MBoC | ARTICLE Glucocorticoids and myosin5b loss of function induce heightened PKA signaling in addition to membrane traffic defects Radia Fortezaa,†, M. Kaimul Ahsanb,†, Fernando Cartón-Garcíac, Diego Arangoc, Nadia A. Ameenb,*, and Pedro J. Salasa,* a Department of Cell Biology, Miller School of Medicine, University of Miami, Miami, FL 33136; bDepartment of Pediatrics, Yale School of Medicine, Yale University, New Haven, CT 06510; cGroup of Biomedical Research in Digestive Tract Tumors, CIBBIM-Nanomedicine, Vall d’Hebron University Hospital Research Institute (VHIR), Universitat Autónoma de Barcelona, 08035 Barcelona, Spain ABSTRACT Loss-of-function mutations in the nonconventional myosin Vb (Myo5b) result in Monitoring Editor microvillus inclusion disease (MVID) and massive secretory diarrhea that often begins at birth. Keith E. Mostov University of California, Myo5b mutations disrupt the apical recycling endosome (ARE) and membrane traffic, San Francisco resulting in reduced surface expression of apical membrane proteins. ARE disruption also results in constitutive phosphoinositide-dependent kinase 1 gain of function. In MVID, Received: Jul 2, 2018 decreased surface expression of apical anion channels involved in Cl− extrusion, such as cystic Revised: Oct 22, 2019 fibrosis transmembrane conductance regulator (CFTR), should reduce fluid secretion into the Accepted: Oct 23, 2019 intestinal lumen. But the opposite phenotype is observed. To explain this contradiction and the onset of diarrhea, we hypothesized that signaling effects downstream from Myo5b loss of function synergize with higher levels of glucocorticoids to activate PKA and CFTR. Data from intestinal cell lines, human MVID, and Myo5b KO mouse intestine revealed changes in the subcellular redistribution of PKA activity to the apical pole, increased CFTR phosphoryla- tion, and establishment of apical cAMP gradients in Myo5b-defective cells exposed to physiological levels of glucocorticoids. These cells also displayed net secretory fluid fluxes and transepithelial currents mainly from PKA-dependent Cl− secretion. We conclude that Myo5b defects result in PKA stimulation that activates residual channels on the surface when intestinal epithelia are exposed to glucocorticoids at birth. INTRODUCTION Microvillus inclusion disease (MVID) is a severe congenital diar- MVID patients will die rapidly due to uncontrolled secretory di- rhea that disproportionately affects infants of Navajo Indians arrhea (SD) unless they receive total parenteral nutrition support in the United States (Oliva et al., 1994; Sherman et al., 2004). for life. Intestinal fluid loss from diarrhea often exceeds 140 ml/ kg per d, resembling SD of cholera (Cutz et al., 1989). At the ultrastructural level the disease is characterized by microvillus This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E18-07-0415) on October 30, 2019. atrophy, subapical PAS-positive vesicles, and large vacuoles † These authors contributed equally to this work. containing fully formed microvilli (microvillus inclusions [MI]) that *Address correspondence to: Pedro J. Salas (psalas@miami.edu); Nadia A. Ameen appear in a fraction of mature enterocytes (Ameen and Salas, (nadia.ameen@yale.edu). 2000). More than 40 loss-of-function mutations in myosin Vb Abbreviations used: AC, adenylate cyclase; ARE, apical recycling endosome; (Myo5b) have been reported to cause MVID (van der Velde CFTR, cystic fibrosis transmembrane conductance regulator; CFTR172, inhibitor of CFTR anion permeability; Cl-, channel; FRET, fluorescence (or Förster) reso- et al., 2013). In addition, homozygous truncating mutations in nance energy transfer; GC, glucocorticoid; kd, knockdown; KO, knockout; MVID, syntaxin 3 (STX3) were also shown to result in a milder late-onset microvillus inclusion disease (OMIM 251850); Myo5b, myosin Vb (unconventional); NHE3, sodium–hydrogen antiporter 3; ROI, region of interest. form of the disease (Wiegerinck et al., 2014). The fact that im- © 2019 Forteza, Ahsan, et al. This article is distributed by The American Society for paired membrane traffic from apical recycling endosomes (AREs) Cell Biology under license from the author(s). Two months after publication it is avail- to the surface is at the core of MVID pathophysiology was fur- able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). ther demonstrated in constitutive (Cartón-García et al., 2015) “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of and conditional (Schneeberger et al., 2015) homozygous Myo5b the Cell®” are registered trademarks of The American Society for Cell Biology. knockout mice, as well as in Rab8- (Sato et al., 2007) and 3076 | R. Forteza, M. K. Ahsan, et al. Molecular Biology of the Cell Rab11- (Sobajima et al., 2014) deficient mice, all of which mimic domain binds 14-3-3 proteins, which increase forward traffic to the human MVID morphological features. cell surface (Liang et al., 2012). The Rab11/Slp4/Myo5b/Munc18 complex carries apically bound We have shown that the phosphoinositide-dependent kinase 1 vesicles to the surface and mediates their binding to STX3, as a first (PDK1) is associated with the ARE in enterocytes (Mashukova et al., step for membrane fusion. This pathway mediates apical delivery of 2012). As ARE vesicles enlarge and become disorganized and a subset of apical membrane proteins including NHE3, the sodium– poorly polarized in MVID, a substantial fraction of PDK1 dissociates hydrogen exchanger that is necessary for sodium absorption in the from the membrane and becomes loosely distributed in the small intestine, CFTR, and other apical membrane proteins (Vogel cytoplasm (Kravtsov et al., 2014). Cytosolic PDK1 can constitutively et al., 2015). Apical surface expression of these proteins is severely activate several kinases, including PKA (Kobayashi and Cohen, decreased in MVID patients (Ameen and Salas, 2000; Kravtsov et al., 1999; Nirula et al., 2006; Pearce et al., 2010; Tobias et al., 2016). 2016). Yet, defects in this exocytic pathway lead to selectivity in Thus, we speculated that increased CFTR secretory activity of a apical protein targeting to the membrane. While there is a consen- smaller number of channels due to cell-autonomous PKA activity sus that NHE3 is not expressed on the apical surface, alkaline could explain the MVID conundrum. phosphatase and sucrase isomaltase appear both on the surface Glucocorticoids (GCs) exert diverse physiological actions and and in subapical compartments (Ameen et al., 2000). Furthermore, ensure salt and water homeostasis by multiple mechanisms: the CFTR, the main Cl− channel responsible for cAMP-dependent SD, is classic slower genomic pathway involves GC binding to intracellular only partially decreased in human MVID intestine (Kravtsov et al., receptors to regulate gene transcription and translation resulting in 2016). It has been proposed that MVID diarrhea results from an protein synthesis. Thus, many GC effects are mediated by genomic imbalance of partially decreased CFTR (secretion) and totally absent up-regulation of serum glucocorticoid kinases in response to stress NHE3 (absorption). However, an absence of NHE3 on the MVID (Lang et al. 2018), which are activated by PDK1. Rapid nongenomic enterocyte apical membrane cannot explain the magnitude of the GC signaling of effector molecules involves activation of membrane- SD in these patients, because NHE3-deficient mice (Schultheis mediated secondary signaling cascades including cAMP and Ca++ et al., 1998) and humans with congenital sodium diarrhea due to (Mitre-Aguilar et al. 2015). Importantly, glucocorticoids dramatically NHE3 mutations (Janecke et al., 2015) display milder diarrhea even increase within days preceding birth in mammalians (Fowden et al. in the presence of normal CFTR expression. In fact, it is counterintui- 1998; Keller-Wood et al. 2009) resulting in profound transcriptional tive that decreased CFTR because of the Myo5b loss of function and signaling changes. For this reason, we focused on GC effects contributes to SD. In an effort to understand the nature of Na+ and on Myo5b-deficient enterocytes as a possible explanation for the Cl− stool losses in MVID diarrhea, the ion transport profile of human perinatal onset of the MVID phenotype. MVID small intestine was investigated by Rhoads and coworkers Accordingly, our goal was to test the hypothesis that there are (Rhoads et al., 1991). In brief, using Ussing chambers and jejunum changes in intracellular signaling arising from Myo5b defects, which tissues ex vivo, these investigators found a 3.2 mEq h−1 cm−2 result in increased apical fluid secretion mediated by PKA and CFTR decrease in net mucosal to serosal Na+ flux, along with a in the presence of the perinatal increase in GCs. 3.9 mEq h−1 cm−2 increase in net serosal-to-mucosal Cl− flux. Basal jejunal Cl− secretion in MVID tissues was found to be close to the RESULTS maximal rate of normal tissues. While these results are totally consis- Two intestinal cell lines deficient in Myo5b display changes tent with a SD, they are still puzzling in the context of decreased in PKA-dependent phosphorylation patterns that mimic CFTR. The decrease in Na+ absorption can be explained by lack of MVID: effect of dexamethasone NHE3 on the cell surface. However, the molecular mechanism for CaCo2 cells (Muller et al., 2008) and the subclone CaCo2BBe the Cl− secretion remains unexplained. (C2BBe; Peterson and Mooseker, 1992), closely resemble villus In summary, there are two unanswered questions regarding the enterocytes of the small intestine ( Collaco et al., 2010; Jakab et al., pathophysiology of MVID. First, it is paradoxical that decreased 2011; Knowles et al., 2014). Effective (>90%) silencing of Myo5b surface expression of CFTR results in massive SD. Second, there are with lentivirus-delivered short hairpin (shRNA) constructs was two forms of MVID: early (shortly after birth) and late onset (during regularly achieved (Figure 1A) after stringent selection in puromycin the first year). Unlike other congenital diarrheas such as down- (Kravtsov et al., 2016). Under these conditions, C2BBe cells display regulated adenoma congenital Cl− diarrhea (Choi et al., 2009) or all the morphologic features of human MVID enterocytes including NHE3 (congenital secretory sodium diarrhea 8; Janecke et al., the loss of microvilli and the appearance of MI (Kravtsov et al., 2015), early-onset MVID presents with low incidence of polyhydram- 2014; Vogel et al., 2015), which can be visualized as a steep de- nios (intrauterine diarrhea; Cutz et al., 1989; Chen et al., 2011). In crease in apical phalloidin signal and intracellular phalloidin-positive other words, it is unclear why SD starts shortly after birth when inclusions in a fraction of the cells (Figure 1B, arrow). the Myo5b mutations and the molecular machinery for intestinal To determine whether the activity of PKA is specifically exerted secretory function are present well before birth. in the apical domain of epithelial cells, where CFTR molecules are The major mechanisms responsible for fluid secretion in SD localized, we used a widely validated antibody recognizing phos- include cAMP/PKA-dependent activation of CFTR in the proximal phorylated PKA consensus target sites. This antibody has been used small intestine (mainly the jejunum) through two mechanisms: in immunohistochemistry, immunofluorescence, and immunoblot 1) direct phosphorylation of CFTR, which increases the open state of assays in more than 40 publications. We validated this antibody by the channel; and 2) exocytic traffic of CFTR-associated subapical immunoblot of forskolin (cAMP)-stimulated C2BBe cell extracts and vesicles to increase the functional CFTR abundance on the brush with a PKA inhibitor (Figure 1C and Supplemental Figure 1A). This border membrane (Ameen et al., 1999; Ameen and Salas, 2000; antibody will be referred to as PKA p-substrate antibody hereafter. Barrett and Keely, 2000; Golin-Bisello et al., 2005). These CFTR As expected, the antibody recognized several bands. Some bands phosphorylations occur in a tandem of at least eight phospho Ser/ appeared only in the forskolin-treated cells, while other bands Thr in the regulatory domain, which are targets of PKA (Bozoky increased in intensity under forskolin stimulation (Figure 1C). et al., 2013). Importantly, once phosphorylated, the CFTR regulatory Because localization of PKA activity is highly regulated, we wanted Volume 30 December 15, 2019 Myo5b defect increases PKA activity | 3077 FIGURE 1: Myosin 5b knockdown and corticoids induce changes in the distribution of PKA activity in C2BBe intestinal epithelial cells. (A) C2BBe cells were transduced with lentivirus expressing a scrambled shRNA (scr, control) or anti- myosin 5b shRNA (Myo5b kd), extracted, and analyzed by immunoblot. (B) C2BBe cells (scr or Myo5b kd) were stained with fluorescent phalloidin (XZ plane from confocal stacks). Arrow, phalloidin-positive inclusion. Scale bar = 10 µm. (C) C2BBe cells were treated with 10 µM forskolin or vehicle (control) for 3 h. Immunoblots were processed with PKA p-substrate antibody (RRXpS/pT) or anti-GAPDH antibody. (D) Parallel cultures were processed for immunofluorescence with the same PKA p-substrate antibody (red channel, XZ plane). Scale bar = 10 µm. to understand the distribution of PKA-phosphorylated targets in in- show mostly single-layered cysts, with only sporadic double-layered testinal cells. C2BBe cells transduced with a scrambled RNA-ex- cells (Supplemental Figure 2B, arrows). Importantly, the cells appear pressing lentivirus (scr) showed mostly lateral membrane localization polarized, but the lumens are usually collapsed, as expected from of PKA-phosphorylated proteins (Figure 1D, arrows) with many cells cells with a net absorptive fluid transport (Supplemental Figure 2B). lacking apical signal. Myo5b knockdown (kd) or dexamethasone We previously used electrophysiologic approaches to demonstrate treatment of scr cells changed this distribution to a broadly cytoplas- stimulated CFTR ion transport activity in Myo5b-deficient T84 cells mic signal but did not result in a clear apical localization. However, (Kravtsov et al., 2016). Accordingly, these cells were used exten- in Myo5b kd cells previously treated with dexamethasone the signal sively in functional studies here (next section). In addition, we used was concentrated and enhanced at the apical domain (Figure 1D, T84 cells in the current studies as a second cell line to decrease the bottom panel). Semiquantitative measurements of pixel intensities chance that the observations may be due to random changes in showed significantly increased signal in the apical domain and de- the genetic background or signaling related to transformation. The creased signal in the basolateral region and cytoplasm (Supplemen- same lentivirus-delivered anti-Myo5b shRNA was used in T84 cells, tal Figure 1B). resulting in a similar steep down-regulation of Myo5b (Figure 2C). Another epithelial intestinal cell line, T84, resembles intestinal Pixel intensities were quantified in regions of interest (ROIs) ran- crypt cells, and has been shown to closely mimic the electrophysio- domly localized at the apical domain by grid overlapping (example logic properties of secretory intestinal epithelia (Tousson et al., shown in the leftmost panel of Figure 2A, and the confocal level and 1996; Silvis et al., 2009). These cells form tight junctions (Supple- depth of ROIs in Figure 2B). Unlike C2BBe cells, only apical PKA p- mental Figure 2A), albeit their differentiation is slower than C2BBe. substrate signal was observed in T84 cells (Figure 2B). PKA-phos- They can also be grown in spheroids, which, after 2 wk in Matrigel, phorylated target showed minimal changes when scr cells were 3078 | R. Forteza, M. K. Ahsan, et al. Molecular Biology of the Cell FIGURE 2: Myosin 5b knockdown and dexamethasone increase apical PKA-phosphorylated targets in T84 intestinal cells. (A) Immunofluorescence localization of proteins phosphorylated by PKA in scr or Myo5b kd (only kd is labeled in the graph) T84 cells were incubated vehicle (control) or with 0.5 µM dexamethasone for 10 d. As a positive control, parallel cultures, expressing scr or kd shRNA were also incubated in 10 µM forskolin for 3 h. Confocal XY images typical of three experiments at the apical focal plane (see B) shown for PKA p-substrate antibody signal (red), and the corresponding images in the transnuclear plane below for DAPI signal. Yellow rectangle: Example of ROI used for quantification in D. Unbiased random distribution of ROIs was ensured by overlapping a grid and placing ROIs at every other intersection. Scale bar = 10 µm. (B) XZ sections from the same experiment. Yellow line shows the focal plane of ROIs shown in A and the approximate focal depth (vertical lines). Scale bar = 10 µm. (C) T84 cells without transduction or selection (wt) or transduced with lentiviral particles expressing scrambled (scr, control) or anti-Myo5b (kd) shRNAs and selected, were analyzed for Myo5b expression by immunoblot to determine knockdown efficacy. (D) Random ROIs were placed on the apical domain in confocal (XY) sections of monolayers as shown in A by overlapping grid intersections. Several ROIs per image were taken. The average pixel intensities were calculated and normalized to the average in scr control cells. n = 3; *, p < 0.05; **, p < 0.01. compared with Myo5b kd cells in the absence of dexamethasone Myo5b expression indicating that PKA is available and in active con- (Figure 2, A and D). However, PKA substrate signal increased 2.5- formation in kd or scr controls. Importantly, average pixel intensities fold when the cells were incubated with forskolin, regardless of significantly increased threefold in Myo5b-deficient cells incubated Volume 30 December 15, 2019 Myo5b defect increases PKA activity | 3079 (Schneeberger et al., 2015). However, when the cells were grown in dexamethasone, the unstimulated Myo5b kd monolayers showed spontaneous fluid secretion at 27% of the maximum secretory flux in forskolin stimula- tion in KD cells, or 11% of the flux in for- skolin-stimulated scr cells (Figure 3A, green arrow). Importantly, this serosal-to-mucosal net fluid flux was abolished by the specific CFTR inhibitor CFTR172 and was indepen- dent of secretagogues. Moreover, our finding of a net secretory flux of 0.8 µl/h per cm2 in the Myo5b kd and dexamethasone condition may seem very small. However, considering that the secretory surface of the newborn small intestine is ∼25,000 cm2 (Struijs et al., 2009), it is consistent with 480 ml fluid secretion per day. That is well FIGURE 3: Myo5b kd and dexamethasone induce a fluid secretory phenotype in live unstimulated intestinal epithelial cells. (A) Transepithelial net fluid secretion in Myo5b kd T84 within the volume range of diarrhea of a cells treated with dexamethasone. Gravimetric determinations of net fluid transport in T84 cells 3 kg MVID patient (Cutz et al., 1989). (grown on filters) show secretion in Myo5b KD cells when incubated in 0.5 μM dexamethasone To independently corroborate fluid (green arrow). This effect was blocked by 10 µM CFTR172. Control vs. pharmacologic secretion, we used spheroid cultures of T84 treatments estimated by ANOVA; *, p < 0.01; **, p < 0.02; ***, p < 0.05; n = 4. NS, not cells in Matrigel. The diameter of the cysts significant. (B) T84 cells expressing scrambled shRNA (scr) or anti-Myo5b shRNA were grown in grown for 10 d did not significantly change Matrigel for 10 d. The volume of the spheroids was measured as maximum caliper diameter at in a 23-h period, either in scr- or Myo5b kd 4 and 27 h after adding 0.5 µM dexamethasone (dexa) or vehicle to the medium. Each dot cells (Figure 3B). Likewise, when scr T84 represents the change in one spheroid. Relative changes in diameter are expressed as the cells were supplemented with dexametha- percentage of increase (or decrease, −). *, p < 0.001; **, p < 0.01 (Kruskal-Wallis). Box-and- sone, the diameter change was <20% whisker plots are shown for categories with n > 5. (Figure 3B and Supplemental Video_­ control). However, in Myo5b kd cultures in the presence of dexamethasone, a level indistinguishable from supplemented with dexamethasone, the cysts significantly grew the forskolin-positive control (Figure 2, A and D). 62% (Figure 3B and Supplemental Video_dexa). These results sup- port the hypothesis that Myo5b-defective cells secrete fluid in the Myo5b-deficient intestinal epithelial cells show apical fluid presence of postnatal physiological levels of GCs. and Cl− secretion in the presence of dexamethasone that To determine whether fluid secretion is due to CFTR-dependent can be reversed by CFTR inhibitors apical Cl− extrusion in epithelial cells deficient in Myo5b, standard Fluid transport was measured in cells grown on filters by a gravimet- electrophysiology experiments in an Ussing chamber were ric method as described before for C2BBe cells (Kravtsov et al., performed. Transepithelial electric current (Isc) is the result of a 2014). These experiments were performed in T84 cells, because subset of cellular ion transport mechanisms which are electrogenic. these cells express CFTR and display electrophysiologic features CFTR-mediated Cl− secretion is among them (Kato and Romero, similar to those in the intestinal epithelium with Cl− secretion 2011). First, T84 cells in tissue culture medium DMEM, were more robust than other epithelial models including CaCo2 cells challenged with forskolin from the apical side. In scrambled shRNA- (Dharmsathaphorn et al., 1984; Resta-Lenert and Barrett, 2002). The expressing cells, stimulation caused a robust increase in Isc, with a gravimetric method showed fluxes of similar magnitude to those transient peak in the range 40–100 µA/cm2, which decreased to a obtained by others with different approaches (Toriano et al., 2001). steady-state plateau after 15–20 min (Figure 4A). The Isc values at The spontaneous absorptive fluid flux average in T84 cells was steady state less than the average of the currents in the absence of 1 µl/h/cm2 (mucosal to serosal; Figure 3A). As a comparison, the stimulation are quantified in Figure 4C. In all cases, Myo5b kd cells absorptive fluxes reported in C2BBe were 3.1 µl/h per cm2 (Kravtsov showed significantly less electrogenic response to forskolin. The et al., 2014). This is consistent with crypt-like T84 cells as compared forskolin response represents the maximum electrogenic activity with villus-resembling absorptive C2BBe cells. As in the case of induced by cAMP. Therefore, it reports on the total available apical C2BBe, Myo5b kd decreased the absorptive flux, but did not revert anion channels responsive to PKA stimulation. Accordingly, this result it into a net secretory (serosal-to-mucosal) fluid flux. As a positive indicated a reduction in the number of channels expected from the control for secretory response, forskolin induced a significant net apical traffic defects resulting from the loss of Myo5b. However, it is fluid secretion in both control and Myo5b kd cells. The forskolin-­ important to notice that under Myo5b kd, forskolin-induced currents induced response was decreased in Myo5b kd cells but not were reduced only to approximately half of the value in controls abolished (Figure 3A). This is expected for cells expressing only a (Figure 4C), indicating that a substantial number of apical anion fraction of the normal CFTR on the apical surface. These findings are channels remain available for transport in Myo5b-­defective cells. consistent with previous electrophysiologic measurements of Because multiple ion transport mechanisms coexist in intestinal forskolin-stimulated CFTR anion currents in T84 cells deficient in epithelial cells, we wanted to simplify the number of electrogenic Myo5b (Kravtsov et al., 2016). They are also consistent with mechanisms by using a modified Ringer medium, without amino decreased swelling of forskolin-stimulated intestinal organoids acids and supplemented with glucose on the basolateral side only, generated from mice deficient in Myo5b grown in low GC levels to supply energy. To eliminate the apical electrogenic Na+/glucose 3080 | R. Forteza, M. K. Ahsan, et al. Molecular Biology of the Cell observed, opposite to the current gener- ated by electrogenic apical Cl− secretion (e.g., Figure 4A, forskolin). Similar changes were observed when cells in normal Cl− were supplemented with the PKA inhibitor Rp-cAMP, which blocks the cAMP binding site and is, therefore, highly specific for PKA (Figure 4B). These results support the notion that unstimulated Myo5b-defective intesti- nal cells display apical anion secretion driv- ing isotonic fluid secretion under PKA sig- naling in the presence of dexamethasone. PKA-dependent phosphorylated targets localize to the apical region in Myo5b KO mice and human MVID enterocytes To confirm whether enhanced PKA activity in the apical domain of intestinal epithelial cells deficient in Myo5b is also observed in vivo, in nontransformed cells, we analyzed a Myo5b KO mouse model and human tissues from MVID patients. The global Myo5b KO mouse model was previously described (Cartón-García et al., 2015). The pups can suck milk but develop massive diarrhea with hypoglycemia and die within 12 h after birth. Accordingly, samples from these mice were obtained at the late fetal stage when intestinal crypts are not FIGURE 4: Myo5b kd and dexamethasone induce a Cl− secretory phenotype in unstimulated yet developed, and only immature villi are intestinal cells. (A) Typical recordings of short circuit current (Isc) in confluent, differentiated T84 observed. Tissue sections were processed cells transduced with scrambled shRNA (blue and purple), or Myo5b antisense shRNA (green for immunohistochemistry (IHC) using the and black traces), grown on Snapwell filters and analyzed in an Ussing chamber in standard PKA p-substrate antibody described in DMEM culture medium. Forskolin (0.5 µM) was added from the apical side only. Some cells had Figure 1, C and D. In wild-type (wt) mice, sig- been incubated in the presence of 0.5 µM dexamethasone for 10 d (blue and black traces). nal appeared in enterocytes near the tip of (B) Typical recordings of Isc in confluent, differentiated T84 cells transduced with scrambled villi. It was distributed to the cytoplasm and shRNA, or Myo5b antisense shRNA (colors as in A), grown on Snapwell filters and measured in − − − an Ussing chamber in normal Cl Ringer solution (129.1 mM Cl ), or low Cl Ringer solution the lateral membrane, and, to some extent, (128 mM sodium gluconate, 1.1 mM Cl−). For both Ringer formulations, the apical solution was to the apical membrane (Figure 5A, arrow). supplemented with 10 mM mannitol while the basolateral solution contained 10 mM glucose. At Conversely, the signal was mostly apical the end of each experiment, the cells were incubated in normal Cl− solution in the presence of (Figure 5B, arrow) in Myo5b KO samples. 40 µM Rp-cAMP. (C) Quantification of experiments like the one shown in B. Measurements were In non-MVID human biopsies, PKA p- taken around 15 min after adding forskolin, in steady state. The bars represent the difference substrate signal was diffuse, with a faint between Isc (in µA/cm2) in stimulated cells minus the baseline in the same experiment. Statistical apical and lateral membrane concentration significance was determined by t test; *, p < 0.05; n = 4. (D) Quantification of the experiments (Figure 5D, arrow). On the contrary, in MVID like the one in C. Bars represent the average difference between nonstimulated Isc (µA/cm2) in biopsies, the signal was restricted to the normal Cl− Ringer and low Cl− (gluconate) Ringer. *, p < 0.04; **, p < 0.02; n = 3. apical membrane (Figure 5E, arrow) and to intracellular apical structures consistent with cotransport, glucose was replaced by mannitol on the apical side. the images of MIs (Figure 5E, arrowhead). In summary, Myo5b KO in Under these conditions, we asked how much of the basal, unstimu- a mouse model of MVID, and MVID in human intestine both result lated current is carried by Cl−. To that end, scrambled or Myo5b in changes in the subcellular distribution PKA-phosphorylated shRNA-expressing cells grown with or without dexamethasone, targets. The redistribution of PKA activity in murine and human were sequentially measured in normal Cl− Ringer, low Cl− Ringer Myo5b loss of function strongly resembles the observations in (gluconate), and back again in normal Ringer. In other words, Cl− Myo5b kd C2BBe (Figure 1D and Supplemental Figure 1B) and T84 concentration was reduced on both sides of the membrane by 100- cells (Figure 2B). Of note, we conclude that the Myo5b defect re- fold. The basal currents in normal Cl− in all cases were small (3 ± sults in apical PKA activity with decreased activity in the cytoplasm 3.2 µA/cm2). There were no statistically significant differences and lateral membrane and increased activity in the apical domain. among all the conditions. Chloride replacement resulted in small changes in basal unstimulated currents, indicating that the current Apical cAMP concentration gradients appear in was not carried by Cl−. The exceptions were Myo5b kd cells incu- Myo5b-deficient cells incubated in dexamethasone bated in dexamethasone (Figure 4, B, black trace, and D). In this If the Myo5b loss-of-function effect on PKA activity has any func- case, a statistically significant change in transepithelial current was tional implication in apical CFTR channel function, it must be Volume 30 December 15, 2019 Myo5b defect increases PKA activity | 3081 FIGURE 5: PKA-phosphorylated substrates concentrate under the apical domain in intestinal epithelia defective in Myo5b in vivo. (A–C) Fetal mouse small intestine, stained with PKA p-substrate antibody (A, B) or isotype control, processed with a similar dilution of nonimmune rabbit serum (C). (B) Sample from Myo5b KO mouse. (A, C) Heterozygous control. (D, E) Human duodenum biopsies from unrelated disease (D, F) or MVID (E). Both patients (6 and 5 mo old, respectively) were subjected to endoscopy and duodenal biopsy as diagnostic procedures for chronic diarrhea. Diagnosis of MVID was reached by PAS and EM (unpublished data), confirming the presence of MI (arrowhead). Arrows point at apical PKA p-substrate signal. Scale bar = 20 µm. localized very close to the membrane. Confocal microscopy does 30 min following treatment with 10 µM forskolin in the same cells not provide adequate resolution in the z-axis to make this assertion. (Figure 7, A and B, scr; examples of the focal plane of ROIs a, Thus, the images in Figures 1 and 2 could represent PKA targets in apical, b, basal, and cytoplasmic are shown in yellow boxes). subapical compartments, which are enlarged in Myo5b-deficient in- Noticeably, cAMP signal was distributed throughout the cytoplasm testinal cells. Therefore, we wanted to examine whether PKA activity and not particularly concentrated under the apical domain in and cAMP localize in the vicinity of the apical CFTR channel (Ameen forskolin-stimulated cells. The cAMP signal was increased in the et al., 2003) in Myo5b loss of function. We confirmed this in the apical domain of Myo5b-deficient cells, especially when preincu- human T84 cells expressing scrambled (scr) and anti-Myo5b bated in dexamethasone (Figure 7, A and B, kd). It is important to shRNA. The latter showed increased colocalization of CFTR and notice that cAMP gradients were inverted from a cytoplasmic/ PKA p-substrate signals (Figure 6A, merge). To verify whether this basal larger than apical in nonstimulated cells, to apical larger than colocalization was within distances enabling molecular interaction, basal when Myo5b deficiency was concurrent with dexamethasone we performed fluorescence resonance energy transfer (FRET) deter- supplementation (Figure 7B, arrows). Also, this increase in apical minations using fluorochromes CY3 and CY5 covalently linked to cAMP was observed in the absence of exogenous stimulation secondary antibodies. FRET efficiency was measured after acceptor other than dexamethasone (serum-free medium and no forskolin). (CY5) photobleaching in random ROIs in the apicalmost confocal This is consistent with the increase in apical PKA p-substrate signal optical section (Figure 6B). FRET efficiency was significantly in- described in the same region of the cell in two intestinal Myo5b kd creased 10-fold in Myo5b-deficient cells, preincubated in dexa- cell lines, Myo5b KO mice, and human MVID enterocytes as shown methasone, as compared with cells expressing scrambled RNA in previous sections. (Figure 6C). While these experiments do not allow calculation of the Förster radius of interaction, we conclude that PKA-phosphorylated Myo5b defect along with dexamethasone increase sites are concentrated within four immunoglobulin G (IgG) molecule PKA-dependent phosphorylation in apical membrane lengths (∼240 nm) of apical CFTR molecules as a result of the Myo5b proteins in intestinal epithelial cells defect and incubation in dexamethasone. To confirm that apical membrane proteins, which would include It is generally accepted that spatially restricted adenylate CFTR, are phosphorylated in PKA targeted sites, cells were screened cyclase activity may result in submicron-sized cAMP microdomains, by vectorial cell surface biotinylation (Rodriguez-Boulan et al., 1989). although the mechanisms to restrict cAMP diffusion remain subject Not all proteins can be biotinylated. Derivatization depends on the to debate (Calebiro and Maiellaro, 2014).To independently test presence of primary amines in the amino acid side chains exposed the hypothesis that PKA activity is increased in the apical domain to the extracellular milieu. Therefore, surface biotinylation provides of Myo5b-deficient cells, we transduced confluent T84 cultures a sampling of apical surface proteins that can be derivatized with with a cAMP upward fluorescence biosensor with high efficiency sulfo-NHS-biotin. As expected from deficient apical membrane using a modified baculovirus that infects mammalian cells. Conflu- traffic, the surface expression of a subset of apical proteins ent fully polarized live cells were imaged 48 h after transduction. decreased in C2BBe Myo5b-deficient cells. Some bands were unde- Low levels of fluorescence, mostly localized to the basolateral tectable in Myo5b kd cells, while others were only very moderately region and cytoplasm, were observed in cells expressing scram- decreased (Figure 8A, stars). This result is consistent with previous bled shRNA. However, the fluorescence increased dramatically observations that loss of Myo5b leads to selective apical traffic 3082 | R. Forteza, M. K. Ahsan, et al. Molecular Biology of the Cell The previous result raises the possibility that CFTR may be a target of PKA in the apical surface in Myo5b-deficient cells. To test that hypothesis, we took advantage of the previous work of various groups show- ing that 14-3-3 proteins associate with ac- tive CFTR through multiple PKA target phosphosites in tandem in the intrinsically disordered regulatory domain (Bozoky et al., 2013) stabilizing the protein (Stevers et al., 2016). Importantly, CFTR coimmunoprecipi- tates with 14-3-3, and co-ip is enhanced by forskolin (Liang et al., 2012). In other words, 14-3-3β and CFTR coimmunopre- cipitation is a reporter of PKA phosphoryla- tion. In enterocytes and intestinal cell lines, 14-3-3β is expressed (Monroy, 2008; Gomez-­Suarez et al., 2016), along with iso- forms ε, θ, ζ, and η. In addition, it has been shown that 14-3-3β heterodimerizes with ζ (Alvarez et al., 2003) and ε (Ghorbani et al., 2016). Because we did not determine the specific isoforms (homo- or heterodimers) involved in this coimmunoprecipitation, we FIGURE 6: CFTR and PKA p-substrate signal colocalize within molecular distances in the apical will generically refer to 14-3-3 proteins domain in cells lacking Myo5b grown in the presence of 0.5 µM dexamethasone for 10 d (dexa). hereafter. These proteins are abundant and T84 cells were fixed and stained with anti-PKA p-substrate (p-s) antibody and anti-CFTR cytosolic. The fraction bound to phosphory- monoclonal antibodies, followed by affinity-purified antibodies labeled with Cy3 or Cy5. (A) Confocal stacks including the apical surface of the cells were imaged in the XZ plane. Scale lated membrane proteins, thus, is expected bar = 10 µm. (B) In similar experiments, the apicalmost confocal section (0.68 µm thick in the to be relatively small. Accordingly, we en- z-axis) showing microvilli was subjected to acceptor (Cy5) bleaching (200 cycles) in random ROI riched our extracts in membrane proteins by and imaged again after bleaching. Scale bar = 4 µm. (C) Average FRET efficiency from three using Triton X-114 extraction, instead of the experiments (30 ROIs) is shown for control (scr) and Myo5b kd cells, incubated or not in standard Triton X-100, followed by temper- dexamethasone (dexa) for 10 d. *, p < 0.001; **, p < 0.04. ature separation of the detergent phase (Bordier, 1981; Taguchi and Schatzl, 2014). defects differentially affecting individual apical membrane protein As predicted, Myo5b kd resulted in a steep increase in CFTR coim- surface expression (Kravtsov et al., 2016). munoprecipitation with 14-3-3 (Figure 8E). Interestingly, when this To extend this result to the other cell line and to understand the co-ip is driven by forskolin, both immature (b) and mature (glycosyl- effect of Myo5b defect and GCs on apical membrane proteins, ated, c) forms of CFTR coimmunoprecipitate with 14-3-3 (Liang similar experiments were conducted. T84 cell cultures were et al., 2012). In the case of Myo5b kd cells, however, it was mostly incubated in dexamethasone (or vehicle) for 10 d. When Triton the c form of CFTR that coimmunoprecipitated with 14-3-3, X-100 extracts of biotinylated (or not, control c) cells were ana- suggesting that phosphorylation occurred in a compartment lyzed, the decrease in apically exposed proteins in Myo5b kd was beyond the Golgi apparatus. This result supports the notion that confirmed, but little or no effect of GCs was noticed (Figure 8B). To the fraction of CFTR that reaches the surface in Myo5b-defective determine whether PKA targeting of biotinylated apical membrane epithelial cells is heavily phosphorylated in the regulatory domain in proteins is affected by loss of Myo5b or corticoids, pull downs with Myo5b-deficient cells, especially in the presence of GCs. streptavidin Sepharose beads followed by PKA p-substrate immunoblot were performed. Pull down resulted in enrichment in DISCUSSION fewer bands phosphorylated by PKA, not readily noticeable in the There is no question that MVID with Myo5b loss-of-function muta- total extract. No major bands were observed in the 140–200 kDa tions is a disease resulting from defects in apically bound membrane range, so we focused the analysis on two major bands with Mr 54 traffic. This work shows that, in addition, the disorganization of the and 60 × 103 (Figure 8C, arrows) in the absence of GC supplemen- ARE compartment (Mashukova et al., 2012; Kravtsov et al., 2014) tation; phosphorylation remained at similar levels in Myo5b- results in changes in intracellular signaling by PKA specifically in the deficient cells. However, for cells grown in the presence of GC, microenvironment below the apical membrane. An apical gradient Myo5b loss of function resulted in a statistically significant increase of cAMP, increased PKA-dependent phosphorylation in apical in phosphorylation (Figure 8, C and D). These results were quanti- proteins, and CFTR–14-3-3 coimmunoprecipitation provide inde- fied by densitometry of the two major bands (Figure 8D). Consid- pendent lines of evidence supporting this idea. Importantly, ering that expression of apical proteins decreases on the surface to increased PKA activity was also confirmed in vivo in human MVID various extents (Figure 8, B and C) because of membrane traffic intestine and mouse Myo5b KO enterocytes, indicating that it is not defects dependent on Myo5b loss of function, these results a consequence of abnormal signaling pathways in transformed cell indicate that proteins reaching the apical surface are exposed to a lines. Furthermore, because PKA activation is highly restricted to the heightened PKA kinase activity in cells deficient in Myo5b and apical membrane, and not global in the cell, it is likely due to effects exposed to physiological levels of GCs. on membrane-bound adenylate cyclases (ACs), phosphodiesterases, Volume 30 December 15, 2019 Myo5b defect increases PKA activity | 3083 physiological concentration. Enteroid de- fined media are not even supplemented with serum or GCs. Accordingly, the results found here have not been characterized in previous publications using cell lines or primary enteroid cultures. Specifically, poor responses to forskolin observed in the volume gain of enteroids derived from Myo5b floxed mice have been interpreted as negative evidence for increased CFTR function in MVID diarrhea. In those experi- ments, Schneeberger and coworkers found a ∼1.5-fold increase in enteroid volume in normal cells as compared with Myo5b KO enteroids (Schneeberger et al., 2015). Bear- ing in mind that the enteroids in that work were not specifically grown in dexametha- sone, those experiments would have to be compared with our T84 cells, expressing scrambled or Myo5b shRNA in the presence of forskolin. In this study, we found that Myo5b kd results in a 47% decrease in the fluid secretion response to forskolin (Figure 3A) compared with the control cells. Like- wise, Myo5b defects resulted in a similar decrease in apical electrogenic anion secre- tion (Figure 4, B and D). Our interpretation is FIGURE 7: Myo5b loss of function induces redistribution of cAMP gradients in intestinal cells in that forskolin represents maximal cAMP the presence of GCs. Cyclic AMP was determined in vivo by transduction of a cAMP “upward” stimulation, thus reflecting functionally the biosensor in T84 cells. Cells expressing a scrambled shRNA (scr) or Myo5b antisense shRNA (kd) partial loss of CFTR on the surface. How- were incubated in dexamethasone or vehicle as described before and transduced with a ever, MVID patients would have no exoge- human-adapted baculovirus expressing cAMP biosensor. Some scr cell cultures (with no nous stimulation for cAMP levels. Instead, dexamethasone) were also preincubated with 10 µM forskolin for 30 min as a positive control for Myo5b loss of function compounded with transduction and biosensor fluorescence. (A) 3D reconstruction of confocal stacks from live T84 GCs at levels that mimic the postpartum cells show DNA counterstain (DAPI, blue) and biosensor fluorescence (red). Yellow rectangles situation result in a cell-autonomous endog- are example focal planes of randomly localized apical (a) or basolateral (b) ROI in the XY sections used for quantification in B. Scale bar = 10 µm. (B) Biosensor fluorescence was quantified in enously driven cAMP apical gradient and positively transduced cells using random ROIs in the transnuclear/basolateral domain (b) and or PKA stimulation that opens CFTR channels − in the apical domain (a) in XY confocal sections. Yellow rectangles show examples of the location and results in Cl extrusion. In summary, the of a and b confocal planes in XZ reconstructions. Arrows point at the only condition in which the need of GCs pathway has been overlooked apical/basolateral cAMP gradient is inverted in unstimulated (no forskolin) cells: Myo5b kd cells as an experimental variable in Myo5b-­ grown in dexamethasone. *, p < 0.001. defective phenotypes and might be key to explain the timing of early-onset MVID. GCs or specifically apical A-kinase anchoring proteins (Mies et al., 2007). exert many transcriptional and nontranscriptional effects. Under- Substantial subapical localization of cAMP determined with a bio- standing which one impacts on PKA activation in the Myo5b-­ sensor strongly supports the first two possibilities. It seems to be defective background is beyond the scope of this work. However, puzzling that Rhoads and coworkers (Rhoads et al., 1991) found that currently there is no therapy for MVID diarrhea. This study suggests octreotide, a known adenylate cyclase inhibitor, had no effect in two that the secretory component of MVID diarrhea could be reverted MVID patients treated for 1 d. This negative result, however, does by modulating such PKA activation. contradict our conclusion. Octreotide inhibits ACs sensitive to Gαi G-proteins, which exclude group II ACs expressed in the intestine MATERIALS AND METHODS (Sabbatini et al., 2014). Octreotide, for example, is not effective to Antibodies and reagents control cholera diarrhea (Farthing, 2002), the paradigm of increased Anti-PKA–phosphorylated substrate sites (PKA p-substrate, anti- cAMP-dependent watery secretion. In summary, we conclude that RRXpS/pT) antibody was obtained from Cell Signaling (cat. # 9621S). the MVID diarrhea arises from the combined consequences of a The sources of other antibodies were as follows: GAPDH complete loss of apical NHE3 (Ameen and Salas, 2000; Kravtsov (Calbiochem; CB1001); tubulin (Abcam; ab52866); Myo5b (Novus et al., 2016) and an incomplete decrease of apical CFTR, which is Biologicals; NBP1-87746); ZO-1 MAb (ZO-1-1A12; ThermoFisher potentiated by PKA increased activity resulting in opening of resi- Scientific). CFTR monoclonal antibodies 596 and 769, against the dent CFTR channels still exposed on the surface. NBD2 (C-terminal) domain (Kreda et al., 2005; Cui et al., 2007) were Epithelial cells in culture commonly used in studies of the effect obtained from the Cystic Fibrosis Foundation, CFTR Antibody of Myo5b mutations (e.g., CaCo2 cells or enteroids) are not regu- Distribution Program, University of North Carolina at Chapel Hill. larly supplemented with GCs. Cell lines would be grown in the pres- Anti-14-3-3 (Proteintech; AB_2218096) was originally developed ence of GCs from the serum, normally diluted to 5–10% of the against full-length 14-3-3θ, but it cross-reacts with other 14-3-3 3084 | R. Forteza, M. K. Ahsan, et al. Molecular Biology of the Cell FIGURE 8: Biotinylated apical membrane protein epithelial cells deficient in Myo5b show increased phosphorylation. (A) C2BBe cells constitutively expressing scrambled shRNA (c and s) or Myo5b antisense shRNA (kd) were grown on filters, biotinylated from the apical side, extracted, pulled down with streptavidin-agarose, and visualized by blot and chemiluminescence with streptavidin. One set of cultures was not biotinylated (control, c). A fraction (10%) of the input was stained with Ponceau S red to show total protein (input). Stars indicate examples of biotinylated bands that remain exposed to the apical chamber in Myo5b-deficient cells (representative experiment out of three). (B) In similar experiments, T84 cells expressing scrambled RNA (s) or Myo5b shRNA (kd) were grown with or without 0.5 µM dexamethasone (dexa), and revealed with streptavidin-HRP. (C) A similar extract was pulled down with streptavidin beads and analyzed by immunoblot with PKA p-substrate antibody. A fraction (5%) of total cell extracts in Triton X-100 (input) was analyzed by immunoblot with anti-tubulin antibody as loading control. (D) Quantification of PKA p-substrate signal in two biotinylated bands. The PKA p-substrate signal relative to the average intensity of the bands in Myo5b- deficient cells with no dexamethasone treatment were plotted for the prominent 54 and 60 kDa bands shown in C. Box plots represent first, third quartile, and median for each group (each dot represents an independent observation). Kruskal-Wallis; *, p < 0.02. (E) T84 cells were treated as described in B. The cells were extracted in 2% Triton X-114 in the cold, and the extracts subjected to detergent condensation to enrich in membrane proteins and their binding partners. 10% of the input was analyzed by immunoblot (i.b.) with the antibodies listed on the right-hand side. The rest was immunoprecipitated with an anti–14-3-3 antibody recognizing multiple isoforms and analyzed for immunoblot with the same antibodies. For 14-3-3 blots, the signal was revealed with an anti-rabbit secondary antibody coupled to HRP that only recognizes the native conformation of IgG. Arrowheads in CFTR immunoblots point at the position of the immature (b) and mature (c) forms. Mr of standards are expressed ×10−3. isoforms (Bai et al., 2014) and has been validated for immunopre- transduction, both cell types were continuously selected in 20 µg/ml cipitation. Fluorescent secondary affinity-purified antibodies with puromycin. For experiments, the cells were kept in 12.5 µg/ml puro- minimal cross-reactivity were obtained from Jackson Immunore- mycin. Cells were kept for up to 10 passages after transduction or search Laboratories. ECL horseradish peroxidase (HRP)–conjugated until the efficiency of knockdown was less than 90%. For experi- affinity-purified antibodies were obtained from Amersham. TrueBlot ments, C2BBe or T84 cells were grown on 3-µm-pore Transwell or HRP–anti-rabbit IgG antibody recognizing native conformation Snapwell filters (Costar) for 2 wk after confluency, when C2BBe cells nonreduced protein was purchased from Rockland (TruBlot; reach full differentiation. In preliminary experiments, we confirmed 18-8816-31). by reverse transcription-quantitative PCR that T84 cells also acquire the highest CFTR expression at that time. Cells and lentivirus T84 three-dimensional cultures (spheroids) in Matrigel (BD Bio- CaCo2 BBe(C2BBe) and T84 cells were obtained from the American sciences) were prepared following the technique described else- Type Culture Collection. Lentivirus particles expressing anti-Myo5b where for Madin–Darby canine kidney cells (Martin-Belmonte et al., shRNA or GFP were described before (Kravtsov et al., 2014). Upon 2008), and adapted for T84 cells (Bentz et al., 2013). The spheroids Volume 30 December 15, 2019 Myo5b defect increases PKA activity | 3085 were used for experiments after 10 d in culture, with changes in tease inhibitors, HG) was used for these experiments as described media every other day. before (Collaco et al., 2010). In this buffer Triton X-114 clouding point is around 30°C. The 6% Triton X-114 HG stock solution was Dexamethasone and pharmacological treatments subjected to at least five cycles of condensation discarding the aque- Synthetic glucocorticoid dexamethasone (Sigma) was kept in di- ous phase and replaced by freshly prepared HG buffer each time methyl sulfoxide (DMSO) stock solution for up to 3 mo at −20°C. (Taguchi and Schatzl, 2014). This precondensed stock was diluted 1:3 Dexamethasone concentration in treatments was established on the in freshly prepared HG buffer before cell extraction (EB). Cell mono- basis of average blood cortisol concentrations in neonates, which layers were washed with PBS on ice and extracted in EB for 15 min. range from 40 µg/l (∼0.1 µM; Jonetz-Mentzel and Wiedemann, The extracts were spun at 12,000 × g in the cold for 15 min and the 1993) to 250 µg/l (∼0.5 µM; Stevens, 1970), depending on the supernatant was warmed to 33°C to separate the Triton X-114 phase assays. Typically, cells were incubated in dexamethasone for 10 d (1 h). The aqueous supernatant was discarded, and the lower deter- before the day of the experiment to mimic the timing of the increase gent phase resuspended in the same volume of cold HG buffer. in serum GCs before birth. Samples of this extract were taken as input. Incubation with the anti- Forskolin, Rp-cyclic 3′,5′-hydrogen phosphorothioate adenosine 14-3-3 antibody was performed in EB overnight at 4°C. Sepharose– (Rp-cAMPS), and CFTR172 were purchased from Sigma. All these protein A beads were prewashed in the same buffer for 1 h, spun, reagents, except Rp-cAMP, were kept in DMSO stock solutions at and added to the cell extracts for 3 h. Upon centrifugation, the beads −20°C. Rp-cAMP stock solution was made in sterile phosphate- were further washed in 1% Triton X-100 in PBS four times (1 h), and buffered saline (PBS) and frozen. H89 was purchased from Tocris. finally in 0.5M Tris buffer, pH 6.8, for 10 min. The protein was eluted Human samples were considered exempt nonhuman research in SDS sample buffer supplemented with 0.1% 2-mercaptoethanol at on the basis that they were deidentified paraffin blocks retrieved 37°C for 10 min. After separating the beads, the procedure was from a repository. repeated one more time and the eluates were pooled. Mouse intestine cAMP biosensor The generation of the Myo5b knockout mouse model used has A cAMP upward mNeon Green cADDis biosensor (Tewson et al., been described before (Cartón-García et al., 2015). Mice heterozy- 2016) expressed under a CMV promoter was obtained from Montana gous for the Myo5b-null allele were crossed and pregnant females Molecular (U0200G) and delivered by transduction with BacMam, a were killed by CO2 asphyxiation at day 20 of gestation. The embryos baculovirus modified to infect mammalian cells (Kost et al., 2007). were obtained, kept on ice-cold PBS, and killed by decapitation. Tail Fully differentiated T84 cell cultures (2 wk after confluency) grown on clips were taken for DNA extraction and genotyping and the whole Transwell filters were transiently transduced with high efficiency by embryos were Formalin-fixed and paraffin-embedded as described this vector. The cells were incubated in the presence of 2 mM so- (Cartón-García et al., 2015). dium butyrate for 48 h after transduction according to the manufac- turer’s specifications before imaging. Six hours before imaging, IHC and immunofluorescence for PKA p-substrate antibody 0.5 µg/ml 4′,6-diamino-2-phenylindole was added to the medium to Immunohistochemistry for the PKA p-substrate antibody was contrast nuclei. Some cultures were supplemented with 10 µM performed using the antigen retrieval protocol described by the forskolin for 30 min as positive control. Live cells were imaged in an manufacturer (www.cellsignal.com/products/primary-antibodies/ inverted confocal microscope using a glass-bottom Petri dish. phospho-ser-thr-pka-substrate-antibody/9621). Published data in- During imaging the cells were kept at 37°C and incubated in serum- clude validation by negative controls using phosphatases before free phenol red–free DMEM buffered with 20 mM HEPES. tissue staining (same website). Independent validation of the use of this antibody for immunohistochemistry was published by others Confocal microscopy (Saitoh et al., 2009). For immunofluorescence, the antibody was Confocal microscopy images were obtained in a Leica SP5 using an used after fixation in 10% trichloroacetic acid as described for other oil immersion 63× objective. For live-cell microscopy a water immer- antibodies against phosphorylated epitopes (Hayashi et al., 1999; sion 63× objective was used. Unless stated otherwise, the sections Wald et al., 2011). were collected at Airy = 0.9 or Airy = 1.2 for live cells to decrease Vectorial apical cell surface biotinylation was performed as de- laser intensity. For Airy 0.9, the section thickness was typically scribed before (Rodriguez-Boulan et al., 1989; Sargiacomo et al., 0.6 µm and section spacing was 0.5 µm. For three-dimensional re- 1989). Briefly, confluent cell monolayers grown on Transwell filters constructions, the stacks were imported into Slidebook software were washed in PBS from the apical side only, leaving normal culture and cropped into 10-pixel-wide images. These images were recon- medium in the basal chamber. The cells were incubated on ice with structed and rotated to show the z-axis. For quantification, ROIs 1 mM sulfo-NHS-LC-biotin (ThermoFisher Scientific; A39256) for were identified at random by overlapping a grid on the image in 15 min, and then extensively washed with ice-cold DMEM. The cells original (XY) images without rotation. To ensure that the measure- were extracted in RIPA buffer in the presence of anti-proteases and ments were performed at the apical domain, all the ROIs used for anti-phosphatases. After centrifugation, the supernatants were incu- quantification comprised images of microvilli. bated in streptavin-agarose beads (Sigma Chemical; S1638) for pull Live-cell microscopy was performed within a warmed chamber at down. It must be noted that RIPA buffer extraction, while efficient for 5% CO2 using a Leica DMI6000 inverted microscope and a 20× most membrane proteins has shown poor yields specifically for CFTR. objective. Time-lapse acquisition was done with LAS X software with Immunoprecipitation of CFTR 14-3-3 complexes was performed images acquired every 15 min. as described before (Kravtsov et al., 2012) but replacing Triton X-100 with 2% Triton X-114 to enrich for transmembrane 14-3-3 interactors, Fluorescence resonance energy transfer as described elsewhere (Bordier, 1981). It must be highlighted that FRET indexes were measured for the pair CY3-CY5 using the accep- CFTR is poorly extracted in RIPA buffer. An ad hoc CFTR extraction tor bleaching technique (Berney and Danuser, 2003; Centonze buffer (25 mM HEPES, 10% glycerol, pH 7.4, supplemented with pro- et al., 2003). The method followed an established protocol for FRET 3086 | R. Forteza, M. K. Ahsan, et al. 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