Unwrapping the ciliary coat: high-resolution structure and function of the ciliary glycocalyx

Abstract

The outermost layer of cilia is a coat of membrane anchored, glycosylated proteins often referred to as glycocalyx. This highly heterogeneous layer provides functions from regulation of adhesion, force transduction and protection to signalling. Despite its importance, there is a lack of studies focusing on the high-resolution molecular architecture of this layer. We describe the structure of the ciliary coat of the green algaChlamydomonas reinhardtiiby cryo-electron tomography and proteomic approaches and present the high-resolution single particle analysis structure of FMG1B via cryo-electron microscopy, the most abundant constituent of theC. reinhardtiiciliary coat. We report FMG1B to be a highly unusual mucin orthologue which lacks the majorO-glycosylation of mammalian mucins, but undergoes significantN-glycosylation. We find that an isoform of FMG1B, FMG1A, previously believed to be not expressed, is present inC. reinhardtiiand differentially regulated from FMG1B. By micro-flow-based adhesion assays we observe increased surface adhesion in the glycocalyx deficient double-mutantfmg1a-fmg1b. We find this mutant to be fully capable of surface-gliding, suggesting that neither isoform is required for extracellular force transduction by intraflagellar transport. Our data provide in-depth structural details of the ciliary coat that functions as the primary contact to the environment and reveal that FMG1 acts primarily as a protective layer with adhesion-regulative properties.