TSC22D,WNKandNRBPgene families exhibit functional buffering and evolved with Metazoa for macromolecular crowd sensing

Abstract

SUMMARYThe ability to sense and respond to osmotic fluctuations is critical for the maintenance of cellular integrity. Myriad redundancies have evolved across all facets of osmosensing in metazoans, including among water and ion transporters, regulators of cellular morphology, and macromolecular crowding sensors, hampering efforts to gain a clear understanding of how cells respond to rapid water loss. In this study, we harness the power of gene co-essentiality analysis and genome-scale CRISPR-Cas9 screening to identify an unappreciated relationship betweenTSC22D2,WNK1andNRBP1in regulating cell volume homeostasis. Each of these genes have paralogs and are functionally buffered for macromolecular crowd sensing and cell volume control. Within seconds of hyperosmotic stress, TSC22D, WNK and NRBP family members physically associate into cytoplasmic biocondensates, a process that is dependent on intrinsically disordered regions (IDRs). A close examination of these protein families across metazoans reveals thatTSC22Dgenes evolved alongside a domain in NRBPs that specifically binds to TSC22D proteins, which we have termed NbrT (NRBPbinding region withTSC22D), and this co-evolution is concomitant with rapid IDR length expansion in WNK family kinases. Our study identifies functions for unrecognized components of the cell volume sensing machinery and reveals thatTSC22D,WNKandNRBPgenes evolved as cytoplasmic crowding sensors in metazoans to co-regulate rapid cell volume changes in response to osmolarity.