The role of Numb/Numblike in lung epithelium

During embryonic development, asymmetric cell division of stem cells plays an important role in generating distinct sibling cell fates. One of the factors involved in asymmetric cell division processes and cell fate determination is the membrane-associated adaptor protein Numb which is known to segregate apically as a cortical crescent in one of the two cleaved daughter cells. The ability of regulating cell fate determination is promoted by inhibition of Notch-signaling thus leading to premature differentiation and depletion of stem- or progenitor cells [1, 2]. Furthermore, there is evidence that both Numb and Notch are implicated in human tumors (non-small-cell lung carcinomas, NSCLCs). Class-1 NSCLCs show a loss of Numb expression while there is simultaneously an upregulated activation of Notch meaning that Numb might have a potential tumor suppressive function [3]. However, the role of Numb/Numblike during lung development has not been investigated so far. In this work we want to investigate if Numb and Numblike are critical factors concerning self-renewal and asymmetric cell division of lung stem cells as well as the complex developmental and differentiation processes of the lung. Therefore we have performed conditional knockout mice for Numb on a Numblike deficient background using the doxycycline inducible SP-CrtTA-tet-O-Cre (surfactant protein C) system which enables us to analyse potential Numb functions in pulmonary stem cell maintenance and/or tumor formation in the distal lung epithelium. SPCrtTApos/tetO-Crepos/Numblox-/-/Numblike-/- mutants are viable and exhibit a complete loss of Numb in the distal lung epithelium. Conditional deletion of Numb/Numblike in the distal lung epithelium under non-disease conditions leads to morphological changes of epithelia cells accompanied by a basal-to-apicolateral translocation of E-Cadherin. These changes indicate a potential function of Numb/Numblike in the lung epithelium polarity and integrity. We have also generated a mouse strain which will allow us to specifically elucidate changes in the transcriptome of conditional mutants by FACS sorting of S-PC positive Alveolar Type II cells (ATll). Due to the fact that Numb/Numblike are multifunctional adapter proteins we have established a SILAC (Stable isotope labeling in cells) based protein interaction screen in HEK 293T cells to identify novel Numb/Numblike interaction partners. In addition to previously described interaction partners of the endocytosis machinery we identified novel interaction partners of Numb/Numblike, which could be confirmed by Co-IP/Western Blotting.

Literature:

[1] Uemura, T., et al., numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos. Cell, 1989. 58(2): p.349–60.

[2] Cayouette, M. and M. Raff, Asymmetric segregation of Numb: a mechanism for neural specification from Drosophila to mammals. Nat Neurosci, 2002. 5(12): p.1265–9.

[3] Westhoff, B., et al., Alterations of the Notch pathway in lung cancer. Proc Natl Acad Sci U S A, 2009. 106(52): p.22293–8.