Requirement for a Uroplakin 3a-like protein in the development of zebrafish pronephric tubule epithelial cell function, morphogenesis, and polarity

Uroplakin (UP)3a is critical for urinary tract development and function; however, its role in these processes is unknown. We examined the function of the UP3a-like protein Upk3l, which was expressed at the apical surfaces of the epithelial cells that line the pronephric tubules (PTs) of the zebrafish pronephros. Embryos treated with upk3l-targeted morpholinos showed decreased pronephros function, which was attributed to defects in PT epithelial cell morphogenesis and polarization including: loss of an apical brush border and associated phospho-ERM proteins, apical redistribution of the basolateral Na+/K+-ATPase, and altered or diminished expression of the apical polarity complex proteins Prkcz (atypical protein kinase C zeta) and Pard3 (Par3). Upk3l missing its C-terminal cytoplasmic domain or containing mutations in conserved tyrosine or proline residues did not rescue, or only partially rescued the effects of Upk3l depletion. Our studies indicate that Upk3l promotes epithelial polarization and morphogenesis, likely by forming or stimulating interactions with cytoplasmic signaling or polarity proteins, and that defects in this process may underlie the pathology observed in UP3a knockout mice or patients with renal abnormalities that result from altered UP3a expression. © 2012 Mitra et al

Structural requirements for PACSIN/Syndapin operation during zebrafish embryonic notochord development.

PACSIN/Syndapin proteins are membrane-active scaffolds that participate in endocytosis. The structure of the Drosophila Syndapin N-terminal EFC domain reveals a crescent shaped antiparallel dimer with a high affinity for phosphoinositides and a unique membrane-inserting prong upon the concave surface. Combined structural, biochemical and reverse genetic approaches in zebrafish define an important role for Syndapin orthologue, Pacsin3, in the early formation of the notochord during embryonic development. In pacsin3-morphant embryos, midline convergence of notochord precursors is defective as axial mesodermal cells fail to polarize, migrate and differentiate properly. The pacsin3 morphant phenotype of a stunted body axis and contorted trunk is rescued by ectopic expression of Drosophila Syndapin, and depends critically on both the prong that protrudes from the surface of the bowed Syndapin EFC domain and the ability of the antiparallel dimer to bind tightly to phosphoinositides. Our data confirm linkage between directional migration, endocytosis and cell specification during embryonic morphogenesis and highlight a key role for Pacsin3 in this coupling in the notochord

Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation

NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing hydrogen peroxide (H2O2) and to a lesser extent O2•-. The ratio of NOX4-derived H2O2 and O2•- can be altered by coenzyme Q (CoQ) mimics. Therefore, we hypothesize that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular endothelial cells, regulates inflammatory activation. To examine endothelial CYB5R3 in vivo, we created tamoxifen-inducible endothelium-specific Cyb5r3 knockout mice (R3 KO). Radiotelemetry measurements of systolic blood pressure showed systemic hypotension in lipopolysaccharides (LPS) challenged mice, which was exacerbated in R3 KO mice. Meanwhile, LPS treatment caused greater endothelial dysfunction in R3 KO mice, evaluated by acetylcholine-induced vasodilation in the isolated aorta, accompanied by elevated mRNA expression of vascular adhesion molecule 1 (Vcam-1). Similarly, in cultured human aortic endothelial cells (HAEC), LPS and tumor necrosis factor α (TNF-α) induced VCAM-1 protein expression was enhanced by Cyb5r3 siRNA, which was ablated by silencing the Nox4 gene simultaneously. Moreover, super-resolution confocal microscopy indicated mitochondrial co-localization of CYB5R3 and NOX4 in HAECs. APEX2-based electron microscopy and proximity biotinylation also demonstrated CYB5R3's localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 knockdown HAECs showed less total H2O2 but more mitochondrial O2•-. Using inactive or non-membrane bound active CYB5R3, we found that CYB5R3 activity and membrane translocation are needed for optimal generation of H2O2 by NOX4. Lastly, cells lacking the CoQ synthesizing enzyme COQ6 showed decreased NOX4-derived H2O2, indicating a requirement for endogenous CoQ in NOX4 activity. In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in NOX4-dependent H2O2 generation via CoQ.This work was supported by National Institutes of Health (NIH) R01 awards [R01 HL 133864 (A.C.S), R01 HL 128304 (A.C.S), R01 HL 149825 (A.C.S), R01 HL 153532 (A.C.S), R01 GM 125944 (F.J.S.), R01 DK 112854 (F.J.S.), 1S10OD021540-01 (Center for Biologic Imaging, University of Pittsburgh), 1S10RR019003-01 (Simon Watkins (S.W.)), 1S10RR025488-01 (S.W.), 1S10RR016236-01 (S.W)]. American Heart Association (AHA) [Established Investigator Award 19EIA34770095 (A.C.S.)], Post-doctoral Fellowship 19POST34410028 (S.Y.)]. American Society of Hematology (ASH) Minority Hematology Graduate Award (A.M.D-O.). Junta de Andalucía grant BIO-177 (P.N.), the FEDER Funding Program from the European Union and Spanish Ministry of Science, Innovation and Universities grant RED2018-102576-T (P.N.)

Wdpcp, a PCP Protein Required for Ciliogenesis, Regulates Directional Cell Migration and Cell Polarity by Direct Modulation of the Actin Cytoskeleton

Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration

Disruption of epithelial polarity in <i>upk3l</i> morphants.

<p>(A–B) Localization of Prkcz (A) or Pard3 (B), Na⁺/K⁺-ATPase, and nuclei in cross-sectioned embryos treated with control (CNT-MO) or <i>upk3l</i> MO (<i>upk3l</i>-MO). (C) Distribution of Na⁺/K⁺-ATPase and nuclei in 2-dpf control or morphant embryos. (D–E) TEM analysis of the proximal segment of PTs from control or morphant embryos. (D) Cross section of tubule show that it is comprised of 5–6 cells surrounding a central lumen. (E) At higher magnification, control lumens were filled with microvilli (mv) and cilia (ci), whereas microvilli were absent in morphants. (F) Immunolocalization of phosphorylated ERM proteins (P-ERM) in frozen sections of embryos. Sections were co-stained for Na⁺/K⁺-ATPase and nuclei. (G) Immunostaining of cilia-associated acetylated-tubulin and nuclei in the PT of laterally oriented embryos. p: proximal tubule, d: distal tubule.</p

Phenotypes of <i>upk3l</i> morphant embryos.

<p>(A–D) Uninjected-, CNT-MO-, or <i>upk3l</i>-MO-injected embryos were examined under a dissecting microscope and photographed. Pericardial edema, when observed, is indicated by a black arrow. (E) Embryo injected with 3 ng <i>upk3l</i>-MO, 100 pg MO-resistant <i>upk3l</i> mRNA, and 50 pg of mCherry mRNA (to mark successful injections). Microinjection of MO-resistant mRNA alone at doses of 50–200 pg did not produce detectable phenotypic abnormalities. (F) Distribution of morphological phenotypes associated with embryos injected with 5 ng CNT-MO, 2–6 ng of <i>upk3l</i>-MO, <i>tp56</i>-MO ± <i>upk3l</i>-MO, or <i>upk3l</i>-MO/<i>upk3l</i> mRNA/mCherry (n = 100).</p

The CT of Upk3l is critical for its function.

<p>(A) Clustal W alignment of the TM and CT region of hUP3a, xUP3a and Upk3l. The amino acids that comprise the TM and CT domain are highlighted, and conserved functional motifs and residues are underlined or shaded, respectively. (B) Phenotypes of embryos injected with CNT-MO, <i>upk3l</i>-MO alone, or <i>upk3l</i>-MO co-injected with mCherry mRNA and mRNAs encoding MO-resistant versions of <i>upk3l</i>ΔCT, <i>upk3l</i>P₂₅₈L, <i>upk3l</i>Y₂₅₁F, or <i>upk3l</i>Y₂₅₁D. Microinjection of MO-resistant mRNA alone at the same doses did not produce detectable phenotypic abnormalities. (C) Morphological phenotypes associated with embryos injected with 5 ng CNT-MO (<i>n</i> = 100), 3 ng of <i>upk3l</i>-MO (<i>n</i> = 100), or 3 ng of <i>upk3l</i>-MO and 100 pg of <i>upk3l</i>, <i>upk3l</i>ΔCT, <i>upk3l</i>P₂₅₈L, <i>upk3l</i>Y₂₅₁F, or <i>upk3l</i>Y₂₅₁D mRNA (n≥50). (D) Localization of FLAG-tagged Upk3l, Upk3lΔCT, Upk3lP₂₅₈L, Upk3lY₂₅₁F, or Upk3lY₂₅₁D in MDCK cells co-stained for actin and nuclei. Upk3lY₂₅₁F and Upk3lY₂₅₁D showed a predominantly intracellular localization and their low levels of expression necessitated an 1.5-fold increase in photomultiplier voltage above that used for the other samples.</p

Pronephric clearance, heart rate, and pericardial area in control and morphant embryos.

<p>(A) Clearance of 70 kDa-TRITC dextran injected into the common cardinal vein of 54-hpf control (top panel) or morphant (bottom panel) embryos. The fluorescence intensity of the sampled area (circled) was measured in each embryo 0, 5, and 24 h following dextran injection. (B) The heart rate, pericardial area, and the dextran retention was recorded for each embryo and the values relative to t = 0 (post dextran injection) were calculated. Data are reported as mean ± SEM (n = 25). (C) Uptake of 500 kDa or 10 kDa FITC-dextran in control or morphant PT epithelial cells. Tubule lumens are marked by asterisks, while internalized dextran is indicated by arrows. Note the incomplete ring of actin in morphant PT cells.</p