Nubp1^(m1Nisw) lungs show altered distribution of Par3 and Numb1.

<p>(A–R) E16.5 lungs were sectioned and stained by immunohistochemistry for the indicated proteins. (A–H) The cellular localization of the polarity protein Par3 is significantly disrupted in Nubp1^(m1Nisw) mutant lung. (I–R) The mutation in Nubp1^(m1Nisw) disrupts the cellular localization of Numb1. Note the defect is restricted to distal epithelial cells. Images K-R are higher magnifications of the regions indicated in images I and J.</p

Line3-2 mutation perturbs cell proliferation and causes increased apoptosis in the lung.

<p>(A–I) In mutant lungs, cell proliferation is decreased during the pseudoglandular stage in mutant lungs, but is higher at the terminal sac stage as detected by EdU incorporation in lung sections. (I) Data presented as percentage of total nuclei. The results are representatives of 6 different sections. (J–R) Apoptosis is markedly increased in mutant lungs. (J–R) Lungs from E14.5 embryos were sectioned and processed for TUNEL staining. (R) The number of TUNEL positive cells were determined on sections and presented as percentage of total nuclei. The results are representatives of 7 different sections. Error bars are presented as SEM. (High) indicates higher magnification view.</p

Nubp1 Is Required for Lung Branching Morphogenesis and Distal Progenitor Cell Survival in Mice

<div><p>The lung is a complex system in biology and medicine alike. Whereas there is a good understanding of the anatomy and histology of the embryonic and adult lung, less is known about the molecular details and the cellular pathways that ultimately orchestrate lung formation and affect its health. From a forward genetic approach to identify novel genes involved in lung formation, we identified a mutated Nubp1 gene, which leads to syndactyly, eye cataract and lung hypoplasia. In the lung, Nubp1 is expressed in progenitor cells of the distal epithelium. Nubp1(m1Nisw) mutants show increased apoptosis accompanied by a loss of the distal progenitor markers Sftpc, Sox9 and Foxp2. In addition, Nubp1 mutation disrupts localization of the polarity protein Par3 and the mitosis relevant protein Numb. Using knock-down studies in lung epithelial cells, we also demonstrate a function of Nubp1 in regulating centrosome dynamics and microtubule organization. Together, Nubp1 represents an essential protein for lung progenitor survival by coordinating vital cellular processes including cell polarity and centrosomal dynamics.</p> </div

Line3-2 contains two missense mutations in the Nubp1 gene.

<p>(A) Schematic representation of the genomic region around the Nubp1 gene, which contains two missense mutations at nucleotide position 743 and 803 of the coding sequence. The mutations leads to a Threonine to Lysine and Proline to Glutamine change in the amino acid sequence of Nubp1. (B) Protein sequence alignment of the indicated genes show the mutations in line3-2 to be in a highly conserved region. Red boxes and asterisks highlight mutated regions. (C–H) E14.5 embryos from a complementation cross between line3-2 and Nubp1 knock-out reveals similar defects in lung (H) and limb development (G) as lin3-2 homozygous mutants (E, F).</p

Line3-2 reveals lung hypoplasia and limb syndactyly.

<p>(A–H) Embryos dissected at E18.5 (A) reveal that line3-2 mutants are slightly reduced in size and have significantly smaller lung lobes (B–F). (G–H) E18.5 embryos fixed and stained for bone (red) and cartilage (blue) in the forelimb demonstrate gross malformation and reduction in the number of digits in the line3-2. (I–N) Histological analysis of E18.5 lung lobes show decreased airway number, increased airway diameter and defects in distal lung epithelial morphology in line3-2. (I–L) E18.5 lungs were dissected, paraffin embedded and sectioned at 10 um. (M) Airway number and (N) the mean linear intercept (MLI) of wild-type and mutant lungs were determined from 4 different embryos at E18.5. Error bars are indicated as standard error of the mean (SEM).</p

Knock-down of Nubp1 causes centrosome multiplication and alteration in centrsome dynamics.

<p>(A–B) A549 cells stably transfected with GFP-Centrin were either transfected with control or Nubp1 specific siRNA. Represented is a time sequence showing the movement of the centriole pair in control cells and the aberrant numbers, increased and irregular centrosome movements in Nubp1 knock-down cells. (C–F) Knock-down of Nubp1 leads to multiple microtubule organization points and decreased acetylated-tubulin. A549 cells were transfected with either control or Nubp1 siRNA and stained for the indicated antigens.</p

Line3-2 mutants show a reduction in distal epithelial cell marker Sftpc but normal proximal differentiation.

<p>(A–J) Lung sections from E18.5 embryos were analyzed by immunohistochemistry for the indicated proximal differentiation markers (A–H) as well as for the distal epithelial cell marker Sftpc (I–J) revealing no significant changes in proximal epithelial differentiation but a marked decrease in Sftpc expression (insets in each panel show higher magnification views). (K) Expression of Sftpc was quantified by qPCR at the indicated stages, confirming the reduced expression of Sftpc in mutant lungs. Error bars represent SEM from 4 different embryos.</p

Rabankyrin-5 Localises to Macropinosomes in NIH3T3 Cells

<div><p>(A) NIH3T3 cells directly fixed and immunostained for endogenous Rabankyrin-5 and EEA1 reveal segregation of Rabankyrin-5 from EEA1-containing structures (arrows) in the cell periphery, while there is colocalisation in the cell centre (arrowheads).</p> <p>(B) Overexpression of Rabankyrin-5 increases the number of peripheral enlarged structures devoid of EEA1. NIH3T3 cells were infected with recombinant adenovirus for Rabankyrin-5 for 18 h. Dextran (2,5 mg/ml) uptake was performed for 3 min at 37 °C, fixed, and immunostained for the indicated antigens.</p> <p>(C and D) Formation of enlarged Rabankyrin-5 structures requires PI3-K activity. NIH3T3 cells either (C) DMSO treated or (D) pretreated for 20 min with wortmannin (WM; 100 nM) were incubated for 8 min with 0,5 μg/ml rhodamine-labelled transferrin and 2,5 mg/ml FITC-labelled dextran (MW, 10.000), fixed, processed for immunofluorescence, and analysed by confocal scanning microscopy.</p> <p>(E) NIH3T3 cells transiently transfected for YFP-Rabankyrin-5 and CFP-actin were imaged using time-lapse video microscopy to visualise the formation of macropinosomes by actin-driven membrane ruffles. Images were taken for the indicated time points. The arrowhead points towards Rabankyrin-5 association to an enlarged vesicle driven by actin dynamics over time. Scale bars represent 10 μm.</p></div

Inhibition of Rab5 Activity Decreases Fluid-Phase Uptake

<div><p>NIH3T3 cells transiently transfected for either (A) GFP-Rab5wt or (B) RN-tre were subjected to a 30-min uptake of rhodamine-conjugated transferrin (1 μg/ml) or dextran (MW, 70.000; 3 mg/ml) at 37 °C and further processed for confocal imaging with indicated antibodies.</p> <p>(A) Rab5wt transfected cells show colocalisation of Rabankyrin-5–labelled macropinocytic structures, indicated by the lack of transferrin accumulation, with Rab5.</p> <p>(B) Cells transiently transfected for RN-tre (asterisk) show a significant reduction of fluid-phase uptake compared to nontransfected cells.</p> <p>(C) Fluid-phase dextran quantification of single cells transfected for RN-tre by measuring internalised fluorescence intensity (<i>p</i> > 0,001). Values shown are means ± standard deviation of at least 15 cells. Scale bars represent 10 μm.</p></div

A Protein of 130 kDa Is a New Rab5 Effector

<div><p>(A) GST-Rab5-GDP and GST-Rab-GTPγS were loaded on beads and incubated with bovine brain cytosol. Bound proteins were eluted and analysed by SDS-PAGE followed by Coommasie Blue staining. The positions of the already known Rab5 effectors (EEA1, Rabaptin-5, hVps34, p110β, and Rabenosyn-5) and of the new Rab5 effector are indicated.</p> <p>(B) Schematic representation of the domain organisation in Rabankyrin-5. ANK, ankyrin repeats.</p> <p>(C) Bovine brain cytosol or HeLa cell cytosol was incubated with GST-Rab5-GDP– or GST-Rab5-GTPγS–loaded beads. Subsequently the beads were washed, and bound proteins were eluted and analysed by Western blotting using anti–Rabankyrin-5 antibodies.</p> <p>(D) GST-Rab4, -5, -7, and -11 fusion proteins were preloaded with GDP or GTPγS and incubated with in vitro-translated ³⁵S-methionine–labelled Rabankyrin-5 full-length protein. As a control, bound and unbound material was analysed by SDS-PAGE followed by phosphoimager analysis.</p> <p>(E) Rabankyrin-5 binds most strongly to PI(3)P. Recombinant full-length Rabankyrin-5 was incubated with liposomes containing 2% of the indicated phosphoinositide. Bound Rabankyrin-5 was detected by Western blotting.</p></div