Fractalkine/CX3CL1 Functions as a Transmembrane Receptor to Promote Transendothelial Migration through glycan-dependent condensation 2257124

Abstract Introduction Biomolecular condensation provides a fundamental mechanism for organizing signaling complexes within cells. However, its occurrence on the cell surface–rich in glycans and exposed to dynamic extracellular forces–remains poorly understood. Fractalkine (CX3CL1), an endothelial membrane-bound chemokine, is heavily O-glycosylated and interacts with the monocyte receptor CX3CR1. Although fractalkine is known to mediate monocyte adhesion and migration, how its glycosylation drives condensate formation and endothelial signaling has not yet been elucidated. Methods We combined live-cell imaging, biochemical reconstitution, and a newly applied mucinase-based O-glycoproteomic approach to dissect the mechanism of fractalkine condensate formation. This strategy enabled site-specific mapping of O-glycosylation within the mucin domain and identification of key sialylated residues critical for condensate assembly. Functional analyses using glycosylation-deficient HUVEC mutants, including transendothelial migration assays, were performed to evaluate the impact of glycans on condensate formation and endothelial signaling. Results We found that endothelial membrane-bound fractalkine forms glycan-dependent condensates at the monocyte—endothelium interface. CX3CR1 engagement induces these condensates in a glycan-dependent manner. Glycoproteomic mapping revealed that a specific site of sialylated O-glycans is critical for these processes. The formation of these endothelial fractalkine condensates promotes the recruitment and activation of VEGFR2, leading to calcium influx and the upregulation of adhesion molecules, thereby enhancing monocyte transendothelial migration. Conclusion Our findings uncover a sialylation-dependent biomolecular condensate that mediates bidirectional fractalkine—CX3CR1 signaling. By recruiting and activating VEGFR2, this condensate links cell-surface glycosylation to endothelial signaling and immune cell migration, revealing a new paradigm of glycan-controlled cell-surface condensation. Funding Source n/a Topic Categories Cytokines and Chemokines and their Receptors (CCR)