Fluorescence Intensities of. GFP-PAC-1 Strains

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1 DOI: /ncb3168 Arbitrry Fluorescence Units full length (1-4) Fluorescence Intensities of GFP-PAC-1 Strins ΔPH GFP-PAC-1 Strins b control c pc-1(3 RNAi) GFP-PAC-1 GFP-PAC-1 Supplementry Figure 1 GFP-PAC-1 trnsgene expression levels nd controls for GFP-PAC-1 N locliztion in the bsence of endogenous PAC-1. () Expression levels of GFP-PAC-1 trnsgenes nd derivtives, mesured by fluorescence intensity (fter bckground subtrction) t the four-cell stge (see Methods), expressed in rbitrry fluorescence units. PAC-1 mino cids present in ech GFP fusion protein re indicted (ΔPH lcks the PH domin only). The box represents first nd third qurtiles, brs represent mximum nd minimum vlues, nd the line within the box is the men fluorescence intensity (full length n= 11 embryos, ΔPH n = 13, n = 11, n= 13, n = 12, n = 14). While expression levels vry between trnsgenes, contct locliztion is evident even from those with the lowest expression levels (ex. full-length GFP-PAC-1 nd GFP-PAC ). Therefore, lck of pprent contct locliztion for GFP-PAC nd GFP-PAC is not due to low expression level. (b) Full-length GFP- PAC-1 in control (empty vector) RNAi embryos loclizes to cell contcts (rrow, 20/20 embryos). (c) In pc-1(3 RNAi) embryos GFP-PAC-1 is not detected (19/19 embryos), indicting tht RNAi effectively depletes PAC-1. Scle brs, 10µm. 1

2 wild type b pc-1 PAR-6 c pc-1; GFP-PAC-1 ΔPH PAR-6 d PAR-6 symmetry 4.0 *** * PAR-6 Polrity Index wild type pc-1 *** pc-1; GFP-PAC-1 ΔPH Supplementry Figure 2 Rescue of pc-1 polrity defects by GFP-PAC-1 ΔPH. () Wild-type, (b) pc-1(xn6), nd (c) pc-1(xn6); GFP-PAC-1 ΔPH 6-8 cell embryos stined for PAR-6. (d) PAR-6 symmetry is lost in pc-1 mutnts but is rescued by expression of GFP-PAC-1 ΔPH. PAR-6 symmetry ws quntified by determining the polrity index, defined s the rtio of PAR-6 levels t contct-free surfces versus hlf of the PAR-6 level t cell contcts (see Methods). Circles represent individul dt points nd the gry line indictes the verge (wild type n = 8 embryos, pc-1 n = 10, pc-1; GFP- PAC-1 ΔPH n = 8). Mesured by Mnn-Whitney U-test, pc-1(xn6) embryos hve polrity index significntly lower thn wild type (***p < 0.001) nd pc-1(xn6); GFP-PAC-1 ΔPH (***p < 0.001). The polrity index of pc- 1(xn6); GFP-PAC-1 ΔPH is mrginlly significntly different from wild type (*p < 0.05). Smples were pooled from two independent experiments. Scle brs, 10µm. 2

3 control b hmp-2(rnai) GFP-PAC-1 N c GFP-PAC-1 N GFP-PAC-1 N d Contcts / cytoplsm PAC-1 N contct enrichment ** ** n.s. control hmp-2(rnai) Supplementry Figure 3 Comprison of GFP-PAC-1 N in hmp-2(rnai) nd embryos. (-c) GFP-PAC-1 N in live embryos of the indicted genotype; imges were cptured t the sme exposure. (d) Quntifiction of GFP-PAC-1 N contct enrichment. Circles represent dt points from individul embryos, nd the verge is gry line. **p < 0.01; n.s. = not significntly different, Mnn-Whitney U test. (Control n = 13 embryos, hmp-2(rnai) n = 9, n = 9). Scle brs, 10µm. 3

4 control b hmp-2(rnai) HMP-1 c wild type HMP-1 d jc-1 e control f hmr-1 GFP- g GFP- h hmr-1 HMP-1 i jc-1 HMP-1 1kb Arm repets xn15 Supplementry Figure 4 Genetic requirements for ctenin locliztion. Control embryos re wild-type embryos fed on bcteri contining empty RNAi vector. (-b) HMP-1 immunostining t cell contcts in control embryos (40/40 embryos) or in the cytoplsm of hmp-2(rnai) embryos (35/35 embryos). (c-d) - ntiserum immunostins cell contcts in wild-type embryos (30/30 embryos) but not in jc-1(xn15) embryos (42/42 embryos). - ntiserum lso stins nuclei non-specificlly, s evidenced by the loss of contct stining, but not nucler stining, in jc-1 mutnt embryos (d). (e-f) GFP- t cell contcts in live control embryo (41/41 embryos) nd in the cytoplsm in live hmr-1 embryo (25/25 embryos). (g) HMP-1 immunostining in embryo. (h) HMP-1 immunostining in hmr-1 embryo, where HMP-1 loclizes to the cytoplsm (45/45 embryos). (i) Schemtic of the jc-1 gene, with exons shown s rectngles nd introns s chevrons. Region encoding the Armdillo (Arm) repets, which contins the peptide used to generte - ntiserum, is indicted. The dshed region indictes the extent of jc-1 deleted in the xn15 llele. Scle brs, 10µm. 4

5 HMP-1-GFP d GFP- g HMR-1-GFP i b wild type jc-1 HMP-1-GFP control e GFP- control h HMR-1-GFP jc-1 jc-1 + j c f k Contcts / cytoplsm Contcts / cytoplsm Contcts / cytoplsm HMP-1 contct enrichment wild type jc-1 contct enrichment control HMR-1 contct enrichment control jc-1 jc-1 + HMR-1-GFP l m HMR-1-GFP control jc-1 + HMR-1 HMR-1 Supplementry Figure 5 Ctenin nd HMR-1 locliztion following ctenin depletion. Control embryos re wild-type embryos fed on bcteri contining RNAi empty vector. (-b) HMP-1-GFP t cell contcts in wild-type nd jc-1 embryos. (c) Quntifiction of HMP-1-GFP contct enrichment in wild-type nd jc-1 embryos (wild type n = 12 embryos, jc-1 n = 12). Circles represent individul vlues nd gry lines the men. HMP-1-GFP levels t contcts in the two genotypes re not significntly different (p = 0.06, Mnn-Whitney U test). (d-e) GFP- t cell contcts in control nd embryo. (f) Quntifiction of GFP- contct enrichment in control nd embryos. (control n = 13 embryos, n = 12). GFP- levels t contcts in the two genotypes re not significntly different (p= 0.3, Mnn-Whitney U test). (g-j) HMR-1-GFP expression in live four-cell embryos of the indicted genotype. (k) Quntifiction of HMR- 1-GFP levels t cell contcts in embryos of the indicted genotype (control n = 18 embryos, n = 33, jc-1 n =17, jc-1 + n = 17). None of the men vlues re significntly different from control (p > 0.05, Mnn-Whitney U test). Control smples nd embryos were pooled from two independent experiments. (l-m) Endogenous HMR-1 immunostining t cell contcts in control (91/91 embryos) nd jc-1(xn15) + (133/133 embryos). Scle brs, 10µm. 5

6 picc-1 1kb b xn14 CCDC85A 64 VSAMLDHSNLIREVNRRLQLHLGEIRGLKDINQKLQEDNQELRDLCCFLDDDRQKGKRVS CCDC85C 43 VGLMLEHGGLMRDVNRRLQQHLLEIRGLKDVNQRLQDDNQELRELCCFLDDDRQKGRKLA CCDC85B 36 LAALVQRGRLMQEVNRQLQGHLGEIRELKQLNRRLQAENRELRDLCCFLDSERQRGRRAA CG IPTANGGGRLTAEQNRQLQSLVNELRALKEQNQRLLDDNQELRDLCCFLDDDRQKGRKLA PICC QKLMRMQSDVVNDANRRVQMHVNEIRMLKEDNRKLAISNKELRDLSCFLDDDRQKTRKLA CCDC85A CCDC85C CCDC85B CG17265 PICC-1 REWQRLGRYTAGVMHKEVALYLQKLKDLEVKQEEVVKENMELKELCVLLDEEK REWQRFGRHAAGAVWHEVARSQQKLRELEARQEALLRENLELKELVLLLDEER RQWQLFGTQASRAVREDLGGCWQKLAELEGRQEELLRENLALKELCLALGEEW REWQRFGRYTASVMRQEVAAYQNKLRQLDDKQQELITDNLELKELCLYLDEER REWQKFGRYTSSLMKQEVDSYHQKMVSIEEKLCTKEREVDELRQLCMYLDEQR c hmr-1 d GFP-PICC-1 GFP-PICC-1 Supplementry Figure 6 picc-1 gene nd regultion of PICC-1 locliztion. () Schemtic of the picc-1 gene, with exons shown s rectngles nd introns s chevrons. The dshed region indictes portion of the picc-1 gene deleted in the xn14 llele. (b) Region of conservtion between PICC-1 nd homologues in humn (CCDC85A-C; CCDC85B is lso known s DIPA) nd Drosophil (CG17265). Blck shding indictes three or more identicl residues; gry shding indictes similr residues. (c-d) GFP-PICC-1 locliztion in hmr-1 mutnt embryo (n = 50 embryos) nd embryo (n = 31 embryos). Compre to wild-type locliztion in Figure 4. Scle brs, 10µm. 6

7 DBD-bit AD-prey HMP-1 PAC-1 N PAC-1 N HMP-1 HMP-2 ++ HMR-1 ICD ++ PICC-1 empty b IP: α-gfp picc-1-gfp IB: α- } IB: α-tubulin c IP: α-ha picc-1-gfp mch-h-pc IB: α-ha IB: α-gfp mch-ha-pac-1 IB: α-tubulin Supplementry Figure 7 Two-hybrid controls nd reciprocl immunoprecipittions. () Control two-hybrid experiments, performed s described in Figure 5 legend. (b) Immunoprecipittion of from embryonic lystes showing co-immunoprecipittion of species. The experiment ws performed seven times, nd representtive exmple is shown. (c) Immunoprecipittion of mcherry-ha-pac-1 from embryonic lystes showing co-immunoprecipittion of. The experiment ws performed four times, nd representtive exmple is shown. Tubulin levels in the input (totl embryonic lyste) re shown s loding control. Uncropped blots re shown in Supplementry Figure

8 Pnels for Figure 5b IP: α- Ctrl IP: α- jc-1 xn15 WT xn15 WT xn15 picc-1-gfp jc-1 picc-1-gfp xn15 WT xn15 WT (Longer exposure) (Shorter exposure) IP: α- Ctrl jc-1 xn15 WT xn15 WT xn15 picc-1-gfp Tubulin Supplementry Figure 8 Uncropped blots for immunoprecipittion experiments shown in Figure 5 nd Supplementry Figure 7. Red boxed res represent regions cropped nd shown in the indicted figure pnels. 8

9 Pnels for Figure 5c IP: α-gfp mch-h-pc picc-1-gfp IP: α-gfp mch-h-pc picc-1-gfp (Shorter exprosure) mch-ha-pac-1 (Shorter exposure) (Longer exposure) mch-ha-pac-1 (Longer exposure) IP: α-gfp mch-h-pc picc-1-gfp Tubulin Supplementry Figure 8 continued 9

10 Pnels for Supplementry Figure 7b IP: α-gfp IP: α-gfp picc-1-gfp picc-1-gfp (Shorter exposure) IP: α-gfp picc-1-gfp (Longer exposure) 50 Tubulin Supplementry Figure 8 continued 10

11 Pnels for Supplementry Figure 7c picc-1-gfp mch-h-pc-1 IP: α-ha Ctrl - - picc-1-gfp IP: α-ha (Shorter exposure) mch-h-pc mch-pac-1-ha (Longer exposure) picc-1-gfp mch-h-pc-1 IP: α-ha Tubulin Supplementry Figure 8 continued 11

12 Supplementry Tble 1. Sttisticl comprisons of GFP-PAC-1 N contct locliztion s jc-1 hmp-1 jc-1; jc-1 + hmp- picc-1 + jc-1; picc-1 picc-1 (RNAi) picc-1 1 (RNAi) hmp-1 + hmp-1 hmr-1 control 1.34e E E E E E E E-09 jc E E E E-06 hmp-1 (RNAi) E E E E E-07 picc E E E E-04 jc-1; picc E E E-04 jc-1 + hmp-1 (RNAi) picc-1 + hmp-1 (RNAi) jc-1; picc-1 + hmp-1 (RNAi) 0.33 p vlues clculted using the Mnn-Whitney U test. Green shding, p 0.001; light green shding, 0.01 p > 0.001, yellow shding, 0.05 p > 0.01; red shding, p > Supplementry Tble 1 Sttisticl comprisons of GFP-PAC-1 N contct locliztion. p vlues clculted using the Mnn-Whitney U test. Green shding, p 0.001; light green shding, 0.01 p > 0.001, yellow shding, 0.05 p > 0.01; red shding, p >

13 Supplementry Tble 2. Primers used for trnsgene construction. Construct Forwrd primer Reverse primer Ppie-1::gfp-pc-1 FL 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTTGAGGAGCACCATCGACGGCTCCAT-3' 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTAAAGCAGTTCATCGCCAGACACCGGTG-3' Ppie-1::gfp-pc-1 ΔPH 5'-CGTAACTAGTGTTGATGAGAATGAATTGTCTACAACTGGC-3' 5'-CGTAACTAGTTCCGTCTCGTTGAAGAATTGTACC-3' Ppie-1::gfp-pc '-GTACCGGCCGAAAGCCTGCTTTTTTGTACAAAGTT-3' 5'-GTACCGGCCGACATCGTCCTCGTCTTCC-3' Ppie-1::gfp-pc '-GGGGACAAGTTTGTACAAAAAAGCAGGCTTTACATCCGATGGTTCCGGTGAGCACAA-3' 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTTAAAGCAGTTCATCGCCAGACACCGG-3 Ppie-1::gfp-pc '-GTACACTAGTCTATCCGTCTCGTTGAAGAATTGTACC-3' 5'-GTACACTAGTACCCAGCTTTCTTGTACAAAGTTG-3' Ppie-1::gfp-pc '-GGGGACAAGTTTGTACAAAAAAGCAGGCTTTATGGAGGAGCACCATCGACGGCTCC-3' 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTTATTTACTTGAATTGAACAACGCAA-3' Ppie-1::gfp-picc-1 5'-AAAAAGCAGGCTTTATGATTATCACAACTCCGAGACGAGC-3' 5'-AGAAAGCTGGGTTTTAATTATGGGTGTGTACATTATTATTATTATTCTTTTCGACGG-3' Ppie-1::gfp-jc-1 5'-AAAAAGCAGGCTTTATGATCTCCTCGGGGTGGATGC-3' 5'-AGAAAGCTGGGTTCTAGACCCAACTATCGTCGATATTCCC-3' Phsp16::h-ph plc1 1 -hmr-1 ICD 5'-GATCGGATCCTATACGAGACGATCGCCTGGTGC-3' 5'-GATCGGATCCTTATTGGGCACTTTCGATACTCTCCAACG-3' Phsp16::h-ph plc1 1 -hmr-1 ICD-M 5'-TAAGGATCCTAAAACCCAGC-3' 5'-GTTCAGCTCATGATCATCTTT-3' Supplementry Tble 2 Primer list for trnsgene construction. 13