawa CSU-X1 spinning disc unit equipped with a dual dichroic mirror set for laser wavelengths 488 nm and 561 nm, 488 nm and 561 nm solid state 50 mW lasers controlled by an Andor revolution 500 series AOTF Laser modulator and combiner, Semrock 512/23 + 630/91 dual band pass emission filter, Semrock 525/30 single band pass emission filter, Semrock 617/73 single band pass filter, Semrock 4800 long pass filter (500–1200 pass), and an Andor iXON DU-885 monochrome EMCCD+ camera. All components were controlled by MetaMorph Microscopy Automation & Image Analysis Software. Live imaging was performed on a Nikon Eclipse-Ti microscope with a Plan Apo VC, ×60, 1.40 NA oil objective (Nikon). The microscope was equipped with an ASI motorized stage MS-2000-XYZ with Piezo Top Plate and a Perfect Focus System (Nikon), and used MetaMorph 7.8 to control the camera and all motorized parts. Confocal excitation and detection was achieved using a 100 mW Cobolt Calypso laser, Yokogawa spinning disc confocal scanning unit (CSU-X1-A1), a GFP emission filter [ET-GFP (49002); Chroma], and a Photometrics Evolve 512 EMCCD camera at a final magnification of 110 nm per pixel, including an additional magnification introduced by an extra intermediate lens 2.0X (Edmund Optics). For FRAP experiments we used a similar microscope setup equipped with an ILas system (Roper Scientific France/PICT-IBiSA, Institut Curie), with the laser set at 100 % laser power and a Plan Apo × 60 NA 1.40 oil lens. Imaging was performed at 1 or 2 frames per second. Microscopy of fixed samples was performed on a Zeiss LSM700 laser scanning confocal microscope equipped with a × 63 Plan-Apochromat 1.4 NA objective; 405 nm, 488 nm, 555 nm, and 633 nm lasers; and the following emission filters: SP490 (400–490 nm), SP555 (400–555 nm), SP640 (400–640 nm), BP490-555 (490–555 nm), LP560 (560–750 nm), LP640 (640–750 nm), and BP592-662 (592–662 nm). The LSM700 was controlled by the Zen software package. Maximum projections were generated from a series of slices of a Z-stack with ImageJ and processed with Adobe Photoshop CS6 and Adobe Illustrator CS6.
FRAP analysis
Images were processed and analyzed in ImageJ. For the FRAP analysis, the average gray value of a 100 × 10 pixel region in the FRAP region or a similar non-bleach area was calculated and background subtracted frame-by-frame by subtracting the average intensity of an empty, non-bleached area. FRAP recovery was calculated as the recovery from the first frame after bleach (set to 0) normalized to the average of the five frames before bleach.
Microtubule depolymerization speed measurement
Microtubule depolymerization speeds were calculated by making kymographs of the acquired movies using the KymoResliceWide plugin in ImageJ and measuring the distance and time of a depolymerization event. Graphs in Additional file 15: Figure S6 show the mean ± standard deviation, as well as individual measurements.