440 Planar cell polarity-disrupting mutation alters Celsr1-mediated cell adhesion and dynamics

Abstract

Planar cell polarity (PCP), the collective polarization of cells along a tissue plane, is fundamental to embryonic development and tissue organization in complex, multicellular organisms. A striking example of PCP is the ordered alignment of body hairs along the mammalian skin. PCP is regulated by an asymmetric junctional complex bridged by the principle PCP component Celsr, an atypical cadherin. Overall, this work seeks to elucidate how Celsr1 mediates cell adhesion to coordinate PCP and how these activities are disrupted by Crash (Crsh), a Celsr1 mutant with characteristic PCP defects such as open neural tube and altered hair follicle orientation in the skin. Crsh harbors a mutation mapping to the cadherin repeats of Celsr1’s extracellular domain, suggesting it may perturb Celsr1 adhesion. Using the mammalian epidermis as a model system and a combination of junctional recruitment and cell aggregation assays, we show that Celsr1 mediates calcium-dependent, homophilic cell adhesion through its extracellular domain. Surprisingly, Crsh mediates cell aggregation comparable to wildtype (WT), demonstrating this variant is competent in mediating trans-adhesive interactions. Yet when mixed, Crsh and WT expressing cells sort into distinct aggregates, indicating this point mutation alters Celsr1 adhesive properties. Crsh displayed more diffuse cell surface distribution and reduced junctional stability as measure by FRAP, suggesting Crsh is defective in lateral clustering. Importantly, chemically induced cis-dimerization of Crsh rescued keratinocyte junctional enrichment, as well as, trans-interactions with WT, indicating a role for cis-interactions in stabilizing Celsr1 adhesion. Finally, we use super-resolution imaging to investigate the subcellular distribution of intercellular PCP complexes and reveal how alterations in Ceslr1 adhesion affect this organization. Collectively, our results support a model in which the Crsh-pertaining region of Celsr1 is responsible for mediating cis-interactions that function to stabilize and reinforce the adhesive complex, a critical prerequisite for PCP organization and functional asymmetry.