Transcytosis Maintains CFTR Apical Polarity in the Face of Constitutive

RESEARCH ARTICLE Transcytosis maintains CFTR apical polarity in the face of constitutive and mutation-induced basolateral missorting Aurélien Bidaud-Meynard1,*, Florian Bossard1, Andrea Schnúr1, Ryosuke Fukuda1, Guido Veit1, Haijin Xu1 and Gergely L. Lukacs1,2,‡

ABSTRACT secretion, slow constitutive internalization, and recycling, as well as Apical polarity of cystic fibrosis transmembrane conductance regulator endo-lysosomal/autophagosomal degradation (Ameen et al., 2007; (CFTR) is essential for solute and water transport in secretory epithelia Fu and Sztul, 2009; Gentzsch et al., 2004; Sharma et al., 2004). Given and can be impaired in human diseases. Maintenance of apical polarity the long cellular and PM half-life of CFTR in epithelia (Okiyoneda in the face of CFTR non-polarized delivery and inefficient apical et al., 2018; Sharma et al., 2004; Varga et al., 2008), a slow internalization rate with a high fidelity of apical recycling is essential retention of mutant CFTRs lacking PDZ-domain protein (NHERF1, also − known as SLC9A3R1) interaction, remains enigmatic. Here, we show for the maintenance of Cl transport capacity by minimizing that basolateral CFTR delivery originates from biosynthetic (∼35%) and premature channel degradation and basolateral missorting. endocytic (∼65%) recycling missorting. Basolateral channels are In polarized epithelia, apical and basolateral PM proteins partition retrieved via basolateral-to-apical transcytosis (hereafter denoted between apical sorting endosomes (ASEs), apical recycling apical transcytosis), enhancing CFTR apical expression by two-fold endosomes (AREs), basolateral sorting endosomes (BSEs), and and suppressing its degradation. In airway epithelia, CFTR transcytosis common recycling endosomes (CREs), respectively (Stoops and is microtubule-dependent but independent of Myo5B, Rab11 proteins Caplan, 2014). Previous studies have shown that apically internalized + + and NHERF1 binding to its C-terminal DTRL motif. Increased CFTR is confined to EEA1 ASEs and recycles via Rab11 AREs in basolateral delivery due to compromised apical recycling and airway cells (Cholon et al., 2009; Swiatecka-Urban et al., 2007), in a accelerated internalization upon impaired NHERF1–CFTR PKA-dependent manner (Holleran et al., 2013), but the molecular association is largely counterbalanced by efficient CFTR basolateral basis of rapid degradation of basolaterally delivered and natively internalization and apical transcytosis. Thus, transcytosis represents a folded CFTR is not known. Nevertheless, forced missorting of CFTR previously unrecognized, but indispensable, mechanism for to the basolateral PM suppressed the transepithelial anion secretion maintaining CFTR apical polarity that acts by attenuating its efficiency (Farmen et al., 2005). constitutive and mutation-induced basolateral missorting. Apical confinement of PM proteins can be achieved either by direct apical targeting from the trans-Golgi network (TGN) (Takeda KEY WORDS: CFTR, Airway epithelium, Polarity, Trafficking et al., 2003), indirectly by transcytosis from the basolateral PM (Anderson et al., 2005) or by non-polarized delivery with selective INTRODUCTION retention at the apical PM and rapid degradation from the basolateral The anion selective channel CFTR, a member (ABCC7) of the ABC PM, as described for CFTR (Swiatecka-Urban et al., 2002). The transporter superfamily, is predominantly expressed at the apical association of postsynaptic density/disc large/ZO-1 (PDZ) domain plasma membrane (PM) of secretory and resorptive epithelia adaptor proteins with PM proteins exposing a PDZ-binding motif can (Riordan, 2008). CFTR maintains the ionic, composition and serve as polarized targeting and/or retention signal for both the apical volume homeostasis of the airway surface liquid, which are and basolateral PM (Brône and Eggermont, 2005). NHERF1 and prerequisites for the physiological regulation of mucocilliary NHERF2 (also known as SLC9A3R1 and EBP50, and SLC9A3R2, clearance and innate immune response of airway epithelia, and to respectively) binding to the CFTR C-terminal PDZ-binding motif prevent uncontrolled infection and inflammation of the lung, the (DTRL1480) has been implicated in the apical channel targeting, primary causes of mortality in cystic fibrosis (CF) (Cutting, 2015; retention, confined lateral diffusion and endocytic recycling (Haggie Rowe et al., 2005). An inherited defect in functional expression of et al., 2004; Moyer et al., 1999; Swiatecka-Urban et al., 2002) by CFTR at the apical PM of epithelia causes CF, while acquired loss of tethering it, via ezrin, to the subapical actin network (Short et al., CFTR expression in airway epithelia and pancreatic duct is 1998; Sun et al., 2000). Deletion of the DTRL motif caused by the associated with chronic obstructive pulmonary disease (COPD) S1455X premature termination codon, however, manifests in the and asthma, and pancreatitis, respectively (Schnúr et al., 2016). isolated elevation of sweat Cl− concentration in the absence of a lung Four vesicular transport routes are recognized as important and pancreatic phenotype in the background of an in-frame deletion determinants of steady-state CFTR PM expression: biosynthetic of exon 14a, which encodes a non-functional CFTR (Mickle et al., 1998). These observations, together with extensive mutagenesis 1Department of Physiology, McGill University, Montréal, QC, H3G 1Y6, Canada. studies (Benharouga et al., 2003; Milewski et al., 2005, 2001; 2Department of Biochemistry, McGill University, Montréal, QC, H3G 1Y6, Canada. Ostedgaard et al., 2003) suggest that CFTR tethering to the subapical *Present address: University Rennes, CNRS, IGDR-UMR 6290, F-35000 Rennes, France. cytoskeleton via NHERF proteins is not essential for the channel apical polarity. To identify additional mechanism(s) that preserve ‡ Author for correspondence ([email protected]) CFTR polarity in the absence of the NHERF tethering function, A.B.-M., 0000-0003-0103-3827; G.L.L., 0000-0003-0900-0675 we investigated candidate trafficking pathways, sorting signals and molecular players that may be involved in CFTR polarity

Received 20 October 2018; Accepted 2 April 2019 development. Journal of Cell Science

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Here, we show that both the limited fidelity of apical CFTR activity at the apical and basolateral PM (236 min versus 242 min biosynthetic secretion and recycling from apical endosomes account in CFBE; 164 min versus 156 min in MDCK, Fig. S1E–G), for the basolateral missorting of the channel. Basolateral CFTR which confirms the non-polarized biosynthetic secretion of CFTR accumulation is effectively counteracted by its rapid internalization in both epithelia (Swiatecka-Urban et al., 2002). Considering that rate and basolateral-to-apical transcytosis (hereafter denoted apical basolateral sorting of CFTR takes place at the TGN, we transcytosis), contributing to the high apical levels of the channel hypothesized that natively folded channels are targeted for apical and its metabolic stability. The microtubule-dependent, but transcytosis instead of lysosomal degradation. Myo5B-independent, CFTR transcytosis in airway cells avoids To assess apical transcytosis, we monitored the postendocytic both apical and basolateral endocytic recycling compartments fate of basolateral CFTR–3HA after labeling the channel with anti- but traverses EEA1+ sorting endosomes. Furthermore, apical HA antibody (Ab) capture from the basolateral compartment of transcytosis offsets the augmented basolateral missorting caused filter-grown CFBE and CR-HBE cells at 37°C for 3 h. CFTR–anti- by C-terminal truncations of the PDZ protein-binding sites in HA-Ab complexes were accumulated at the apical PM as well as CFTR, providing a plausible explanation for the isolated resorbtive, intracellularly (Fig. 2A, left panel), visualized by Alexa Fluor 488 but not secretory, defects in the sweat gland and respiratory (AF488)-conjugated secondary Ab and laser confocal fluorescence epithelia, respectively, of individuals harboring the S1445X–CFTR microscopy (LCFM). Following a 2 h chase, the intracellular CFTR mutant. staining was diminished (Fig. 2A, middle panel). No staining was detected in the absence of CFTR (Fig. S1H), indicating that the RESULTS primary Ab staining cannot be attributed to non-specific binding or Human airway epithelial models for studying CFTR polarized CFTR-independent Ab transcytosis. We used similar assays to sorting confirm CFTR transcytosis by electron microscopy (EM) with To investigate polarized trafficking, CFTR containing a 3HA tag in post-embedding secondary Ab-decorated nanogold labeling. Anti- its fourth extracellular loop was expressed in CF bronchial epithelial HA–CFTR complex accumulation at the apical or basolateral PM cells (CFBE41o– or CFBE) under the control of the tetracycline was detected following anti-HA labeling from contralateral transactivator (Ehrhardt et al., 2006; Veit et al., 2012). Filter-grown compartments only in CFTR-expressing CFBE cells (+dox, 3 h, CFBE cells were cultured for 4–5 days post-confluence and CFTR 37°C) (Fig. 2B,C; Fig. S2A), suggesting that CFTR undergoes expression was induced with doxycycline (+dox) to reach similar or bidirectional transcytosis. lower expression to that in Calu-3 cells, a lung adenocarcinoma epithelia cell line endogenously expressing CFTR (Fig. 1A). The Determination of CFTR apical transcytotic flux mass difference between the endogenous and tagged CFTR can To estimate the CFTR transcytotic flux, we labeled basolateral be attributed to 3HA tag and altered complex glycosylation, channels by anti-HA Ab capture and detected the transcytosed CFTR– demonstrated by the mobility shift observed after glycosidase Ab complexes by PM-ELISA at the apical PM (Fig. S2B, right). The digestion (Fig. S1A), but had no significant impact on the channel amount of transcytosed CFTR amount was expressed as the percentage folding, trafficking, stability and function (Peters et al., 2011). As a of the steady-state apical CFTR density (Fig. S2B, left). CFTR second model, CFTR–3HA was expressed in conditionally accumulation at the apical PM increased with increasing basolateral reprogrammed human primary bronchial epithelial cells (CR- Ab-labeling time (0.5–3 h) at 37°C (Fig. 2D). After 3 h of Ab capture, HBE) differentiated under an air–liquid interface for ≥4 weeks 32±3% and 14±3% (means±s.e.m.) of the apical PM pool was labeled, after lentiviral transduction (Fig. 1B). Hallmarks of airway epithelia indicating it had undergone transcytosis, in CFBE and CR-HBE differentiation were assessed: tight-junctional localization of ZO-1 cells, respectively (Fig. 2E). These values, owing to constitutive (also known as TJP1), mucin 5 accumulation in goblet cells and internalization of transcytosed CFTR–anti-HA Ab complexes, are accumulation of acetylated tubulin in cilia, as well as development likely underestimates. The channel transcytosis flux remained of transepithelial electrical resistance (>500 Ω/cm2), airway surface unaltered at ∼50% reduced CFTR PM density, achieved by reduced liquid (ASL, ∼10 µm) and polarized H+/K+ ATPase and transferrin dox induction (0.05 µg · ml−1), which adjusted the mature CFTR receptor (TfR) expression (Fig. 1C; Fig. S1B–D and see Materials expression to ∼25% of that in Calu-3 cells (Fig. 1A; Fig. S2C). and Methods). Apical localization and function of exogenously Several lines of evidence validate the specificity of the ELISA- expressed CFTR–3HA was demonstrated by immunostaining and based transcytosis assay. First, transepithelial or paracellular Ab the presence of dox-inducible and CFTR inhibitor (Inh172)-sensitive transport was ruled out based on the lack of HRP-conjugated Ab PKA-activated short circuit current (Isc) (Fig. 1D,E, see also translocation from the basolateral into the apical compartment at Fig. 6I). 37°C (Fig. S2D,E). Second, neither non-specific mouse nor rabbit Ab was apically transcytosed, indicating that FcRn- or pIgR- CFTR is constitutively transcytosed in human airway mediated Ab transport cannot contribute to the transcytotic signal epithelial cells (Fig. S2F). Likewise, the contribution of fluid-phase transcytosis of Newly synthesized CFTR is targeted randomly to both apical and anti-HA Ab was negligible (Fig. S2F; Ap HA versus Ap+Bl HA). basolateral PM in MDCK cells (Swiatecka-Urban et al., 2002). To Third, CFTR transcytosis was completely abolished on ice or in the study CFTR biosynthetic targeting in CFBE cells, we determined absence of CFTR expression (Fig. 2F). Fourth, we showed the kinetics of domain-specific appearance of a cohort of newly previously that binding of primary anti-HA and secondary Abs do translated CFTR molecules, containing a horseradish peroxidase not alter CFTR turnover (Sharma et al., 2004). Finally, we (HRP) tag in its fourth extracellular loop (Veit et al., 2014). HRP confirmed that endogenous transferrin and EGF receptors (TfR activity, as a surrogate measure of CFTR PM appearance, was and EGFR) also undergo apical transcytosis in CFBE cells (Fig. 2E) monitored by measuring the amount of Amplex Red oxidation to despite the fact that they display highly polarized expression at the the fluorescent resorufin after dox-induction of CFTR–HRP basolateral PM. Jointly, these results suggest that CFTR belongs to a transcription in CFBE and MDCK cells. We could not detect a handful of PM proteins [e.g. TfR, aquaporin 2 (AQP2) and the Cu- significant difference in the delivery kinetics of CFTR–HRP ATPase] whose polarized missorting is ‘corrected’ by transcytosis. Journal of Cell Science

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Fig. 1. Characterization of human CF bronchial epithelial models to study polarized CFTR sorting. (A,B) Immunoblot analysis of CFTR–3HA expression (A) in CFBE cells after 3 days induction with the indicated amount of doxycycline and (B) following lentivirus transduction of CR-HBE cells. For comparison, an equal amount of Calu-3 cell lysate was loaded (A). Black and white arrowheads indicate complex- and core-glycosylated CFTR, respectively. Na+/K+ ATPase was probed as a loading control. (C) CR-HBE cells were differentiated at an air–liquid interface (ALI) and stained for tight junctions (ZO-1, red), mucin 5 (muc5A/C, green), cilia (acetylated-tubulin, green) and nuclei (DAPI, blue). Cells were visualized by laser confocal fluorescence microscopy. The upper and lower panels are vertical and horizontal optical sections, respectively. Ap, apical surface. (D,E) WT-CFTR–3HA is apically expressed in CFBE (D) and CR-HBE (E) cells. Cells were stained for WT-CFTR–3HA (green), actin (red) and DAPI (blue). Scale bars: 5 µm. Representative of at least three independent experiments.

Contribution of biosynthetic and endocytic missorting pulse-labeling that after 24 h dox washout (dox-OFF), CFTR to CFTR basolateral delivery translation was completely inhibited (Fig. 3A). This resulted in Basolateral delivery of newly synthesized CFTR may occur CFTR clearance of the biosynthetic pathways and a 52±3.3% from the endoplasmic reticulum by non-conventional trafficking reduction in channel apical density (Fig. 3B). Considering that the (Gee et al., 2011; Yoo et al., 2002), the TGN (Swiatecka-Urban CFTR transcytosis rate (expressed as percentage of its PM density) et al., 2002) and/or, in principle, from the apical PM via is independent of the apical expression of the channel (Fig. S2C), basolateral (apical-to-basolateral) transcytosis. To evaluate the the 33.6±4.8% attenuation of CFTR transcytosis in dox-OFF biosynthetic pathway contribution to CFTR basolateral delivery, CFBE (Fig. 3C) can be attributed to the lack of basolateral CFTR apical transcytosis was measured following termination of delivery from the TGN. CFTR apical transcytosis was similarly

CFTR transcription/translation. We confirmed by metabolic reduced (∼40–50%) after translational arrest in polarized kidney Journal of Cell Science

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Fig. 2. CFTR is apically transcytosed in human airway epithelial cells. (A) CFTR–3HA was labeled by anti-HA Ab capture (3 h, 37°C) from the basolateral (Bl) or the apical (Ap) compartment in filter-grown CFBE or CR-HBE cells. Cells were chased for 2 h (37°C) in the absence of anti-HA Ab. CFTR–anti-HA Ab complexes were visualized with AF488-anti-mouse Ab and laser confocal fluorescence microscopy. Lower panels represent z-sections. Scale bars: 5 µm. (B,C) Monitoring CFTR transcytosis with immuno-EM. (B) Immuno-EM detection of CFTR-3HA at the apical PM after basolateral labeling with anti-HA (3 h, 37°C) in CFBE. CFTR-anti- HA complexes labeled with secondary Ab conjugated to nanogold (arrow). The inset represents a higher magnification of the indicated area. Scale bar: 0.5 µm. (C) CFTR-expressing or non-expressing (no dox) CFBE cells were labeled either by apical (Ap) or basolateral (Bl) anti-HA Ab capture for 3 h (37°C). CFTR–anti-HA antibody complexes were visualized with anti-mouse Ab conjugated to nanogold particles as in B. The number of nanogold particles per micrometer was counted along the apical and basolateral PM segments (n=2–3 independent experiments, Mann–Whitney U-test). (D) CFTR apical transcytosis was measured over time as in Fig. S2B. (E) Apical transcytosis of CFTR–3HA, and endogenous EGFR and TfR were measured as described in the Materials and Methods in the indicated cells. (F) Apical transcytosis of CFTR is abolished when the channel is basolaterally labeled by anti-HA Ab for 3 h on ice or when CFTR transcription is not induced (- dox) (n=4). Data are means±s.e.m. on each panel. **P<0.01, parentheses indicate the number of independent experiments (D,E) or regions of interest (C).

(MDCK, LLC-PK1) and airway (H441) epithelial cells (Fig. 3C), Under steady-state conditions, we detected only a small fraction of underscoring cell-type-independent biosynthetic polarized the total CFTR–HRP (2.4±0.6%) or CFTR–3HA (3.3±0.4% in missorting of the channel. CFBE, 0.5±0.2% in CR-HBE and 2.7±1.1% in LLC-PK1) expression Journal of Cell Science

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Fig. 3. Apical transcytosis retrieves basolaterally missorted CFTR from the biosynthetic and apical recycling pathways. (A) Onset of CFTR translational arrest in dox-OFF CFBE. Translational inhibition was measured by metabolic pulse labeling with 35S-metionine/35S-cysteine (30 min) after 0–24 h of dox-OFF (i.e. after washout) by fluorography. (B,C) CFTR apical density in CFBE cells (n=12) (B) and apical transcytosis after 24 h of dox-OFF in the indicated epithelial cells (C). (D) CFTR endocytosis and recycling in CFBE cells monitored by PM-ELISA (two-tailed unpaired t-test). (E) Apical CFTR labeling after basolateral-to-apical transcytosis. CFTR was labeled by continuous basolateral anti-HA capture at 37°C. Apical CFTR-Anti-HA Ab complexes were measured by PM-ELISA. (F,G) CFTR basolateral transcytosis detection by immuno-EM in CFBE cells after apical capture of anti-HA Ab (3 h, 37°C). The micrograph on the right is a higher magnification of the selected area. Arrows, CFTR-anti-HA-nanogold-anti-mouse complex. Scale bar: 1 µm. (G) Quantification of the number of nanogold particles at the basolateral PM after apical or basolateral anti-HA Ab uptake (data extracted from Fig. 2C, n=2–3 independent experiments, Mann–Withney U-test). (H) ‘Round-trip’ CFTR transcytosis was monitored by simultaneous exposure of CFBE to anti-HA and anti-mouse Fab–HRP in the apical and basolateral compartment, respectively, for 4 h or 24 h. Transcytosed CFTR was quantified by PM-ELISA (n=3). (I) Inhibition of transcytosis accelerates apical CFTR turnover. After blocking newly synthesized CFTR arrival (dox-OFF, 24 h), CFBE cells were exposed basolaterally to 0.5% tannic acid (15 min, 37°C) or mock treatment. Apical CFTR density was measured after 1 h incubation at 37°C by PM-ELISA (n=3) and expressed as a percentage of control. Ap, apical; Bl, basolateral. Data are means ±s.e.m. on each panel, parentheses indicate the number of independent experiments (C–E) or regions of interest (G). *P<0.05, **P<0.01, ****P<0.0001. at the basolateral PM by assessing HRP activity and PM ELISA, that at least 75% of the apical CFTR pool could be labeled with anti- respectively (Fig. S3A). The modest steady-state basolateral density of HA Ab from the basolateral compartment after 12 h incubation CFTR can be attributed to the concerted result of the surprisingly (Fig. 3E). rapid basolateral (41.8±7.3%/5 min) and the >5-fold slower apical Based on our previous results, we inferred that, of the (8.0±1.0%/5 min) internalization rate, in concert with efficient apical basolaterally delivered and then apically transcytosed CFTR, endocytic recycling (40±2.2%/10 min) of CFTR (Fig. 3D). These ∼33% originates from the TGN (Fig. 3B) and ∼66% comes from processes are complemented by CFTR constitutive apical transcytosis the apical PM/endosomes (Figs 1D and 2A) through apical-to- to preserve the channel polarity, as substantiated by the observation basolateral transcytosis. To visualize CFTR apical-to-basolateral Journal of Cell Science

5 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs226886. doi:10.1242/jcs.226886 transcytosis, the channel was apically labeled by anti-HA Ab EEA1+ ASEs and segregate from TfR+ CREs and Rab11+ AREs for capture (3 h, 37°C), and followed by immunoelectron microscopy apical transcytosis. (immuno-EM). Apically endocytosed CFTR–Ab complex was detectable at the basolateral PM (Fig. 3F,G). Missorting of internalized CFTR for basolateral transcytosis If CFTR is basolaterally missorted from apical endosomes, a To confirm CFTR transcytotic itinerary in CFBE cells and assess ‘round-trip’ transcytosis of apically labeled CFTRs should be the origin of CFTR-loaded apical-to-basolateral transcytotic observed. To this end, we simultaneously exposed CFTR- vesicles, we knocked down putative or proven regulators of CFTR expressing CFBE cells to apical anti-HA Ab and basolateral HRP- endocytic trafficking by means of siRNA (Fig. S4A,B). Although conjugated secondary Fab at 37°C. We monitored the time-dependent ablation of Rab5a and Rab11 affected CFTR recycling (Gentzsch appearance of the HRP activity at the apical PM (Fig. 3H). The results et al., 2004) (Fig. 5A, top; Fig. S4C), they failed to influence CFTR confirmed that apical CFTR-anti-HA Ab complexes successively transcytosis (Fig. 5B). Similar results were observed after ablation underwent apical-to-basolateral targeting, association with HRP– of SNX4, a Rab11-interacting protein, ACAP1, a coat protein Fab, and then basolateral-to-apical transcytosis. associated with recycling endosomes, and SNX17, which have been Jointly, these results suggest that constitutive apical transcytosis implicated in E-cadherin, Glut4 and integrin, but not in CFTR preserves the CFTR apical polarity, which would be compromised recycling (Li et al., 2007; Solis et al., 2013; Steinberg et al., 2012) in the absence of trancytosis. To test this conjecture, we inhibited (Fig. 5A,B). CFTR transcytosis also remained unaltered upon cargo delivery to and retrieval from the basolateral PM by short Rab4a and Rab8a depletion, confirming our immunostaining results tannic acid (TA) crosslinking (15 min, 37°C) (Polishchuk et al., (Figs 4A and 5B). These results suggest that basolateral missorting 2004). Basolateral TA exposure accelerated the loss of apical CFTR of internalized CFTR occurs before its Rab5-dependent entry into to 11.3±1.2%/h as compared to control (∼5.5%/h) in dox-OFF the ASEs and confirm that CFTR apical transcytosis is independent CFBE cells (Fig. 3I). This translates to a reduction of CFTR half-life of the Tf+ CREs and Rab11+ AREs in CFBE, in contrast to other from ∼12.4 h to ∼5.8 h, assuming an exponential decay kinetic in transcytosed cargoes (e.g. pIgR), which transit the CREs and AREs the absence of apical transcytosis, and underscores the significance prior to exocytosis (Jerdeva et al., 2010). of transcytosis in CFTR apical PM stability. Only syntaxin 3 (STX3) knockdown decreased CFTR apical transcytosis significantly (Fig. 5B; Fig. S4A,B). Depletion or The CFTR transcytotic route in airway epithelia cells mutation of the apical targeting signal of this t-SNARE enhances To delineate the transcytotic route of CFTR in CFBE cells, the basolateral mistargeting of NHE3 and GFP-tagged p75 (neurotrophin channel was basolaterally labeled with anti-HA Ab (45 min, 37°C), receptor) (Sharma et al., 2006; Vogel et al., 2015) but did not induce a while HA detection at the cell surface was prevented with anti- global inversion of epithelial (Vogel et al., 2015) or CFTR (Fig. S4D) mouse Fab. CFTR localization was then assessed by examining polarity. Interestingly, a fraction of STX3 was localized at the colocalization with organelle markers for ASEs (EEA1) and AREs basolateral PM and its retrieval through an ubiquitin-dependent (Rab11, herein referring to both Rab11a and Rab11b), as well as mechanism facilitates the recruitment of cargoes into apical exosomes CREs and BSEs (Tf), by indirect immunostaining. Quantification (Giovannone et al., 2017). Considering that biosynthesis arrest of the colocalization between transcytotic CFTR and these abrogated the effect of STX3 siRNA on CFTR transcytosis (Fig. 5C, organelle markers in the 3D-volume of z-stacks was evaluated in dox-OFF), we suggest that STX3, besides controlling CFTR apical three volume clusters, representing the basal, middle and apical exocytosis (Collaco et al., 2010), might be involved in the delivery third volume of epithelia. The results show that basolaterally labeled and/or retrieval of basolaterally targeted channels from the CFTR colocalized with Tf in basal and middle layers, and with biosynthetic pathway. EEA1 in middle and apical layers of the cells, while it marginally colocalized with Rab11 throughout the cells (Fig. 4A,B). CFTR transcytosis requires microtubules After 1 h of apical labeling, CFTR preferentially accumulated in To test the contribution of myosin VB (Myo5B) and microtubules EEA1+ endosomes and, after a 3 h chase, showed modest (MTs), which are important proteins for transport of transcytotic colocalization with Rab11. This is consistent with the longer cargoes (Jerdeva et al., 2010; Tzaban et al., 2009), MTs were residency time for internalized CFTR in sorting rather than disrupted with nocodazole and Myo5B expression was silenced by recycling endosomal compartments (Fig. S3B–E). We could not means of siRNA. While Myo5B knockdown modestly decreased resolve significant colocalization of CFTR with the fast recycling CFTR apical expression as a consequence of reduced entry of apically endosome marker Rab4a or Rab8a (Fig. 4A). This is compatible endocytosed channel into AREs (Swiatecka-Urban et al., 2007), it with the model that basolaterally retrieved CFTR enters a TfR- failed to interfere with apical transcytosis (Fig. 5D; Fig. S4A,E). containing compartment but segregates from this compartment and Nocodazole, however, profoundly decreased CFTR transcytosis, primarily accumulates in EEA1+ endosomes en route to the apical similar to what was observed for TfR (Fig. 5D; Fig. S4F). This cannot PM. To verify functionally whether transcytotic CFTR traverses be attributed to the impeded biosynthetic basolateral transport of TfR-containing endocytic compartments, basolateral endosomes CFTR, as comparable inhibition of transcytosis (54.7±6.7% versus were loaded with HRP–Tf (30 min) to target the CREs (and to a 50.9±6.3%) was observed in dox-OFF cells (Fig. 5D; Fig. S4G). much lesser extent the BSEs) and these compartments were Considering that the channel endocytosis and recycling rates functionally inactivated by HRP-mediated cross-linking (Cresawn remained unaltered upon MT disruption (Fig. S4H), the decreased et al., 2007; Henry and Sheff, 2008). This previously validated CFTR apical expression could be attributed to its impeded protocol efficiently abrogated basolateral recycling and apical transcytosis (13.4±4% in 3 h) (Fig. S4I and Fig. 5D). transcytosis of TfR but failed to alter CFTR transcytosis (Fig. 4C, D). In contrast, ablation of BSEs by using HRP-conjugated wheat Transcytosis counteracts CFTR missorting upon disruption germ agglutinin (HRP–WGA) profoundly decreased CFTR of the interaction with PDZ proteins transcytosis (Fig. 4D). Overall, these results indicate that N- and O-glycans, glycosylphosphatidylinositol (GPI) anchors, and basolaterally internalized CFTRs predominantly accumulate in cytosolic and transmembrane segments have been identified as Journal of Cell Science

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Fig. 4. See next page for legend. apical sorting determinants (Cholon et al., 2009; Kundu et al., 1996; motifs of transmembrane cargoes, represented by tyrosine- (YxxΦ, Lisanti et al., 1989; Potter et al., 2006; Sharma et al., 2006). NPxY, where Φ represents a hydrophobic amino acid) and

Basolateral targeting signals may partly overlap with internalization di-leucine-based (D/ExxxLL) motifs (Stoops and Caplan, 2014). Journal of Cell Science

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Fig. 4. Apical transcytotic route of CFTR in polarized CFBE. Δ6-CFTR was also augmented in MDCK and LLC-PK1 cells (A) Immunocolocalization of transcytotic CFTR. CFTR was labeled by (Fig. 6G). basolateral anti-HA Ab capture (45 min, 37°C), while staining of apically Remarkably, Δ6-CFTR displayed similar biochemical and delivered CFTR-anti-HA complexes were blocked with goat anti-mouse Fab. Then, the colocalization of transcytotic CFTR–anti-HA (green) with EEA1, functional apical PM expression to WT-CFTR despite its Rab11, Rab4 or Rab8 (red) was determined by indirect immunostaining and accelerated internalization and impeded apical recycling, as laser confocal fluorescence microscopy at the apical, middle, and basal planes measured through immunostaining, PM ELISA and short-circuit of filter-grown CFBE cells (lower panels). Vertical optical sections are shown as current (Isc) determination, respectively (Fig. 6H,I). Finally, we top panels. TfR was labeled by means of Cy3–Tf uptake (45 min). Inserts could not resolve a detectable difference in the PM turnover of Δ6- ∼ represent a 3.5-fold magnification of the selected area. Ap, apical PM; F, filter. and WT-CFTR by PM-ELISA or a biotinylation assay (Fig. 6J; Arrows indicate colocalization. (B) Quantitative colocalization of transcytosed ’ Fig. S5D), suggesting that 2-fold augmented round-trip transcytosis CFTR with organellar markers. The Manders colocalization coefficient was Δ calculated on each slice of 3D-volumes [n=15 (EEA1), 20 (Rab11) and 16 (Tf) maintains the WT-like apical PM stability of 6-CFTR and z-stacks from three independent experiments] that were divided into three highlights the sorting capacity and significance of transcytotic approximately equal sections (apical, middle and basal). Two-tailed unpaired pathway to preserve CFTR polarity. t-test. (C) Functional inactivation of TfR-containing endosomes. Tf–HRP was basolaterally loaded (30 min, 37°C) and incubated with H2O2 and DAB or DAB NHERF1 association attenuates CFTR basolateral alone (control). Apical (Ap) transcytosis or basolateral (Bl) recycling of Tf–HRP missorting and transcytosis was measured as described in the Materials and Methods and expressed as percentage of control (n=3). (D) CFTR apical transcytosis is largely To search for additional proteins that may contribute to the recycling independent of the TfR trafficking route but intersects with BSEs. Following fidelity of apically internalized CFTR, the consequence of siRNA- functional ablation of Tf–HRP- or HRP–WGA-loaded endosomes, CFTR mediated depletion of CFTR-interacting PDZ domain proteins was apical transcytosis was measured for 3 h at 37°C. Tf–biotin was used as measured (Figs S4B and S5F). The apical transcytosis of WT- but control. Parentheses indicate the number of independent experiments. Data not Δ6-CFTR or TfR was increased only by NHERF1 knockdown are means±s.e.m. on each panel. *P<0.05; **P<0.01; ***P<0.001; (Fig. 7A,B). NHERF1 siRNA accelerated the internalization and ****P<0.0001; n.s., not significant. inhibited the recycling of WT-, but not Δ6-CFTR, at a moderately altered CFTR apical density (Fig. 7C–E), consistent with previous To identify targeting motifs that may influence CFTR reports (Benharouga et al., 2003; Ostedgaard et al., 2003). Finally, transcytosis efficacy, we inactivated three potential polarized we showed that NHERF1 was partly colocalized with internalized sorting signals (Fig. 6A): first, three di-leucine and tyrosine-based CFTR in subapical vesicular compartments (Fig. 7F,G; Fig. S5G, endocytic motifs (Hu et al., 2001) were mutated to alanine residues H), as well as with the EEA1+ ASEs and, to a lesser extent with the (K3-CFTR); second, two N-linked glycosylation sites (N894D- and Rab11+ AREs (Fig. 7H–I). These results are consistent with N900D-CFTR) were inactivated in the fourth extracellular loop; the physiological relevance of the NHERF1–CFTR interaction in and, finally, considering the role of PDZ proteins in apical targeting, the channel apical retention and endosomal recycling via the tethering and recycling of CFTR, we truncated the PDZ-binding ASEs/AREs compartments in CFBE cells, and provide a plausible motif (DTRL) by eliminating the last six residues (Δ6-CFTR) at the explanation for the augmented basolateral missorting seen for C-terminus. All of these CFTR variants were stably expressed in CFTR upon preventing NHERF1–CFTR association, either by CFBE cells (Fig. S5A). CFTR C-terminal truncation or NHERF1 ablation. Surprisingly, apical transcytosis of Δ6-CFTR, but not K3- or N900D-CFTR, was increased by 49.5±7.8%, suggesting that the Δ6 DISCUSSION mutation compromised the fidelity of CFTR apical biosynthetic Here, we report that apical and basolateral transcytosis represent targeting, endocytic recycling or both processes (Fig. 6B). The previously unrecognized membrane trafficking pathways that comparable expression level of Δ6- and wild-type (WT)-CFTR– significantly contribute to the development and maintenance of 3HA and the inability of Δ6-, but not WT-CFTR to associate with CFTR apical polarity, as well as the phenotypic suppression of the GST–NHERF1 fusion protein were confirmed (Fig. S5B,C). If CFTR C-terminal truncations in multiple epithelial cells, including the amplified basolateral arrival Δ6-CFTR was caused by increased immortalized and primary human airway cells. We show that CFTR missorting at the TGN, inhibition of biosynthetic secretion would apical transcytosis and highly efficient basolateral internalization, affect its apical transcytosis by a greater extent than for WT-CFTR. jointly, play a role in counteracting the constitutive basolateral However, we found that transcytotic flux of Δ6-CFTR, in contrast to missorting of the channel and maintaining a ∼30-fold higher that of WT-CFTR, was insensitive to translation inhibition (Fig. 6C, amount of CFTR at the apical than at the basolateral PM in CFBE dox-OFF), which suggests that the amplified basolateral arrival of cells (Figs S5E and S6). The physiological significance of CFTR Δ6-CFTR likely emanates from augmented missorting at apical apical transcytosis is illustrated by the >2.1-fold reduction of its endosomes and not from the biosynthetic secretion. This inference half-life in conditions with preserved apical internalization but is supported by the increased apical internalization (18.3±3.1 versus inhibition of basolateral retrieval. CFTR basolateral accumulation in 7.4±1.2%/5 min) and impeded recycling (20.5±1.8 versus 35.3± the absence of apical transcytosis would decrease apical Cl− 2.0%/10 min) of Δ6-CFTR relative to WT-CFTR (Fig. 6D,E), secretion by reducing basolateral Cl− entry, causing attenuated which both increase the intracellular pool of the mutant channel. coupled water secretion and mucociliary clearance of airway These data are also in line with CFTR tethering to the subapical epithelia (Ballard et al., 2002; Farmen et al., 2005). actin cytoskeleton and enhanced channel recycling by the PDZ By taking advantage of a Tet-ON-inducible CFTR expression domain-containing NHERF proteins (Haggie et al., 2004; system and the relatively rapid termination of CFTR biosynthesis, Swiatecka-Urban et al., 2002), as well as with the ∼1.5-fold while maintaining ∼50% of the mature CFTR pool, we show that increased basolateral density of Δ6-CFTR (Fig. 6F). The accelerated ∼65% of the basolaterally delivered CFTR originates from apical basolateral transcytosis of Δ6-CFTR mirrors the stimulated apical endocytic vesicles via reversed transcytosis. A similar phenomenon transcytosis of TfR upon inhibition of its basolateral recycling after prevails for endosomal missorting of TfR and EGFR at basolateral

AP-1B ablation (Perez Bay et al., 2013). The apical transcytosis of endosomes (Cotton et al., 2013; Gravotta et al., 2007). In addition, Journal of Cell Science

8 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs226886. doi:10.1242/jcs.226886

Fig. 5. Modulation of CFTR transcytosis. (A,B) CFTR recycling, apical density and transcytosis were measured in CFBE cells silenced for the indicated protein. Results are expressed as a percentage of that seen with the control siRNA (CTL). (C) STX3 may contribute to the basolateral missorting of newly synthesized CFTR. CFTR apical transcytosis was measured after 24 h dox-OFF (i.e. after washout) in CFBE cells transfected with control (CTL) or STX3 siRNAs (siSTX3). (D) CFTR apical transcytosis is MT dependent. CFTR and TfR transcytosis was determined in CFBE cells transfected with control (CTL) or Myo5B-specific siRNAs (siMyo5B 1 or 2) or pretreated for 30 min and during transcytosis with 33 µM nocodazole (Noco) or DMSO. Data are means±s.e.m. on each panel, parentheses indicate the number of independent experiments. *P<0.05; **P<0.01; n.s., not significant. missorting of newly synthesized CFTR from the TGN fuels ∼35% to proceed at CREs (Gravotta et al., 2012), commitment of CFTR to of the basolateral channel delivery, likely in a STX3-dependent apical recycling likely takes place before its Rab5-mediated entry into manner. Interestingly, the contribution of apical transcytosis to ASEs. This inference is supported by the observation that accelerated CFTR polarity and the basolateral channel expression varies endocytosis of CFTR lacking its PDZ-binding motif diplays enhanced between epithelia, suggesting cell-specific alterations in the basolateral targeting and subsequent apical transcytosis, whereas fidelity of CFTR polarity maintenance. Notably, the reduced rate silencing of proteins along CFTR endocytic sorting (Rab5, EEA1 or of CFTR transcytosis in MDCK cells and the limited efficiency of SNX17) or recycling (Rab11, ACAP1, or SNX4) pathways failed to CFTR biotinylation relative to an ELISA-based assay may offer an do so. Second, the results of CFTR mutagenesis and siRNA screens explanation for the previously undetected transcytosis of CFTR or showed that transcytosis of CFTR in CFBE cells is independent of EGFR (Cotton et al., 2013; Swiatecka-Urban et al., 2002). N-glycans, AP-1B, Rab3b, Rab17, Rab25 and the exocyst complex, Based on colocalization and functional endosomal ablation studies, which are essential for the apical transcytosis of pIgR, TfR and FcRn we propose that basolaterally internalized CFTR enters transcytotic (Hunziker and Peters, 1998; Nelms et al., 2017; Perez Bay et al., 2013, endosomes (TEs), which are EEA1+ and show reduced TfR content, 2014; Tzaban et al., 2009; van IJzendoorn et al., 2002). Finally, while that fuel the transcellular migration of CFTR in a MT-dependent, but we cannot rule out a small contribution of the AREs (Fig. 4A,B), Myo5B-independent, pathway (Fig. S6). Several observations suggest transcytotic CFTR seems to mostly avoid the CRE and ARE that CFTR apical transcytosis has some unique characteristics in compartments that are commonly used by other transcytotic cargoes

CFBE cells. First, unlike basolateral sorting of TfR, which is thought (Jerdeva et al., 2010; Lalioti et al., 2016). Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs226886. doi:10.1242/jcs.226886

Fig. 6. The effect of sorting signal and PDZ-domain-binding motif mutation on CFTR polarized sorting and function in epithelia. (A) Mutagenesis of sorting signals that may influence CFTR transcytosis. Endocytic consensus sites Tyr1424 (Y) and Leu1430-1431 (L) were mutated to Ala (A). NN represents Asn895 and Asn900, two N-linked glycosylation sites in the MSD2. DTRL, PDZ-domain-binding motif at the C-terminus; MSD, membrane spanning domain; NBD, nucleotide- binding domain; R, regulatory domain. (B) Deletion of the DTRL motif (Δ6) increases CFTR apical transcytosis relative to WT-CFTR, but the K3 and N900D mutations do not. (C) Biosynthetic basolateral missorting of CFTR depends on the binding of PDZ-domain protein(s). CFTR apical transcytosis was measured in control (+dox) or dox-OFF (24 h after washout) cells. (D) Apical but not basolateral CFTR internalization is accelerated upon deletion of the PDZ-binding motif (two-tailed unpaired t-test). (E) The Δ6 mutation impairs CFTR apical recycling (n=6), as measured by PM-ELISA. (F) WT- and Δ6-CFTR basolateral cell-surface density was measured by PM-ELISA (two-tailed unpaired t-test). (G) The effect of the Δ6 mutation on CFTR apical transcytosis in the indicated cell line. (H) Δ6-CFTR is predominantly detected at the apical PM in CFBE by indirect immunostaining, performed as in Fig. 1D. Scale bar: 5 µm. (I) The apical PM density and channel function of WT- and Δ6-CFTR were measured by PM-ELISA and by determining the 10 µM forskolin-stimulated short-circuit current (Isc), respectively, and corrected for their relative mRNA level (n=3). The CFTR-mediated Isc was quantified after inhibition with CFTRinh 172. (J) Apical stability of WT- and Δ6-CFTR is similar. CFTR was labeled with anti-HA (1 h, ice) and chased for the indicated time before PM-ELISA (two-tailed unpaired t-test). Data are mean±s.e.m. on each panel, parentheses indicate the number of independent experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; n.s. not significant.

Interestingly, constitutive TfR transcytosis appears to preferentially exocytosis in certain epithelia, as proposed for the TfR in MDCK bypass the Rab11+ AREs in CFBE cells, which express AP-1B cells (Perez Bay et al., 2013). These alternative, but parallel, pathways (Figs S7A–C and S4J), similar to that of the neonatal Fc receptor in may be able to compensate for each other activity upon loss of MDCK cells (Tzaban et al., 2009). Hence, it is plausible that both the function, which would prevail for the EEA1-independent CFTR transcytotic and endocytic/recycling pathways for CFTR and other transcytosis (Fig. 5B). The activation of an alternative slow recycling cargoes interconnect at a common compartment with sorting capacity pathway may also explain the surprising inability of Rab11 silencing for direct or indirect cargo targeting through the AREs prior to to reduce the apical density of CFTR, in contrast to what is seen with Journal of Cell Science

10 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs226886. doi:10.1242/jcs.226886

Fig. 7. NHERF1 ablation phenocopies the Δ6-CFTR cellular phenotype. (A) CFTR transcytosis was assayed after siRNA-mediated knockdown of the indicated PDZ proteins in CFBE and expressed as the percentage of that seen with the control siRNA (CTL). (B–E) NHERF1 silencing mirrors the effect of the PDZ-binding motif truncation of CFTR. CFBE cells were transfected with control siCTL or NHERF1 1 siRNA and then assayed for (B) CFTR and TfR transcytosis following 3 h basolateral anti-HA or Tf-HRP capture, (C) CFTR internalization, (D) CFTR recycling, (E) CFTR apical PM density (n=10), measured as described in the Materials and Methods. (F) NHERF1 colocalizes with the apical endocytosed CFTR. CFTR was labeled by anti-HA capture (1 h, 37°C) from the apical compartment. Residual CFTR apical PM was blocked with goat anti-mouse Fab on ice. NHERF1 (red) and CFTR (green) were visualized by indirect immunostaining and laser confocal fluorescence microscopy in permeabilized cells. The upper panel represents a z-section. The insert is a 4-fold magnification of the indicated area. Scale bar: 5 µm. (G) Quantification of colocalization between NHERF1 and subapical endocytosed WT- and Δ6-CFTR in CFBE cells (two- tailed unpaired t-test). (H,I) NHERF1 can localize to both ASEs and AREs. Fixed and permeabilized filter-grown CFBE cells were stained for NHERF1 (green) and EEA1 (red, H) or Rab11 (red, I). Arrows indicate colocalization. Scale bars: 1 µm. Data are mean±s.e.m. on each panel, parentheses indicate the number of independent experiments (A–D) or number of regions of interest from two independent experiments (G). *P<0.05; **P<0.01; ****P<0.0001; n.s. not significant.

Rab5a or ACAP1 depletion, despite inhibition of the channel et al., 2005, 2007). In addition, the combined effect of the lysosomal endocytic recycling (Fig. 5A), which is consistent with the operation and basolateral targeting of some internalized CFTRs and the short of alternative recycling pathways described for CFTR in other duration of the recycling assay (10 min), to minimize re- epithelia (Saxena et al., 2006; Silvis et al., 2009; Swiatecka-Urban internalization of exocytosed channels, are jointly responsible for Journal of Cell Science

11 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs226886. doi:10.1242/jcs.226886 the underestimation of CFTR recycling efficiency in our assay. This Wolde et al., 2007). Considering that deletion of the PDZ motif inference is in line with the observation that Rab11 silencing only decreased CFTR biosynthetic delivery to the basolateral PM enhances CFTR intracellular accumulation by 10% after 30 min (Fig. (Fig. 6C, dox-OFF), we speculate that NHERF1 overexpression S4C and Materials and Methods). Improved assay sensitivity, may rescue ΔF508 not only by increasing its retention/recycling at permitting shorter recycling time and correction for apical CFTR the apical PM but also by promoting its biosynthetic over endocytic re-internalization, will be necessary to achieve a more-precise basolateral missorting, which may further reduce the lysosomal assessment of the role of Rab11 in CFTR trafficking in CFBE cells. degradation propensity of the channel upon reducing its basolateral Comparison of the exocytotic machinery of apical CFTR in transcytosis. relation to other transcytotic cargoes derived from biosynthetic, recycling and transcytotic pathways of airway epithelia remains to MATERIALS AND METHODS be investigated. Intriguingly, we were unable to detect a significant Antibodies and reagents reduction of CFTR apical transcytosis and PM density upon Antibodies used in this study are listed in Table S1. Nocodazole, sodium 2- ′ ablation of Myo5B, which has been implicated in maintaining mercaptoethanesulfonate (MESNA), 3,3 -diaminobenzidine tetrahydrochloride CFTR apical expression along recycling and secretory pathways in (DAB), horseradish peroxidase (HRP) and tannic acid were purchased from Sigma-Aldrich. Y-27632 was from Tocris. HRP–Tf and Ultroser-G were CFBE and CaCo2 cells (Swiatecka-Urban et al., 2007; Vogel et al., purchased at Jackson Immunoresearch and Pall Corporation, respectively. 2015). Recent studies were also unable to detect profound changes in functional CFTR expression in intestinal epithelia cells Cell culture and medium expressing an inactive Myo5B or following Myo5B knockdown CFBE, Calu-3 and 3T3-J2 cells are generous gifts from Dieter Gruenert (Kravtsov et al., 2016). Furthermore, Myo5B-independent apical (University of California-San Francisco, USA), John W. Hanrahan (McGill − polarization of CFTR is in line with the augmented Cl secretion University, Canada) and Bob Scholte (Erasmus MC, Rotterdam, The seen in microvillus inclusion disease, which is caused by loss of Netherlands), respectively. LLC-PK1 and NCI-H441 were purchased from function of Myo5B (Kravtsov et al., 2016). the ATCC. MDCKII was described before (Benharouga et al., 2003; Veit Our results expand the established role of NHERF1 in CFTR et al., 2012). CFBE cells were propagated in minimal essential medium traffic at multiple cellular locations. It is known that by tethering (MEM) supplemented with fetal bovine serum (FBS), L-glutamine and CFTR to the subapical cytoskeleton, NHERF1 enhances the HEPES (Invitrogen) on coated plastic flasks as described previously (Veit et al., 2012) and were seeded and differentiated for ≥3 days on coated plastic channel apical retention (Haggie et al., 2004; Swiatecka-Urban wells or polyester permeable supports (Transwell filters, Corning). Both et al., 2002). Importantly, CFTR retention at basolateral PM is MDCKII and LLC-PK1 cells were cultured in Dulbecco’s modified Eagle’s compromised as a consequence of modest expression level of medium (DMEM; Invitrogen) supplemented with 10% FBS, and NCI-H441 NHERFs, ezrin and cortical actin (Reczek et al., 1997; Short et al., cells were cultured in RPMI-1640 (Gibco) supplemented with 10% FBS and 1998), accounting for the unprecendently fast CFTR internalization, 10 mM Hepes, and were differentiated on polyester permeable supports for ensuring its low basolateral PM density. Here, we provide evidence >3 days post-confluence. In these cell types, CFTR displayed a similar for enhanced colocalization of NHERF1 with the endocytic pool of polarized expression at the apical PM (Benharouga et al., 2003; Veit et al., WT-CFTR relative to that of Δ6-CFTR (Fig. 7F,G), supporting the 2012) (Fig. S3A). Human lung adenocarcinoma Calu-3 cells were cultured paradigm that NHERF1-mediated sorting directs the channel in DMEM/F12 (Invitrogen) supplemented with 10% FBS. All cells were towards recycling at subapical endosomes (ASEs and/or AREs) maintained in a 37°C incubator under 5% CO2. CFBE, MDCK, NCI-H441 and LLC-PK1 cell lines expressing inducible WT- and Δ6- CFTR with a (Cardone et al., 2015; Cushing et al., 2008). Finally, we demonstrate 3HA or HRP tag were generated using the ClonTech pLVX-Tight-Puro that PDZ motif elimination has a negligible influence on CFTR lentivirus technology, as described previously (Veit et al., 2014, 2012, apical expression, transport activity and stability. Increased CFTR 2018) and induced with 250–500 ng/ml dox. MDCKII cells, stably basolateral internalization and apical transcytosis flux largely offset expressing WT- or Δ6-CFTR-3HA, were generated by transduction with the accelerated removal of the channel and decreased recycling at retroviral particles (Benharouga et al., 2003). the apical PM. These results expand on previous observations obtained on CFTR variants with compromised binding for PDZ CR-HBE characterization and expression of CFTR-3HA proteins (Benharouga et al., 2003; Ostedgaard et al., 2003) and offer Primary human bronchial epithelial cells (HBE) cells were isolated in Walter a plausible explanation for the isolated elevation of sweat Cl− E. Finkbeiner’s laboratory under approval from the University of California, concentration in the absence of pancreatic and lung phenotype in San Francisco Committee on Human Research as well as purchased from the individuals harboring a single copy of CFTR lacking the C-terminal CF Canada Primary Airway Cell Biobank at McGill University. For both 26 residues (S1455X-CFTR) (Mickle et al., 1998). Finally, primary HBE sources, informed consent was obtained for all lung tissue donors and all clinical investigation was conducted according to the principles increased apical transcytosis of CFTR may also explain the mild expressed in the Declaration of Helsinki. HBE were conditionally CF cellular phenotype (Ameen et al., 2007) of the naturally reprogrammed and differentiated according to a modified protocol of Liu occurring N287Y-CFTR mutant, exhibiting accelerated apical et al. (Liu et al., 2012). Briefly, HBE cells were cultured on irradiated 3T3-J2 internalization (Silvis et al., 2003). fibroblasts in proliferation F-Medium (Liu et al., 2012) with 10 µM of ROCK Whether transcytotic CFTR routes are affected by CF-causing inhibitor Y-27632. After expansion, cells were plated on collagen IV-coated mutations and can influence a non-native channel degradation via transwell filters and differentiated in Ultroser-G medium (Neuberger et al., ubiquitin-dependent lysosomal targeting from the PM, such as 2011) at the air–liquid interface for at least 4 weeks with the basolateral ΔF508 (Okiyoneda et al., 2018), remains to be explored. medium being changed every 2-3 days. CR-HBE cells constitutively – Interestingly, the CF-causing folding mutant P67L-CFTR expressing WT-CFTR 3HA were developed by transducing the cells with lentiviral particles encoding the WT-CFTR–3HA in pTZV4-CMV-IRES- (Bagdany et al., 2017; Sabusap et al., 2016) displays considerably puro (Open Biosystems) as described previously (Veit et al., 2012) during reduced transcytotic flux that can be restored to that of WT upon proliferation, followed by 2 days of puromycin selection before seeding on folding correction with VX-809 (data not shown). Intriguingly, filter supports. The Air Surface Liquid (ASL) height was determined after overexpression of NHERF1 or impeding lysosomal CFTR by staining the ASL with 2 mg/ml Tetramethylrhodamine-conjugated dextran ablation of CFTR-associated ligand (CAL, also known as GOPC), (D1819, 70,000 MW, Neutral, Invitrogen), dispersed in a low boiling point partly rescues the PM expression of ΔF508 (Guerra et al., 2005; perfluorocarbon (Fluorinert FC-72, boiling point 56°C, 3 M Company) and Journal of Cell Science

12 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs226886. doi:10.1242/jcs.226886 the epithelia with Cell tracker (Invitrogen). The ASL height was determined in 37°C. Then cells were washed with buffer H (154 mM NaCl, 3 mM KCl, 3–10 areas of two filters using a Zeiss700 upright fluorescence laser confocal 10 mM Hepes pH 7.8, 1 mM MgCl2, 0.1 mM CaCl2 and 10 mM glucose) microscope equipped with an environmental chamber at 37°C and 5% CO2. and incubated further in buffer H, supplemented with Amplex Red at 37°C. Apical and basolateral media were sampled for Amplex Red fluorescence ∼ CFTR cell surface ELISA and transcytosis assay every 5 min for 20 min. Non-induced cells were used to determine the CFTR apical density and apical transcytosis were measured by performing background fluorescence. PM-ELISA. Cells were incubated with anti-HA antibody (1:1000) in the apical (apical density) or basolateral (transcytosis) compartment, respectively, Basolateral PM turnover inhibition by tannic acid at 37°C for 0.5–4 h. After washing of the apical PM with ice-cold PBS The basolateral membrane turnover was inhibited by tannic acid, using a (Gibco) supplemented with 1 mM MgCl2 and 0.1 mM CaCl2 (PBSCM), all modified protocol of Polishchuk et al. (2004). CFBE cells were treated for the cells were incubated apically with HRP-conjugated anti-mouse-IgG or 15 min with 0.5% tannic acid in serum-free medium, then washed two times Fab (1:1000) in PBSCM with 0.5% bovine serum albumin (PBSCM-BSA) in serum-free medium and once in full medium followed by PM-ELISA. (1 h, on ice). Cells were washed with PBSCM and the HRP activity in the Reversibility of the tannic acid treatment was ascertained by measurement of apical compartment was determined in the presence of Amplex Red CFTR–anti-HA complex basolateral uptake after an 8 h chase (data not fluorogenic substrate (Invitrogen) at 544 nm excitation and 590 nm shown). emission wavelengths, using a POLARstar OPTIMA (BMG Labtech) or a Tecan Infinite M1000 (Tecan Group) fluorescence plate reader. CFTR apical Inactivation of endosomal compartments by HRP-mediated transcytosis was expressed relative to its apical density, measured in parallel ablation [percentage Ap transcytosis=(Ap signaltranscytosis/Ap signalapical density)×100]. Tf-containing endosomes were functionally ablated as described previously Non-specific Ab binding was determined by using either non-expressing (Cresawn et al., 2007) with the following modifications. Tf-depleted CFBE CFBE cells or replacing the anti-HA with non-specific mouse IgG under the cells were loaded with HRP–Tf (5 µg/ml) for 30 min (37°C) from the same experimental conditions. EGFR transcytosis was measured by using the basolateral chamber. Cells were then washed with ice-cold PBSCM same PM-ELISA assay, with anti-EGFR instead of anti-HA antibody. To and incubated twice for 5 min with 150 mM NaCl and 20 mM citric acid, monitor TfR apical transcytosis, endogenous Tf was first depleted in serum- pH 5.0 to remove cell surface-bound HRP–Tf. WGA-labeled endosomes free medium (1 h), before incubation with HRP–Tf or HRP (both 5 µg/ml) in were functionally inactivated after 20 min internalization of WGA–HRP the apical or basolateral compartment. After 3 h, the peroxidase activity was (10 µg/ml, Sigma) from the basolateral compartment. WGA–HRP remaining measured on the apical PM and in the medium. TfR transcytosis was at the cell surface was removed with 3×10 min incubation with 100 mM N- expressed as the signal intensity in the apical compartment relative to that of acetyl-D-glucosamine. Endosomal functional ablation was induced by the steady-state TfR density at the basolateral PM. ‘Round-trip’ transcytosis initiating crosslinking upon the addition of 0.1 mg/ml DAB and 0.025% was measured by incubating the cells simultaneously with mouse anti-HA H2O2 for 1 h on ice in the dark. The reaction was quenched with PBSCM antibody (1:1000) in the apical chamber and with HRP-conjugated anti- containing 1% BSA. As controls, DAB or H2O2 were omitted, or Tf–biotin mouse-Fab (1:1000) (Jackson Immunoresearch) in the basolateral chamber and WGA–Alexa Fluor 647 were used instead of Tf–HRP and WGA–HRP, for 4 to 24 h at 37°C. Cells were then extensively washed with ice-cold respectively. PBSCM and assessed for HRP activity in the apical compartment. siRNA-mediated inhibition of gene expression CFTR internalization and recycling assays Stealth siRNAs and siRNAs were purchased from Invitrogen, Origene and The CFTR internalization rate was measured by determining the rate at which Qiagen, respectively (Table S2). To limit off-target effects, the siRNAs were CFTR–Ab complexes disappeared from the apical PM by ELISA, as described used at a final concentration of 25 nM and phenotypic screens were carried previously (Glozman et al., 2009). Apical CFTR was labeled with anti-HA out using pools of two to four siRNAs. For filter-based experiments, CFBE (1:1000) for 1 h on ice. After washing with ice-cold PBSCM, one plate was cells were electroporated using the Neon Transfection system (Invitrogen) incubated for 5 min at 37°C (denoted P5′), while the control plate was kept on ice with three 10 ms 1500 V pulses using 100 µl Neon pipette tips according to (denoted P0). The amount of CFTR remaining at the PM was determined using the manufacturer’s protocol. Cells on coated plastic were transfected with HRP-conjugated secondary Ab and Amplex Red in both plates. The Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s internalization rate was expressed as the percentage of the initial amount of protocol and then were polarized for 4 days in presence of dox-induced CFTR removed in 5 min [percentage CFTR internalization=(P0−P5′)×100/P0]. CFTR expression. The CFTR recycling rate was assessed by monitoring the exocytosis of internalized CFTR–Ab complexes at the apical PM. To this end, CFTR- Immunofluorescence microscopy and colocalization expressing CFBE cells were seeded on four plates (P1–P4). After anti-HA Differentiated filter-grown CFBE cells were fixed in 4% paraformaldehyde binding to apical CFTR (1 h on ice, P1–P4), the CFTR–Ab complex was and permeabilized with 0.2% Triton X-100. After blocking in PBSCM-BSA, allowed to internalize for 30 min at 37°C (P2, P3 and P4) while a control plate cells were incubated with primary Ab, washed with PBSCM and incubated was kept in cold medium (P1). The anti-HA antibody remaining on the PM with Alexa Fluor-conjugated secondary Abs. Extensively washed and cut-out was blocked with anti-mouse monovalent Fab (Jackson ImmunoResearch, pieces of filters were mounted between a glass slide and coverslip. For 1:75, 1 h, on ice, P3 and P4). Recycling of endocytosed CFTR was elicited at monitoring intracellular CFTR colocalization, CFTR was labeled by anti-HA 37°C for 10 min (P4), while a blocking efficiency plate was kept in cold capture at 37°C for the indicated time. When indicated, PM CFTR staining was medium (P3). All plates were apically incubated with HRP-conjugated anti- blocked with anti-mouse Fab prior to fixation (1 h, on ice). TfR was visualized mouse-IgG or Fab (1:1000) for 1 h on ice, washed and peroxidase activity was by exposing the cells to Cy3–Tf (20 µg/ml, Jackson Immunoresearch, 45 min– measured. The recycling efficiency in 10 min was expressed as a percentage 1 h at 37°C) in the basolateral compartment after depletion of the endogenous of the endocytosed pool of CFTR, taking into consideration the blocking Tf in serum-free medium (1 h, 37°C). Nuclei were stained with DAPI. efficiency of the anti-mouse Fab [percentage apical recycling=(P4−P3)×100/ Horizontal optical sections (20–30) were acquired using a LSM-710 or LSM- (P1−P2)]. In addition, we also took into consideration that the recyclable 780 LFCM equipped with a Plan-Apochromat 63×/1.40 oil objective (Carl endosomal CFTR pool was slightly reduced due to missorting of internalized Zeiss), reconstituted using the Zen 2012 software package and representative CFTR to the basolateral PM (∼5.6%) and lysosomes (∼5.6%) (Fig. 3D). vertical sections are shown. For colocalization study, the Manders’ colocalization coefficient was calculated using the JACOP plugin of Fiji Time course of polarized delivery of newly synthesized software (Bolte and Cordelieres, 2006) after background subtraction and CFTR–HRP thresholding from 3D volumes of z-stacks. Then, the colocalization coefficient To measure the kinetics for delivery of newly translated CFTR to the apical of all the slices of a z-stack was sorted in three groups to evaluate the – ∼ colocalization at the apical, middle and basal volume of the cells.

and basolateral PM, CFTR HRP expression was induced for 3.5 h at Journal of Cell Science

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Monitoring CFTR apical stability by cell surface biotinylation and was immunoprecipitated with a mixture of M3A7 and L12B4 anti-CFTR immunoblotting Abs. Following autoradiography, radioactivity incorporated into CFTR was Apical surface proteins of filter-grown CFBE cells were biotinylated for quantified by phosphorimage analysis, using a Typhoon workstation (GE 15 min on ice with 1 mg/ml EZ Link sulfo-NHS-SS-biotin (Thermo Fisher Healthcare). Scientific) in buffer H (as above). Excess biotin reagent was quenched with 100 mM glycine in PBSCM. Cells were then shifted to 37°C for 0, 4, 12 or EndoH and PNGase F digestion of CFTR 24 h before being lysed in RIPA buffer (150 mM NaCl, 20 mM Tris-HCl, CFBE and Calu-3 cells expressing CFTR–3HA and endogenous CFTR 1% Triton X-100, 0.1% SDS and 0.5% sodium deoxycholate, pH 8.0) were grown on 6-cm coated plastic dishes for 4–5 days post confluency. containing protease inhibitor (5 µg · ml–1 leupeptin, 5 µg · ml–1 pepstatin, CFTR expression in CFBE cells was induced by treatment with 250 ng/ml 500 µM PMSF, all from Sigma-Aldrich). Biotinylated proteins were isolated dox for 4 days. Cells were lysed (0.3% Triton X100, 150 mM NaCl, 20 mM from postnuclear lysates with streptavidin–agarose beads (Invitrogen) at 4°C Tris-HCl pH 8.0) and after centrifugation (at 4°C, 12,000×rpm for 10 min), with end-over-end rotation. Proteins were eluted with 5× Laemmli sample the supernatants were digested with EndoH or PNGase F enzyme according buffer and visualized by immunoblotting with anti-HA antibody or a to the manufacturer’s protocol. Samples were immunoblotted with anti- mixture of L12B4 and M3A7 mouse monoclonal anti-CFTR Abs followed CFTR antibodies (L12B4, M3A7). by either IRDye 800-conjugated anti-mouse-IgG Abs (Licor Biosciences, Lincoln, NE) with the Odyssey Infrared Imaging System (Licor RT-qPCR Biosciences) or using enhanced chemiluminescence as described For WT- and Δ6-CFTR mRNA expression, total RNA was extracted from (Benharouga et al., 2001). CFBE lysed in Qiazol and analyzed using the one-step QuantiFast SYBR Green RT-PCR kit (Qiagen, 204154) as recommended by the manufacturer. Electron microscopy Briefly, total RNA was extracted from polarized CFBE grown on coated plastic Filter-grown CFBE cells were incubated with anti-HA antibody (1:500) in in 24-well plates using the miRNeasy Mini Kit (Qiagen, 217004). Reverse the basolateral or apical compartment for 3 h at 37°C. Cells were washed, transcription and PCR amplification was performed sequentially in a fixed, permeabilized and incubated with 1.4 nm nanogold-conjugated anti- Stratagene Mx3005P real-time thermocycler (Agilent, 401513) during the mouse Fab fragment (1:50) (Nanoprobes, Yaphank, NY) for 1 h on ice. Cell same thermocycler protocol on 50–100 ng total RNA, as determined by monolayers were then post-fixed with 1% osmium tetroxide in 0.1 M measuring its Nanodrop UV-Vis light absorbance. The abundance of phosphate buffer for 1 h at 4°C. Samples were dehydrated through a series of transcripts was determined using a SYBR Green fluorescence amplification graded ethanol baths and embedded in epon resin. Semi-thin sections of curve and its intersection with a preset threshold, yielding a Ct value. Data were ∼1 µm were obtained with a diamond knife on an ultramicrotome (Ultracut analyzed by efficiency-corrected comparative quantification with MxPro E, Reichert-Jung) and stained with 1% Toluidine Blue. Then, 60-nm-thick QPCR software (Agilent) and the variations in initial RNA loading amount was sections were cut and counterstained with 4% uranyl acetate and Reynold’s normalized by using GAPDH as a reference gene. mRNA expression lead citrate. Sections were observed under a Philips CM120 electron differences between samples were reported as the percentage abundance microscope equipped with a Gatan digital camera. The number of nanogold relative to a reference sample (e.g. WT-CFTR). PDZ protein downregulation particles per micrometer of PM was calculated using the ImageJ software. was evaluated with a Quanti-Tect reverse transcription kit (Qiagen) as Post-labeling steps and imaging were performed at the EM facility of the previously described (Veit et al., 2012). Primers are given in Table S3. Department of Pharmacology, McGill University. Statistical analysis Short-circuit current measurement Results are presented as mean±s.e.m. of the number of independent The Isc of CFTR-expressing CFBE cells was measured on cells differentiated experiments indicated in the figure legends, as biological replicates. Unless on 12 mm Snapwell filters (Corning) mounted in Ussing chambers and specified, P-values were calculated with the means of at least three bathed in Krebs-bicarbonate buffer (140 mM Na+, 120 mM Cl−, 5.2 mM K+, independent experiments by two-tailed paired Student’s t-test and P<0.05 − 2+ 25 mM HCO3 , 2.4 mM HPO4, 0.4 mM H2PO4, 1.2 mM Ca ,1.2mM was considered significant. Normal distribution of data and homogeneity of Mg2+ and 5 mM glucose, pH 7.4) at 37°C in the presence of an apical-to- variance were validated by calculating the skew factor (−2> skew <2) and basolateral chloride gradient. To functionally isolate apical PM, the performing the F-test, respectively. For non-normal data, a Mann-Withney contralateral domain was permeabilized with 100 μMamphotericinB.In U-test was used for calculating the P-values, as indicated in the figure each experiment, 100 µM amiloride and 20 µM forskolin were added legends. For normal distributions with non-homogenous variances, the sequentially to the apical and basolateral side to inhibit the epithelial Na+ Welch correction was applied to two-tailed unpaired t-test to calculate the channel and activate CFTR, respectively. CFTR activity was inhibited by P-values, as indicated in the figure legends. All ELISA-based assays were 20 µM of channel blocker CFTRinh 172 (Tocris Bioscience, Ellisville, MO). performed using two to four technical replicates, except for CFTR–HRP polarized delivery (one or two wells assayed per timepoint). Short-circuit Recombinant NHERF1 purification and pulldown current measurement and quantitative PCR (qPCR) were performed with GST or GST–NHERF1 was expressed in the BL21 Escherichia coli by two technical replicates. means of a pGEX-4T plasmid. Bacterial pellets were resuspended in 50 mM Tris-HCl pH 8, 50 mM NaCl, 5 mM EDTA, 0.5% NP-40, 5% glycerol Acknowledgements (50 µl/ml) and sonicated. The lysate was centrifuged at 23,700 g (30 min, We are grateful to J. W. Hanrahan, B. Scholte and to the late D. Gruenert for cell lines 4°C) and passed through a Dowex 50X2-400 ion-exchange resin (Acros and W. E. Finkbeiner for primary HBE. We thank G. Bertolin and M. Tramier for Organics). The flow through was incubated with glutathione–Sepharose 4B helpful discussions on colocalization quantification, R. Robert and R.G. Avramescu beads (GE Healthcare) for 2 h at 4°C. After three washes, beads were for help with initial Isc and qPCR measurements, respectively, and D. da Fonte for TEER measurement. Post-labeling steps and EM imaging were performed at the EM incubated with the post-nuclear supernatant of RIPA lysate (obtained as Δ facility of the Department of Pharmacology, McGill University. The core facility of above) from CFBE cells expressing WT- or 6-CFTR for 2 h at 4°C under W. E. Finkbeiner (Department of Pathology, University of California, San Francisco) rotation. After three washes with RIPA medium, co-isolated CFTR was was supported by grants from National Institutes of Health [DK072517] and Cystic immunoblotted. Fibrosis Foundation Research and Translational Core Center [VERKMA15R0].

Metabolic pulse labeling Competing interests Post-confluent filter-grown CFBE cells were incubated with methionine- The authors declare no competing or financial interests. and cysteine-free α-MEM medium for 45 min at 37°C. Cells were then pulse 35 35 labeled in the presence of [ S]-methionine and [ S]-cysteine (0.1 mCi/ml; Author contributions Perkin Elmer, Waltham, MA) from the basolateral compartment for 30 min Conceptualization: A.B.-M., F.B., G.L.L.; Methodology: A.B.-M., F.B., G.L.L.; at 37°C in a humid chamber. After washing with ice-cold PBSCM, CFTR Validation: A.B.-M., G.L.L.; Formal analysis: A.B.-M., F.B., A.S., R.F., G.V., H.X.; Journal of Cell Science

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Investigation: A.B.-M., F.B., A.S., R.F., G.V., H.X.; Writing - original draft preparation: epithelia: low levels of expression are sufficient to correct Cl− transport and A.B.-M., G.L.L.; Writing-review and editing: A.B.-M., G.L.L.; Visualizatioin: A.B.-M., overexpression can generate basolateral CFTR. Am. J. Physiol. Lung Cell. Mol. G.L.L.; Supervision: G.L.L.; Project administration: G.L.L.; Funding acquisition: Physiol. 289, L1123-L1130. doi:10.1152/ajplung.00049.2005 G.L.L. Fu, L. and Sztul, E. (2009). ER-associated complexes (ERACs) containing aggregated cystic fibrosis transmembrane conductance regulator (CFTR) are degraded by autophagy. Eur. J. Cell Biol. 88, 215-226. doi:10.1016/j.ejcb.2008. Funding 11.003 This work was supported by the the Canadian Institutes of Health Research (MOP- Gee, H. Y., Noh, S. H., Tang, B. L., Kim, K. H. and Lee, M. G. (2011). Rescue of 142221), National Institute of Diabetes and Digestive and Kidney Diseases ΔF508-CFTR trafficking via a GRASP-dependent unconventional secretion (5R01DK075302) and the Cystic Fibrosis Canada. A.B.-M. was supported by a pathway. Cell 146, 746-760. doi:10.1016/j.cell.2011.07.021 travel grant from Cystic Fibrosis Canada. F.B. was a recipient of a Richard & Edith Gentzsch, M., Chang, X.-B., Cui, L., Wu, Y., Ozols, V. V., Choudhury, A., Pagano, Strauss Fellowship, McGill Univeristy. G.L.L. is a Canada Research Chair. R. E. and Riordan, J. R. (2004). Endocytic trafficking routes of wild type and Deposited in PMC for release after 12 months. ΔF508 cystic fibrosis transmembrane conductance regulator. Mol. Biol. Cell 15, 2684-2696. doi:10.1091/mbc.e04-03-0176 Supplementary information Giovannone, A. J., Reales, E., Bhattaram, P., Fraile-Ramos, A. and Weimbs, T. Supplementary information available online at (2017). 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