Antoniades I , Stylianou P , Christodoulou N , Skourides PA .

	Fig. 1 FAK is recruited at CAs through its FERM and FAT domains Merged and single channel (for GFP) maximum intensity projections (MIPs) of multiciliated cells, expressing centrin RFP (red in merged images) and GFP FAK or GFP-fused FAK deletion mutants. (a-a’) Full length FAK displays strong localization at CAs and each basal body is associated with a punctum of GFP FAK. CA complexes appear as bright puncta throughout the cell, displaying an anteroposterior intensity gradient. (b-b’) FAK δ FAT does not associate with the basal bodies as efficiently as FAK and there are no bright CA-puncta, showing that the FAT domain is necessary for proper localization at CA complexes. (c-c’) The FAT domain is not sufficient to target FAK at CAs and only few faint CA-puncta are present in the cell. (d-d’) GFP FAK δ FERM localizes strongly on the rootlets while the apical CA localization displayed by full length FAK (a-a’) is absent. (e-e’) The FERM domain is not sufficient to drive FAK at CAs and only few faint puncta, corresponding to CAs, are present. (f-f’) GFP FF displays strong localization at CAs similarly to full length FAK showing that combination of the FERM and FAT domains is sufficient for proper localization of FAK. Scale bars: 5 μM.
	Fig. 2 Interaction with paxillin is not the sole determinant for FAK’s CA localization Merged and single channel (for GFP) maximum intensity projections (MIPs) of multiciliated cells, expressing centrin RFP (red in merged images) and GFP FAK or GFP-fused FAK mutants. (a-a’) Full length FAK displays strong localization at CAs. (b-b’) Talin binding-deficient FAK (E1015A) displays strong localization at CAs indicating that interaction with talin is not necessary for correct localization. Bright puncta in close association with basal bodies are present throughout the cell. (c-c’) FAK L1034S displays strong localization at CAs, while FAK I936E/I998E (d-d’) fails to associate with the basal bodies. (e-j) Optical sections of intercalating multiciliated cells expressing centrin RFP and GFP-fused FAK mutants. (e-e’) Wild type FAK localizes at CAs during basal body migration. CA-localized FAK appears as bright puncta associated with the basal bodies. Neither GFP FAK δ FAT (f-f’) nor GFP FAT (g-g’) associate with migrating basal bodies, suggesting that neither the FAT domain nor the FERM domain (present in GFP FAK δ FAT) are individually sufficient for localization at CAs. (h-h’) GFP FAK E1015A localizes at CAs similarly to wt FAK. GFP FAK L1034S (i-i’) associates with the migrating basal bodies while GFP FAK I936E/I998E (j-j’) fails to do so. Scale bars: 5 μM.
	Fig. 3 FAK’s function during ciliogenesis depends on the FERM and FAT domains and interaction with paxillin, but is kinase independent Comparison of the ability of different mutants to rescue the FAK MO elicited ciliogenesis defects. Surface view (MIPs) of the multiciliated epidermis of Xenopus tadpoles, immunostained against acetylated tubulin which stains the cilia (red). Tadpoles have been injected with a FAK MO together with centrin RFP (grey) and different FAK constructs. Targeted cells are identified by expression of centrin RFP. (a) In control embryos cells appear normal with numerous cilia projecting from their apical surface. (b) In morphant embryos cells display severe ciliogenesis defects and have fewer or even no cilia. (c) Expression of wt FAK rescues the phenotype and cells project cilia normally. (d) Expression of FAK δ FAT fails to rescue the phenotype showing that the FAT domain is necessary for proper function of FAK during ciliogenesis. (e) Expression of FAK E1015A partially rescues the phenotype and the majority of cells project cilia normally, showing that interaction of FAK with talin may only play a minor role during ciliogenesis. (f-g) Expression of two paxillin binding-deficient mutants cannot rescue the ciliogenesis defects as the majority of cells have very few or no cilia, indicating that interaction with paxillin is critical for proper ciliogenesis. (h) Expression of FAK δ FERM does not rescue the defects showing that the FERM domain of FAK has an important role for its function in ciliogenesis. (i) Expression of FF leads to partial rescue of the phenotype. (j) Expression of kinase inactive (KD) FAK rescues similarly to wild type FAK showing that kinase activity is dispensable during ciliogenesis. (k) Expression levels of the FAK constructs used in rescue experiments analyzed in Western Blots. All constructs are stable and at the expected molecular weight. Scale bars: 10 μμ.
	Fig. 5 Interaction with paxillin is necessary while FAK’s kinase activity is dispensable for the generation of ciliary flow (a-f) Representation of the flow of QDs as it was tracked over the skin of tadpoles. (a) Control embryos display robust and directional flow. FAK morphants (b) display impaired flow, while expression of wt FAK (c) partially rescues the phenotype leading to improved flow. Expression of FAK δ FAT (d) does not improve the flow, while in FAK I936E/998E expressing tadpoles (e) the flow is still impaired. (f) Expression or kinase inactive FAK leads to improved flow along the tadpoles showing that the kinase activity of FAK is dispensable for proper function in cilia.
	Fig. 7 The interaction of FAK with paxillin is not the sole determinant for FAK’s FA localization (a-c) HeLa cells expressing RFP paxillin (FA marker) and GFP FAK (a-a’), GFP FAK L1034S (b-b’) or GFP FAK I936E/I998E (c). FAK L1034S localizes at FAs while FAK I936E/I998E does not. (d) Representative blots showing immunoprecipitated GFP FAK, GFP FAK L1034S and GFP FAK I936E/I998E and blotted for GFP and paxillin. Wt FAK co-precipitates paxillin while none of the paxillin-binding mutants does. (e-e’) Paxillin -/- and reconstituted (marked with yellow asterisk) cells immunostained against FAK. In both types of cells FAK is recruited at FAs showing that interaction with paxillin is not the only determinant for localization. Scale bars: 5 μμ.
	Fig. 8 The FAK-Paxillin interaction is partially responsible for FAK’s CA localization (a-a’’) Multiciliated cell expressing centrin CFP, mKate FAK and GFP paxillin. FAK co-localizes with paxillin at CAs. Each basal body displays strong localization of both paxillin and FAK. (b-b’’) Multiciliated cell expressing centrin CFP, mKate FAK and GFP paxillinC. FAK can still localize at CAs together with paxillinC but less efficiently. (c) Quantification of the ratio of GFP/mKate signal on separate CAs, from two tailed unpaired t-test. Error bars represent S.E.M. While in control cells (expressing GFP paxillin) the mean ratio is 1.2 ± 0.026 (Mean ± SEM) (n=226 CAs from 9 embryos) in cells expressing GFP paxillinC the mean ratio is increased, more than two fold, to 2.8 ± 0.052 (Mean ± SEM) (n=207 CAs from 11 embryos) (***, p<0.0001). Scale bars: 2 μμ.
	Fig. 9 The FERM-Kinase interaction is disrupted at CAs in a kinase activity independent manner (a-c) Quantification of the YFP to CFP intensity ratio of the wt (a) and the kinase inactive (K454R) FAK biosensor (b and c) in control (b) and Y15 treated embryos (c), at CAs and in the cytosol. Results were analyzed with two tailed unpaired t-test. Error bars represent S.E.M. (a) The YFP/CFP ratio at CAs is higher than in the cytosol (***, p<0,0001, n=37 CAs and n=33 cytosolic regions from 3 embryos) suggesting that FRET is specifically elevated at CAs. (b-c) The ratio remains higher in the absence of any enzymatic activity showing that the conformational change at CAs is kinase activity independent (***, p<0,0001, n=40 CAs and n=30 cytosolic regions from 5 embryos in b; and ***, p<0,0001, n=28 CAs and n=24 cytosolic regions from 3 embryos in c). (a’, b’ and c’) Color coded images of the YFP/CFP intensity ratio of the FAK biosensors, showing higher ratio at CAs and suggesting that FAK is in the open conformation at these sites. (d-e) Epidermal region of embryos immunostained for p-Y576 FAK and acetylated tubulin. (d) FAK is phosphorylated at cell-cell contacts and dense cilia project from the apical surface of multiciliated cells in control embryos. (e) Treatment with the Y15 inhibitor blocks phosphorylation on tyrosine 576 and no signal is detected at cell-cell contacts while cilia project normally. Treatment of embryos with the inhibitor, during ciliogenesis stages, does not affect the density of multiciliated cells along the epidermis (g) or the docking and spacing of basal bodies (i) compared to control embryos (f and h). (j-k) Tracking of the QD flow over the skin of control or inhibitor treated tadpoles. Like controls (j), embryos treated with the Y15 inhibitor (k) produce robust posteriorward flow, showing that inhibition of FAK’s kinase activity does not affect either ciliogenesis or the function of cilia and their ability to generate flow. Scale bars: 2 μμ in a’, b’ and c’; 10 μμ in d and e; 5 μμ in h and i.

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