The apical protein Apnoia interacts with Crumbs to regulate tracheal growth and inflation

Most organs of multicellular organisms are built from epithelial tubes. To exert their functions, tubes rely on apico-basal polarity, on junctions, which form a barrier to separate the inside from the outside, and on a proper lumen, required for gas or liquid transport. Here we identify apnoia (apn), a novel Drosophila gene required for tracheal tube elongation and lumen stability at larval stages. Larvae lacking Apn show abnormal tracheal inflation and twisted airway tubes, but no obvious defects in early steps of tracheal maturation. apn encodes a transmembrane protein, primarily expressed in the tracheae, which exerts its function by controlling the localization of Crumbs (Crb), an evolutionarily conserved apical determinant. Apn physically interacts with Crb to control its localization and maintenance at the apical membrane of developing airways. In apn mutant tracheal cells, Crb fails to localize apically and is trapped in retromer-positive vesicles. Consistent with the role of Crb in apical membrane growth, RNAi-mediated knockdown of Crb results in decreased apical surface growth of tracheal cells and impaired axial elongation of the dorsal trunk. We conclude that Apn is a novel regulator of tracheal tube expansion in larval tracheae, the function of which is mediated by Crb

Yorkie controls tube length and apical barrier integrity during airway development

Epithelial organ size and shape depend on cell shape changes, cell-matrix communication, and apical membrane growth. The Drosophila melanogaster embryonic tracheal network is an excellent model to study these processes. Here, we show that the transcriptional coactivator of the Hippo pathway, Yorkie (YAP/TAZ in vertebrates), plays distinct roles in the developing Drosophila airways. Yorkie exerts a cytoplasmic function by binding Drosophila Twinstar, the orthologue of the vertebrate actin-severing protein Cofilin, to regulate F-actin levels and apical cell membrane size, which are required for proper tracheal tube elongation. Second, Yorkie controls water tightness of tracheal tubes by transcriptional regulation of the δ-aminolevulinate synthase gene (Alas). We conclude that Yorkie has a dual role in tracheal development to ensure proper tracheal growth and functionality

YAP1 Recruits c-Abl to Protect Angiomotin-Like 1 from Nedd4-Mediated Degradation

Tissue development and organ growth require constant remodeling of cell-cell contacts formed between epithelial cells. The Hippo signaling cascade curtails organ growth by excluding the transcriptional co-activator Yes Associated Protein 1 (YAP1) from the nucleus. Angiomotin family members recruit YAP1 to tight junctions [1], but whether YAP1 plays a specific role outside of the nucleus is currently unknown.The present study demonstrates that the E3 ubiquitin ligase Nedd4.2 targets Angiomotin-like 1 (AMOTL1), a family member that promotes the formation of epithelial tight junctions, for ubiquitin-dependent degradation. Unexpectedly, YAP1 antagonizes the function of Nedd4.2, and protects AMOTL1 against Nedd4.2-mediated degradation. YAP1 recruits c-Abl, a tyrosine kinase that binds and phosphorylates Nedd4.2 on tyrosine residues, thereby modifying its ubiquitin-ligase activity.Our results uncover a novel function for cytoplasmic YAP1. YAP1 recruits c-Abl to protect AMOTL1 against Nedd4.2-mediated degradation. Thus, YAP1, excluded from the nucleus, contributes to the maintenance of tight junctions

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication and protect, repair and restart damaged forks. Here we identify downstream neighbor of SON (DONSON) as a novel fork protection factor and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilizes forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATM- and Rad3-related (ATR)-dependent signaling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity and the potentiation of chromosomal instability. Hypomorphic mutations in DONSON substantially reduce DONSON protein levels and impair fork stability in cells from patients, consistent with defective DNA replication underlying the disease phenotype. In summary, we have identified mutations in DONSON as a common cause of microcephalic dwarfism and established DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability

Depletion of endogenous YAP1 or Nedd4.2 affects endogenous AMOTL1 levels.

<p>HEK293T cells were transiently transfected with either YAP1 or Nedd4.2 shRNA. Even a small reduction of YAP1 results in almost complete absence of AMOTL1. However depletion of Nedd4.2 maintains AMOTL1 levels. Actin was used as a loading control.</p

Nedd4.2 interacts with AMOTL1 and mislocalizes AMOTL1 from tight junctions.

<p><i>A</i>, Nedd4.2 reduces AMOTL1 protein levels. Co-expression of myc-tagged Nedd4.2 (myc.Nedd4.2) decreased Flag-tagged AMOTL1 (F.AMOTL1), occasionally to very low protein levels (lane 2). <i>B</i>, AMOTL1 interacts with Nedd4.2. HEK 293T cells were transfected with constructs encoding F.AMOTL1, myc.GFP, or myc.Nedd4.2. Cell lysates were probed with anti-myc and anti-Flag antibodies. Precipitation of F.AMOTL1 immobilized myc.Nedd4.2, but not myc.GFP, while precipitated F.GFP did not bind myc.Nedd4.2. <i>C</i>, Binding of endogenous AMOTL1 to Nedd4.2 was detected with anti-AMOTL1 and anti-Nedd4.2 antibodies, respectively. CEP164 was used as a negative control. <i>D</i>, AMOTL1 co-localizes mostly at the plasma membrane with Nedd4.2 in HEK293T cells. <i>E</i>, MDCK cells were transfected either with empty vector or Nedd4.2. Cells were then stained for endogenous AMOTL1 (grayscale) and the tight junction marker, ZO-1 (red). AMOTL1 is localized to tight junctions together, with ZO-1. Note that in dividing cells (yellow arrowheads) less ZO-1 as well as AMOTL1 are observed at junctions. When Nedd4.2 is added, endogenous ZO-1 as well as AMOTL1 are mislocalized from tight junctions and appear mostly in the cytoplasm. Scale bars represent 20 µm.</p

The WW3 domain of Nedd4.2 interacts with AMOTL1.

<p><i>A</i>, The two PPxY motifs of AMOTL1 (PY1: PPEY, amino acid 310–313, and PY2: PPEY, amino acid 367–370) are important for the interaction with Nedd4.2. Precipitation of wild type F.AMOTL1 immobilized both myc.Nedd4.2 and V5.YAP1 (left panel), while precipitation of mutant F.AMOTL1PY1/2, lacking both PPxY motifs, immobilized V5.YAP1, but only small amounts of myc.Nedd4.2 (right panel). <i>B</i>, The WW1, WW2, WW3 and WW4 domains of Nedd4.2 were mutated, and co-expressed with Flag-tagged AMOTL1. Only the interaction between the myc.Nedd4.2 WW3 and AMOTL1 was decreased, indicating that this domain interacts with AMOTL1.</p

Phosphorylation of Nedd4.2 by c-Abl at tyrosine 71 and 457 inhibits its E3 ligase activity.

<p><i>A</i>, Myc-Nedd4.2 proteins purified from HEK293T cells, co-expressing either wild type or kinase-dead (KD) c-Abl, were analyzed by nanoLC-MS/MS after in-gel digestion and phosphopeptide enrichment. Two singly Y-phosphorylated peptides (Y71 and Y457) were identified. <i>B</i>, Nedd4.2 proteins with Y71F and Y457F substitutions exhibit moderately reduced levels of c-Abl-mediated tyrosine phosphorylation. HEK293T cells were transfected with plasmids encoding myc-tagged wild type (WT) Nedd4.2 or mutants (Y71F, Y457F, or Y71F/Y457F), together with Flag-tagged AMOTL1 and WT or kinase-dead c-Abl, as indicated. Nedd4.2 proteins were precipitated with anti-myc antibodies and blotted with antibodies, as indicated. c-Abl and AMOTL1 were detected in the lysates with anti-Flag antibody. <i>C</i>, c-Abl restricts the ability of Nedd4.2 to extend the poly-ubiquitin chains of AMOTL1. Flag-tagged AMOTL1 (F.AMOTL1) and HA.Ubiquitin were co-expressed with either myc.Nedd4.2 alone, or with myc.Nedd4.2 and V5.cAbl. Ubiquitylated AMOTL1 species, detected by anti-HA antibodies, were shifted from higher molecular weights (poly-Ubi-AMOTL1) above 220 kDa towards lower molecular weight complexes between 100 kDa and 220 kDa (oligo-Ubi-AMOTL1) which are no longer degraded (see text for details). Nedd4.2^Y71F/Y457F is resistant to c-Abl phosphorylation. While c-Abl reduced the molecular weight of ubiquitylated AMOTL1 (left panel), there was no detectable change in molecular weight of ubiquitylated AMOTL1 (right panel) when c-Abl was co-expressed with the double tyrosine Nedd4.2 (Nedd4.2^Y71F/Y457F) mutant. <i>D</i>, The dual functions of YAP1. Upper panel: AMOTL1 recruits cytoplasmic YAP1 and c-Abl to tight junctions to curtail the ability of Nedd4.2 to poly-ubiquitylate AMOTL1. Lower panel: In cells primed to undergo proliferation, YAP1 translocates to the nucleus. Now, Nedd4.2 poly-ubiquitylates AMOTL1 and targets it for degradation.</p

c-Abl phosphorylates Nedd4.2 which forms a triple complex with AMOTL1 and YAP1.

<p><i>A</i>, Flag-tagged c-Abl (F.cAbl), immobilized on M2 sepharose beads, precipitates Nedd4.2. Immunoprecipitates were blotted with anti-myc antibody. AIP4, E3 ligase was used as a negative control. <i>B</i>, Protein-protein interactions among AMOTL1, Nedd4.2 and YAP1. HEK293T cells transfected with Flag-tagged AMOTL1, myc.Nedd4.2, V5.YAP1 and V5.cAbl plasmids were lysed and immunoprecipitation was performed using M2 beads. Precipitates were blotted with the antibodies indicated. Increased levels of AMOTL1 were detected in the presence of YAP1 and c-Abl. <i>C</i>, HEK293T cells were co-transfected with myc.Nedd4.2 and either wild type or kinase-dead (KD) c-Abl. Immunoprecipitation was performed with anti-myc antibody. Samples were blotted with anti-phosphotyrosine antibody to test for Nedd4.2 phosphorylation. <i>D</i>, Tyrosine phosphorylation of Nedd4.2 by c-Abl inhibits AMOTL1 degradation. Flag-tagged AMOTL1 (F.AMOTL1) and myc-tagged Nedd4.2 (myc.Nedd4.2) were co-expressed with either wild type or kinase-dead Flag-tagged c-Abl (F.cAbl). Precipitates, immobilized with anti-myc antibody, were probed with anti-phosphotyrosine and with anti-myc antibody show equal amounts of Nedd4.2; staining with anti-Flag revealed AMOTL1 protein levels.</p

YAP1-AMOTL1 binding requires the AMOTL1 motifs LPTY and the PPEY.

<p>Either of Flag-tagged AMOTL1^188–191A (LY: LPTY, amino acids 188–191), AMOTL1 ^{188–191A/310–313A} (LY/PY1: LPTY, amino acids 188–191 and PPEY, amino acids 310–313) or AMOTL1 ^{188–191A/367–370A} (LY/PY2: LPTY, amino acids 188–191 and PPEY, amino acids 367–370) mutants were used to precipitate YAP1. Immunoblotting was carried out using the indicated antibodies.</p