Single-Cell Transcriptomic Profiling of Pluripotent Stem Cell-Derived SCGB3A2+ Airway Epithelium.

Lung epithelial lineages have been difficult to maintain in pure form in vitro, and lineage-specific reporters have proven invaluable for monitoring their emergence from cultured pluripotent stem cells (PSCs). However, reporter constructs for tracking proximal airway lineages generated from PSCs have not been previously available, limiting the characterization of these cells. Here, we engineer mouse and human PSC lines carrying airway secretory lineage reporters that facilitate the tracking, purification, and profiling of this lung subtype. Through bulk and single-cell-based global transcriptomic profiling, we find PSC-derived airway secretory cells are susceptible to phenotypic plasticity exemplified by the tendency to co-express both a proximal airway secretory program as well as an alveolar type 2 cell program, which can be minimized by inhibiting endogenous Wnt signaling. Our results provide global profiles of engineered lung cell fates, a guide for improving their directed differentiation, and a human model of the developing airway

The Effect of Arrestin Conformation on the Recruitment of c-Raf1, MEK1, and ERK1/2 Activation

Sergio Coffa is with Vanderbilt University; Maya Breitman is with Vanderbilt University; Susan M. Hanson is with Vanderbilt University; Seunghyi Kook is with Vanderbilt University; Vsevolod V. Gurevich is with Vanderbilt University; Kari Callaway is with UT Austin; Kevin N. Dalby is with UT Austin.Arrestins are multifunctional signaling adaptors originally discovered as proteins that “arrest” G protein activation by G protein-coupled receptors (GPCRs). Recently GPCR complexes with arrestins have been proposed to activate G protein-independent signaling pathways. In particular, arrestin-dependent activation of extracellular signal-regulated kinase 1/2 (ERK1/2) has been demonstrated. Here we have performed in vitro binding assays with pure proteins to demonstrate for the first time that ERK2 directly binds free arrestin-2 and -3, as well as receptor-associated arrestins-1, -2, and -3. In addition, we showed that in COS-7 cells arrestin-2 and -3 association with β2-adrenergic receptor (β2AR) significantly enhanced ERK2 binding, but showed little effect on arrestin interactions with the upstream kinases c-Raf1 and MEK1. Arrestins exist in three conformational states: free, receptor-bound, and microtubule-associated. Using conformationally biased arrestin mutants we found that ERK2 preferentially binds two of these: the “constitutively inactive” arrestin-Δ7 mimicking microtubule-bound state and arrestin-3A, a mimic of the receptor-bound conformation. Both rescue arrestin-mediated ERK1/2/activation in arrestin-2/3 double knockout fibroblasts. We also found that arrestin-2-c-Raf1 interaction is enhanced by receptor binding, whereas arrestin-3-c-Raf1 interaction is not.Funding was provided by National Institutes of Health grants GM081756, GM077561, and EY011500 (VVG), and GM059802 and the Welch Foundation (F-1390) (KND). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Pharmac

Biological Role of Arrestin-1 Oligomerization

Members of the arrestin superfamily have great propensity of self-association, but the physiological significance of this phenomenon is unclear. To determine the biological role of visual arrestin-1 oligomerization in rod photoreceptors, we expressed mutant arrestin-1 with severely impaired self-association in mouse rods and analyzed mice of both sexes. We show that the oligomerization-deficient mutant is capable of quenching rhodopsin signaling normally, as judged by electroretinography and single-cell recording. Like wild type, mutant arrestin-1 is largely excluded from the outer segments in the dark, proving that the normal intracellular localization is not due the size exclusion of arrestin-1 oligomers. In contrast to wild type, supraphysiological expression of the mutant causes shortening of the outer segments and photoreceptor death. Thus, oligomerization reduces the cytotoxicity of arrestin-1 monomer, ensuring long-term photoreceptor survival.SIGNIFICANCE STATEMENT Visual arrestin-1 forms dimers and tetramers. The biological role of its oligomerization is unclear. To test the role of arrestin-1 self-association, we expressed oligomerization-deficient mutant in arrestin-1 knock-out mice. The mutant quenches light-induced rhodopsin signaling like wild type, demonstrating that in vivo monomeric arrestin-1 is necessary and sufficient for this function. In rods, arrestin-1 moves from the inner segments and cell bodies in the dark to the outer segments in the light. Nonoligomerizing mutant undergoes the same translocation, demonstrating that the size of the oligomers is not the reason for arrestin-1 exclusion from the outer segments in the dark. High expression of oligomerization-deficient arrestin-1 resulted in rod death. Thus, oligomerization reduces the cytotoxicity of high levels of arrestin-1 monomer

The effect of different β2AR ligands on ERK2 binding to arrestins and ERK2 activation.

<p>HA-tagged ERK2 was co-expressed with Flag-tagged WT arrestin-2 (<b>A,B,C</b>), or arrestin-3 (<b>D,E,F</b>) in COS-7 cells. Cells were serum starved 24 hours after transfection and stimulated for 10 min at 37°C with 10 µM of indicated β2AR ligands. Arrestins were immunoprecipitated with anti-Flag antibody, and co-immunoprecipitated ERK2 was visualized with anti-HA antibody. The binding of ERK2 to arrestin-2 (<b>B</b>) or arrestin-3 (<b>E</b>) was significantly increased by treatment with ligands. <b>C,D.</b> ERK1/2 activation in cell lysates was determined by Western blot with anti phospho-ERK1/2 antibody. Means ± SD of 3–4 independent experiments are shown in bar graphs; representative blots are shown in panels <b>A</b> and <b>D</b>. ANOVA with Bonferroni post-hoc test revealed the following differences: *, p<0.05; **, p<0.01; ***, p<0.001, as compared to untreated cells.</p

Free non-visual arrestins enhance ERK2 phosphorylation by MEK1.

<p><b>A, B.</b> ERK2 (12 pmol) was incubated with MEK1 (2 pmol) in 0.1 ml of 50 mM Hepes-Na, pH 7.2, 100 mM NaCl, and 0.1 mM [γ-³²P]ATP in the absence (control) or presence of 4.4 pmol of arrestin-2 (Arr2), arrestin-3 (Arr3), or arrestin-3-(1–393) (Arr3-(1–393)) for 30 min at 30°C. The reaction was stopped by MeOH-precipitation of the proteins. The pellet was dissolved in SDS sample buffer and subjected to SDS-PAGE. The gels were stained, dried, and exposed to X-ray film to visualize radiolabeled bands (panel <b>A</b>). ERK2 bands were cut out and ³²P incorporation was quantified by scintillation counting (panel <b>B</b>). Means ± SD of four independent experiments are shown. (**) p<0.01, as compared to control.</p

ERK2 binding to arrestin-1 and both non-visual arrestins is direct.

<p><b>A</b>. Active (phosphorylated at T183 and Y185 by MEK1) or inactive ERK2 (30 pmol) was pre-incubated with or without 30 pmol of indicated arrestin for 20 min at 30°C, then phosphorylated rhodopsin (50 pmol) was added and incubated in the light (to produce P-Rh*) in 0.1 ml for 5 min. Rhodopsin-containing membranes were pelleted through 0.2 M sucrose cushion and dissolved in SDS sample buffer. ERK2 in the pellet (1/300 of each sample) was quantified by Western blot using anti-ERK antibodies (Cell Signaling) and known amounts of purified ERK2 to generate calibration curve. Abbreviations: Arr1, visual arrestin-1, Arr2, arrestin-2, Arr3, arrestin-3. Representative blot is shown. <b>B</b>. Quantification of ERK2 binding to P-Rh*-associated arrestins. <b>C</b>. CNBr-activated Sepharose (30 µl) containing 9 µg of covalently attached active phosphorylated (without or with 1 mM ATP) or inactive ERK2 was incubated with 3 µg of indicated purified arrestin in 60 µl of binding buffer (50 mM Tris-HCl, pH 7.4, 100 mM KCl, 1 mM EGTA, 1 mM DTT) for 20 min at 30°C. The beads were washed twice with 1 ml of ice-cold binding buffer supplemented with 0.01 mg/ml BSA. Bound arrestins were eluted with SDS sample buffer and quantified by Western blot, where known amounts of respective arrestins were run alongside samples to generate calibration curves. Means ± SD of three independent experiments are shown in panels <b>B</b> and <b>C</b>.</p

WT and Δ7 mutant of arrestin-2 rescue β2AR-mediated ERK activation in response to ICI118551 in DKO MEFs.

<p>DKO MEFs were infected with retrovirus encoding GFP (control, -), or untagged WT arrestin-2 (A2-WT), arrestin-2-3A (A2-3A), or arrestin-2-Δ7 (A2-Δ7). The cells were serum-starved 48 hours post-infection for 2 hours, stimulated with 1 µM ICI118551 for 10 min at 37°C, lysed, and analyzed by Western blot. Means ± SD of 3–4 independent experiments are shown in bar graphs; representative blots are shown below. *, p<0.05; **, p<0.01.</p

Impaired Lysosomal Integral Membrane Protein 2-dependent Peroxiredoxin 6 Delivery to Lamellar Bodies Accounts for Altered Alveolar Phospholipid Content in Adaptor Protein-3-deficient pearl Mice

Kook S, Wang P, Young LR, et al. Impaired Lysosomal Integral Membrane Protein 2-dependent Peroxiredoxin 6 Delivery to Lamellar Bodies Accounts for Altered Alveolar Phospholipid Content in Adaptor Protein-3-deficient pearl Mice. JOURNAL OF BIOLOGICAL CHEMISTRY. 2016;291(16):8414-8427.The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo. Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients