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

ABSTRACT: The outermost layer of eukaryotic 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 signaling. We describe the structure of the ciliary coat of the green alga Chlamydomonas by cryo-electron tomography and proteomic approaches and present the high-resolution single particle structure of FMG1B via cryo-electron microscopy, the most abundant constituent of the ciliary coat. We report FMG1B to be a highly unusual mucin orthologue, which lacks the major O-glycosylation of mammalian mucins, but undergoes significant N-glycosylation. We find that an isoform of FMG1-B, FMG1-A, previously believed to be not expressed, is present in Chlamydomonas and differentially regulated with FMG1-B. By micro-flow-based adhesion assays, we observe increased surface adhesion in the glycocalyx deficient double-mutant fmg1a-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. Data deposited here include label free quantification data on whole cells or isolated cilia confirming single knockout of FMG1B and high abundance of FMG1A in CC4533fmg1b (combined_protein_WC_CC4533fmg1b.tsv ) and double knockout of FMG1A and FMG1B in M32fmg1afmg1b (combined_proteinCilia_M32_M32fmg1afmg1b.tsv). The latter dataset was further analyzed for abundance changes in the cilia proteome (Log2_LFQ_ratios_M32_M32fmg1afmg1b.csv). Further, isolated cilia of wild type M32 were analyzed under non-reducing conditions to confirm structure predicted disulfide bridges via LC-MS/MS in FMG1B (FMG1B_intraprotein_disulfides.xlsx). Finally, isolated cilia of M32 were subjected to N-glycopeptide analyses using InSource-CID (FMG1B_HexNAc_modified_peptides.xlsx).

INSTRUMENT(S):

ORGANISM(S): Chlamydomonas Reinhardtii

TISSUE(S): Photosynthetic Cell

SUBMITTER: Lara Melanie Hoepfner  

LAB HEAD: Michael Hippler

PROVIDER: PXD055593 | Pride | 2025-04-29

REPOSITORIES: Pride