Mike Boxem (website)
Polarization of epithelial cells into apical and basolateral domains is essential for the functioning of epithelia as selectively permeable barriers. Loss of epithelial polarity contributes to epithelial diseases like polycystic kidney disease and retinal dystrophies. Moreover, epithelial cancers are characterized by loss of cell polarity and epithelial integrity, and many polarity regulators are mutated or deregulated in cancer.
The establishment of epithelial polarity relies on mutually antagonistic interactions between cortically localized polarity regulators. The Par (Par3/Par6/aPCK) complex and the Crumbs (Crumbs/Sdt/PATJ) complex together promote apical domain identity, while the Scribble proteins (Scrib, Lgl, Dlg) promote basolateral domain formation. However, the exact mechanisms that organize cell polarity vary between different tissues and organisms. For example, in Drosophila, C. elegans, and mouse embryos the requirements for Par3, Par6, Cdc42, aPKC and Crumbs all vary between epithelial tissues. A full understanding of epithelial polarity establishment can only be achieved therefore by studying the components involved under different conditions and in multiple cell types.
- Investigate the requirements for the apical Par complex in larval epithelia.
- Investigate the composition and functioning of the Crumbs complex.
Summary of Results
PAR-6, but not PAR-3, plays an essential role in the C. elegans epidermal epithelium
The apical Par complex components each play essential roles during embryonic development, but their functioning in larval tissues remains largely unknown. To analyze the role of the apical Par proteins PAR-3 and PAR-6 specifically in larval epithelia, we made use of the auxin-inducible protein degradation (AID) system . We identified a critical role for PAR-6, but not PAR-3, in development of the epidermis. The C. elegans epidermis consists of a large multinuclear syncytial cell (hyp7) that covers most of the animal’s body. Embedded within this cell is a row of epithelial cells, termed seam cells, which contributes additional nuclei to hyp7 through asymmetric divisions in which one daughter cell differentiates and fuses with hyp7 (Figure 1).
Degradation of PAR-6 in hyp7 and the seam cells resulted in numerous defects, including long delays, failure to divide, and aberrant cell fates (Figure 2). The exception was the first (L1) division, which occurred normally even without detectable PAR-6 present. These results demonstrate that PAR-6 plays an essential role in the normal developmental pattern of divisions of the seam cells.
The C. elegans Crumbs protein CRB-3 can induce apical membrane formation
In contrast to Drosohila, the C. elegans crumbs genes do not play an essential role in cell polarity [2–4]. To investigate whether C. elegans Crumbs proteins can promote apical domain identity, we overexpressed a CRB-3 mCherry fusion protein in the intestinal epithelium. Overexpression of this protein resulted in the appearance of bulges in the apical domain of the intestine (Figure 3). These results indicate that CRB-3 can promote apical domain formation.
MAGU-2 is the likely C. elegans homolog of Stardust
The closest C. elegans homolog of the Crumbs complex component Stardust is MAGU-2. However, this protein had not yet been characterized. Using the yeast two-hybrid system, we demonstrated a physical interaction between MAGU-2 and CRB-1. Moreover, we observed apical localization of an endogenous MAGU-2::GFP fusion protein above the level of cell junctions in embryonic intestinal cells (Figure 4).
Finally, we no longer observed apical enrichment of MAGU-2 in the embryo, indicating that MAGU-2 depends on Crumbs for its localization (Figure 5). Together, these results indicate that while the C. elegans Crumbs proteins are not essential for polarity establishment, several aspect of their functioning are conserved. These include the binding to the Stardust homolog MAGU-2, and the ability to induce apical domain formation when overexpressed.
- Zhang, L., Ward, J.D., Cheng, Z., and Dernburg, A.F. (2015). The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans. Dev. Camb. Engl. 142, 4374–4384.
- Waaijers, S., Ramalho, J.J., Koorman, T., Kruse, E., and Boxem, M. (2015). The C. elegans Crumbs family contains a CRB3 homolog and is not essential for viability. Biol. Open 4, 276–284.
- Bossinger, O., Klebes, A., Segbert, C., Theres, C., and Knust, E. (2001). Zonula adherens formation in Caenorhabditis elegans requires dlg-1, the homologue of the Drosophila gene discs large. Dev Biol 230, 29–42.
- Segbert, C., Johnson, K., Theres, C., van Furden, D., and Bossinger, O. (2004). Molecular and functional analysis of apical junction formation in the gut epithelium of Caenorhabditis elegans. Dev Biol 266, 17–26.
Riga, A., Castiglioni, V.G., and Boxem, M. (2019). New insights into apical-basal polarization in epithelia. Current opinion in cell biology. In Press