Supplementary Information

Transcription

1 Supplementary Information Supplementary Figure 1: Luminal localization of CCM-3. (a) The CCM-3::GFP fusion protein localizes along the apical (luminal) surface of the pharynx (b) as well as the lumen of the intestine (arrowheads), and rachis of the germline (arrow) (c), and the cytoplasm of oocytes (asterisks). (d) Within the canal, CCM-3 maintains strong apical and cytoplasmic distribution, but frequently shows enrichment at the growing tip (top panel), sometimes in the form of a short trailing cytoplasmic extension (bottom panel). (e) Although over-expression of CCM-3 using the canal-specific exc-9 promoter rescued canals in most ccm- 3(-/-) worms, about 20% of ccm-3(+/-) heterozygotes (indicated by the bright yellow signal in the pharynx - asterisk) expressing this transgene had severe canal truncations (top panel) and enlarged lumen with cystic tips (bottom panel). While such over-expression caused canal defects, it did not alter the apical localization of CCM-3 (f). White and yellow arrowheads indicate the terminal (posterior) ends of the canal and the worm body, respectively. Scale bars are indicated for whole worm and high magnification canal sections in the first appropriate panel, and are modified only in panels where the magnification is different. a and e (top) are 100x magnification; b, c, d, e (bottom) and f are 630x magnification.

2 Supplementary Figure 2: kri-1/ccm1 does not regulate canal extension or morphology. The kri-1(ok1251) deletion allele (a), unlike ccm-3(tm2806), does not affect morphology of the excretory canal. Under the control of the endogenous kri-1 promoter, KRI-1::GFP (b) is expressed in the pharynx (c) and intestine (d), but not the excretory canals, indicating some overlap with CCM-3. (e) Ablation of ccm-3 by RNAi in kri-1(ok1251) mutants results in

3 synthetic lethality. Graph shows average ± s.e.m., n 40, * = p <0.05 from control (Student s t- test). In panel (a) the asterisk denotes the location of the excretory cell (at the anterior/head of the worm), and white and yellow arrowheads denote the posterior tips of the canal and the worm body, respectively. Images a and b at 100x, c and d at 630x magnifications.

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5 Supplementary Figure 3: Ablation of ccm-3 by RNAi phenocopies the ccm-3(tm2806) mutation. All panels show mcherry tagged markers on control (empty vector) RNAi (top) versus ccm- 3(RNAi) (bottom). Below canal images are light intensity readings, where peak heights correspond to the relative fluorescence of the mcherry-tagged protein. CDC-42 (a) and its activated marker GBDwsp-1 (b) both show reduced puncta in canals of worms grown on ccm- 3(RNAi). Golgi bodies (c), likewise, are severely diminished in the absence of ccm-3. Punctate distribution of RAB-5 (early endosomes) (d) and RAB-7 (late endosomes) (e), however, are not affected by ablation of ccm-3, while recycling endosomes (RAB-11) are reduced (f). All images are taken from the same posterior region of the canal at 630x magnification, and are to be viewed moving left to right, from anterior to posterior on the worm.

6 Supplementary Figure 4: Over-expression of ERM-1 cannot rescue loss of ccm-3.

7 (a) ERM-1 is highly expressed in the excretory canals. Over-expression of ERM-1::GFP was unable to rescue canal truncations in ccm-3(-/-) mutants (a, middle panel, and b), nor did it improve canal integrity; lumen defects were still apparent (a, bottom panel). Graph shows average ± s.e.m., n = 50, * = p <0.05 from control (Student s t-test). White and yellow arrowheads indicate the terminal (posterior) ends of the canal and the worm body, respectively. a top and middle panels are 100x magnification; a bottom panel is 630x magnification.

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9 Supplementary Figure 5: ccm-3 mutant canals accrue more damage towards the posterior tip. Sequential 50μm cuts of the canal starting just below the pharynx and terminating near the midline (a) were taken. Pharynx (black), excretory cell body (red), and excretory canals (orange) have been overlaid to indicate relative positioning of the cuts. The wild-type (N2) canal (B) retains rounded shape, organelle content and CV numbers. A varicosity can be seen at the 100µm position, and a multivesicular body is also present at the 150µm position (indicated by the black arrowhead). Conversely, the ccm-3(-/-) canal (c) progressively deteriorates as it reaches the posterior end. The basolateral membrane becomes progressively more distended towards the posterior end, as the lumen expands and CVs aggregate. CV numbers remain high and Golgi/E.R. are absent in all but the anterior most ccm-3(-/-) canal section. Image (a) is at 100X magnification. All canal images are taken at 19,000X magnification.

10 Supplementary Figure 6: Loss of the exocyst complex gene exoc-8 affects canal growth and causes cyst formation. Canal extension and morphology also requires components of the exocyst. Ablation of exoc-8 (a)

11 causes truncation of canals by approximately 20% (d). Concurrent loss of ccm-3 does not exacerbate this truncation (b, d). The exoc-8(ok2523) deletion allele also causes discontiguity of the lumen, and leads to an accumulation of expanded vesicles in the cytoplasm (c, left panels) as well as severe distal tip cyst formation (c, right panels). Asterisks denote location of the excretory cell (at the anterior/head of the worm), white and yellow arrowheads denote canal, and total worm length, respectively. All canals are oriented with their posterior tips pointing to the right. a and b are 100x magnification, c panels are 630x magnification. Graph shows average ± s.e.m., n 20, * = p<0.05 from control (Student s t-test).

12 Supplementary Figure 7: RHO-1 does not affect canal length, and cannot restore RE markers (a)rnai to the Rho GTPases, rho-1 (RhoA) and chw-1(rhou/v), middle and bottom panels, did not cause truncations in the excretory canal (b). RhoA is implicated as a target of the ternary CCM complex, in which CCM proteins normally suppress its activity. (c) Increased Rho activity is caused by CCM gene mutations in mammals, but ablation of rho-1 did not restore RAB-11 puncta in ccm-3(-/-) canals. Asterisks denote location of the excretory cell (at the anterior/head of the worm), white and yellow arrowheads denote canal, and total worm length, respectively. All canals are shown with their posterior tips pointing to the right. a panels are 100x magnification, c panels are at 630x magnification. Graph shows average ± s.e.m., n 30.

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14 Supplementary Figure 8: Over-expression of actin can rescue loss of ccm-3. Canal lengths of ccm-3 mutant worms were measured relative to the total length of the worm from the pharynx to the anus (a); indicated by the broken lines at 0 and 100%. ccm-3(+/-) have an average canal length of 89% relative to wild-type (Red), while ccm-3(-/-) have an average length of 63% (Blue). Canal lengths were quantitated as 70%, 80% and 90% of wild-type length (White). Over-expression of ACT-5::GFP in ccm-3(-/-) worms caused a significant rescue of truncated canals (c). (e) ACT-5::GFP over-expression increased populations of worms with canal lengths over 70%, 80% and 90% by ~2, 3, and 4 fold respectively. Asterisks denote location of the excretory cell (at the anterior/head of the worm), white and yellow arrowheads denote canal, and total worm length, respectively. b-d are 100x magnification. Graph shows average ± s.e.m., n 50, * = p <0.05 (Student s t-test) from control ccm-3(-/-) canals.

15 Supplementary Figure 9: Un-cropped western blots (a) Un-cropped blot represented in Figure 2a. (b) Un-cropped blot represented in Figure 6b. Molecular weight markers are indicated between the input and IP lanes in panel (a).

16 Supplementary Table 1: Relative frequency of canal phenotypes in ccm-3(-/-) mutants. Lumen expansion Discontiguous/ectopic lumen Presence of cysts Cytoplasmic trailing at tip Frequency of a canal having at least one trait 61% 59% 41% 67% 95% N = 100 Supplementary Table 2: Relative quantification of discreet puncta for endocytic markers. CDC-42 GBDwsp-1 GRIP RAB-5 RAB-11 Control 13.6 ± ± ± ± ± 3.8 ccm-3(-/-) 3.0 ± ± ± 1.0 * 18.3 ± ± 0.7 * ccm ± ± ± 1.0 * 18.4 ± ± 1.6 * (RNAi) gck-1(rnai) 5.1 ± ± 1.3 * 5.4 ± 1.6 * cash ± ± 0.7 * 5.5 ± 1.3 * (RNAi) cdc ± ± 0.6 * 1.3 ± 0.7 * (RNAi) mrck ± ± 0.8 * 3.9 ± 0.6 * (RNAi) ccm-3(-/-) ; 9.4 ± 1.5 * ACT-5 (++) N=10, *= P 0.005, = P (Student s t-test) Note 1; Discreet puncta were quantified as those reaching or surpassing a fixed fluorescence intensity rating. Puncta numbers were quantified from stretches of excretory canal measuring approximately 100µm. Note 2; Significance in difference is compared to the control value, except in the case of RAB- 11 puncta in the ccm-3(-/-); ACT-5(++) line, which have been analyzed to compare RAB-11 puncta numbers in ccm-3(-/-) mutants.

17 Supplementary Table 3: Relative quantification of organelles in N2 and ccm-3(-/-) canals Mitochondria Golgi ER N2 1.9 ± ± ± 0.4 ccm-3(-/-) 4.9 ± 0.9 * 0.3 ± 0.1 * 0.4 ± 0.3 * N 8, * = P 0.05 (Student s t-test) Canal sections from sequential cuts taken just below the pharynx, totalling approximately 1µm in length, were scored for number of organelles (i.e., a score of 1 mitochondria could be attributed to a small section of mitochondria only seen in the first of the stacked images). Supplementary Table 4: Canal phenotype of STRIPAK, GTPase signalling and Canal formation gene mutants Worm Gene Gene ID Human Gene Canal Phenotype ccm-3 C14A4.11 CCM3/PDCD10 Strong truncation, abnormal lumen, cystic, cytoplasmic trailing at canal tips gck-1 T19A5.2 MST4 Strong truncation, abnormal lumen, cystic, cytoplasmic trailing at canal tips gck-4 C04A11.3 SLK Weak truncation cash-1 K07C5.8 STRN3 Strong truncation, abnormal lumen, cystic, cytoplasmic trailing at canal tips C30A5.3 C30A5.3 MOB3 Healthy canals farl-11 F10E7.8 STRIP1/2 Strong truncation, abnormal lumen, cystic, cytoplasmic trailing at canal tips C49H3.6 C49H3.6 CTTNBP2/NL Healthy canals mrck-1 K08B12.5 MRCKβ Strong truncation, abnormal lumen, cystic, cytoplasmic trailing at canal tips cdc-42 R07G3.1 CDC42 Strong truncation, abnormal lumen, cystic, cytoplasmic trailing at canal tips rho-1 Y51H4A.3 RHOA Healthy canals exoc-8 Y105E8B.2 EXOC8 Strong truncation, abnormal lumen, cystic, cytoplasmic trailing at canal tips erm-1 C01G8.5 EZR/MSN/RDX Very strong truncation, strongly cystic, severe lumen defects aqp-8 K02G10.7 AQP10 Weak truncation, abnormal lumen, cystic, cytoplasmic trailing at canal tips rab-11 F53G12.1 RAB11 Strong truncation, abnormal lumen, cystic, cytoplasmic trailing at canal tips

18 Supplementary Table 5: Strains used Genotype Strain Description bgis312 {P vha-8 ::GFP} UG756 Excretory canal marker dpy-5(e907)i; sex13892 {P ccm-3 ::GFP + dpy-5(+)} BC13892 ccm-3 transcriptional reporter onis3 {P ccm-3 ::GFP::CCM-3; unc-119(+)}ii; unc- WD356 CCM-3 translational reporter 119(ed3) III N2; onex62[p exc-9 ::ccm-3::gfp; P myo-2 ::mcherry; P myo-3 ::mcherry; P rab-3 ::mcherry] WD416 Canal specific CCM-3 translational reporter C14A4.11(tm2806)/+II FX02806 Unbalanced ccm-3 (C14A4.11) mutant WD119 ccm-3 mutant let(gk838)]ii let(gk838)]ii; bgls312{p vha-8 ::GFP} WD188 ccm-3 mutant expressing canal marker let(gk838)]ii; bgls312 {P vha-8 ::GFP}; onex15{p ccm3 ::ccm-3; P myo-2 ::mcherry; P rab- 3::mCherry} WD204 ccm-3 mutant expressing extrachromsomal ccm-3(+) rescuing array let(gk838)]ii; onex55[p exc-9 ::ccm-3; P exc- 9::mCherry + P myo-2 ::mcherry] let(gk838)]ii; onex64[p exc-9 ::ccm-3::gfp; P myo- 2::mCherry; P myo-3 ::mcherry; P rab-3 ::mcherry] WD382 WD418 ccm-3 mutant expressing ccm- 3(+) rescuing array in canal ccm-3 mutant expressing canal specific CCM-3 translational reporter gck-1(km15)v/nt1[qis51](iv; V) JS345 gck-1 mutant gck-1(km15)v/nt1[qis51](iv; V); bgls312{p vha- 8::GFP} WD195 gck-1 mutant expressing canal marker gck-1(km15)v/nt1[qis51](iv; V); bgls312{p vha- 8::GFP}; onex36{p gck-1 ::gck-1; P myo-2 ::mcherry} WD243 gck-1 mutant expressing extrachromosomal gck-1(+) gck-1(km15)v/nt1[qis51](iv; V); bgls312{p vha- 8::GFP}; onex45{p gck-1 ::GCK-1(K62R, T186A); P myo-2 ::mcherry} gck-1(km15)/nt1[qis51](iv; V); onex66[p exc- 9::gck-1::mCherry; rol-6(su1006)] let(gk838)]ii; bgls312{p vha-8 ::GFP}; onex29{p gck- 1::GCK-1; P myo-2 ::mcherry} gck-1(km15)v/nt1[qis51](iv; V); onex67[p exc- 9::ccm-3::GFP; P myo-2 ::mcherry; P myo-3 ::mcherry; P rab-3 ::mcherry] N2; onex69[p exc-9 ::ccm-3::gfp; P exc-9 ::gck- 1::mCherry; rol-6(su1006)] WD278 WD420 WD236 WD421 WD423 rescuing array gck-1 mutant expressing extrachromosomal gck-1(+) rescuing array (kinase dead) gck-1 mutant with canalspecific GCK-1 translational reporter ccm-3 mutant expressing extrachromosomal gck-1(+) rescuing array gck-1 mutant with canalspecific CCM-3 translational reporter Canal specific CCM-3 and GCK-1 translational reporters

19 muex344 {P kri-1 ::GFP::kri-1acDNA; odr- CF2282 KRI-1 translational reporter 1::mCherry} kri-1(ok1251)i; bgls312{p vha-8 ::GFP} WD187 kri-1 mutant expressing canal marker mrck-1(ok586)v/nt1[qis51](iv;v) WH556 mrck-1 mutant mrck-1(ok586)v/nt1[qis51](iv;v); bgis312{p vha- 8::GFP} WD309 mrck-1 mutant expressing canal marker exoc-8(ok2523)i RB1928 exoc-8 mutant exoc-8(ok2523)i; bgls312{p vha-8 ::GFP} WD276 exoc-8 mutant expressing canal marker qpis103[p exc-9 ::mcherry::grip] BK220 Golgi marker (GRIP domain) (canal specific) qpis96[p exc-9 ::mcherry::cdc-42] BK204 CDC-42 marker (canal specific) qpis104[p exc-9 ::mcherry::gbd wsp-1 ] BK262 Activated CDC-42 marker (canal specific) qpis99[p exc-9 ::mcherry::rab-5] IV BK209 RAB-5 marker (canal specific) qpis100[p exc-9 ::mcherry::rab-7] BK210 RAB-7 marker (canal specific) qpis97[p exc-9 ::mcherry::rab-11]v BK205 RAB-11 marker (canal specific) qpis102[p exc-9 ::mcherry::chc-1] BK219 CHC-1 marker (canal specific) fgex13[p erm-1 ::erm-1::gfp, rol-6p::rol-6(su1006)] VJ402 ERM-1 marker fgex12 [P act-5 ::act-5::gfp] VJ268 Actin marker ccm-3(tm2806)/unc-4(e120) sqt-1(sc13)ii; WD324 ccm-3 mutant expressing canal qpis103[p exc-9 ::mcherry::grip] marker, with canal-specific let(gk838)]ii; bgls312[p vha-8 ::GFP]; qpis96[p exc- 9::mCherry::cdc-42] let(gk838)]ii; bgls312[p vha-8 ::GFP]; qpis104[p exc- 9::mCherry::GBD wsp-1 ] let(gk838)]ii; bgls312[p vha-8 ::GFP]; qpis99[p exc- 9::mCherry::rab-5]IV let(gk838)]ii; bgls312[p vha-8 ::GFP]; qpis97[p exc- 9::mCherry::rab-11] let(gk838)]ii; bgls312{p vha-8 ::GFP}; fgex13[p erm- 1::erm-1::gfp, rol-6p::rol-6(su1006)] WD369 WD373 WD375 WD396 WD402 Golgi marker ccm-3 mutant expressing canal marker, with canal-specific CDC-42 reporter ccm-3 mutant expressing canal marker, with canal-specific activated CDC-42 marker ccm-3 mutant expressing canal marker, with canal-specific RAB-5 marker ccm-3 mutant expressing canal marker, with canal-specific RAB-11 marker ccm-3 mutant expressing canal marker, with ERM-1 marker WD370 ccm-3 mutant expressing canal let(gk838)]ii; bgls312{p vha-8 ::GFP}; fgex12[p act- marker, with actin marker

20 5::act-5::GFP] let(gk838)]ii; bgls312{p vha-8 ::GFP}; qpis97[p exc- 9::mCherry::rab-11]; fgex12[p act-5 ::act-5::gfp] N2; qpis102[p exc-9 ::mcherry::chc-1]; onex70[p exc- 9::ccm-3::GFP; P myo-2 ::mcherry; P myo-3 ::mcherry; P rab-3 ::mcherry] WD431 WD425 ccm-3 mutant expressing canal marker, with canal-specific RAB-11 and actin markers Canal specific CCM-3 translational reporter, with canal specific CHC-1 marker