Multiscale coordination of planar cell polarity

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PROJECT SUMMARY Cells have a remarkable capacity to self-assemble into organ-like structures in vitro. However, current in vitro-derived organs lack proper size and higher-order patterning, features that require organism-level information not present in a dish. Notably, the collective polarization and unidirectional alignment of cells across a tissue, a phenomenon known as planar cell polarity (PCP), is lacking in vitro organs, yet is essential for proper organ formation and function. Thus, to fulfill the promise of tissue engineering to generate functional organs in vitro we must understand how cells establish long-range collective polarization. We have established the murine skin as a model system to investigate the multiscale coordination of PCP in an expansive and regenerative tissue. By developing methods to perform ex vivo culturing, long-term live imaging, biophysical perturbations, and organotypic reconstitution of the epidermis, we have made key new discoveries about PCP establishment at the tissue, cellular and molecular scales. We discovered that uniaxial tissue deformation acts as a symmetry breaking cue that defines the major axis of PCP alignment in the epidermis. We further showed that that primary keratinocytes grown in the absence of global cues establish spontaneous, locally aligned domains of planar polarity de novo. Through super-resolution imaging and mapping the adhesive interactions of PCP components, we identified a role for cadherin-mediated cis- interactions in the clustering and sorting of asymmetric PCP complexes. The broad goal of this work is to build on these previous discoveries and technological developments to decipher how PCP is organized across different biological length scales. Using the mammalian skin epidermis as a model system, Specific Aim 1 will determine how long-range mechanical cues bias and align planar cell polarity across the epidermis. Specific Aim 2 will investigate the mechanisms by which cells skin cells spontaneously generate PCP through self- organization. Specific Aim 3 will decipher the nanoscale architecture and biochemical interactions of PCP complexes. Using technical innovations recently developed in my laboratory to perform live and super-resolution imaging of endogenously-tagged PCP proteins in both native and organotypic tissues, this work will provide fundamental new insights into the multiscale coordination of PCP.