Study On The Mechanism Of Chemotaxis CCL5 Effect Of Chemokine Phase Separation On Cell Surface

Chemotokines produced by various cells,control the directed migration of immune cells by forming concentration gradients in tissues.It plays a key role in the recruitment of immune cells in humoral immune and inflammatory responses.However,how chemokines form concentration gradients in tissues remains controversial.The mainstream studies believe that chemokines form concentration gradients through molecular diffusion,but they are unable to support the directional cell migration of immune cells due to their instability.Furthermore,upon interaction with glycosaminoglycans,most chemokines are anchored on the cell membrane surface and thus can not form concentration gradients through molecular diffusion.The aim of this project is to study how chemokines perform chemotaxis function by forming stable concentration gradients in vivo.We proved that chemokine CCL5 co-phase-separated with glycosaminoglycans on cell surface to form stable concentration gradients and mediate the directional migration of immune cells.A novel source-sink model based on chemokine phase separation is proposed,which elucidates a new mechanism of directional migration of immune cells based on chemokine concentration gradient.In this paper,pET-28a-CCL5,pET-28a-CCL5-EGFP and related mutant plasmids were constructed,and active CCL5,CCL5-EGFP and related mutant proteins were obtained by expression and purification.Through confocal microscopy,enzymoscope,fluorescence bleaching recovery and Transwell chemotaxis experiments,it was revealed that the liquid phase separation of CCL5 was driven by the electrostatic interaction between its ⁴⁴RKNR⁴⁷domain and heparin,and thus enhanced its chemotaxis ability.In vitro diffusion experiments showed that it was the CCL5 diffusion driven by weak interaction promote its chemotaxis.In addition,in a source-sink model that mimics the release of CCL5 in tissues,CCL5 can form stable concentration gradients in tissues in the form of co-phase separation with glycosaminoglycans on the cell surface,exhibiting strong chemotactic function.In conclusion,this study reveals the molecular mechanism by which CCL5 forms a stable concentration gradient in tissues and enhances its chemotactic function through cophase separation with glycosaminoglycan,and proposes a source-sink model based on phase separation,which provides a new perspective for the function of chemokines in tissues and answers the question of how chemokines function in tissues.