Galectin-3-Induced Glycan Lattices as Biophysical Modulators of Membrane Phase Behavior in Live Cells

The plasma membrane is an important interface that integrates extracellular biochemical input with biophysical organization to regulate cell behavior. Galectin-3, a multivalent glycan-binding protein, can influence both events through the formation of extracellular glycan lattices on the surfaces of glycosylated cells. Although such lattices have been proposed to reshape membrane organization, their impact on nanoscale membrane phase behavior has remained difficult to quantify. Here, we establish a link between Galectin-3 lattice formation and the remodeling of plasma membranes by using imaging fluorescence correlation spectroscopy (ImFCS) to measure diffusion coefficients of a series of fluorescently labeled probes that partition into ordered or disordered regions of the cell membrane. Across several human cell types (BeWo, BxPC3, THP-1, and HEK293T), we observed that Galectin-3 induced significant changes in the lateral mobility of membranes in a manner dependent on the capacity of Galectin-3 to oligomerize and bind glycans, and that specific glycoproteins can play outsized contributing roles. Membrane regions enriched in Galectin-3 exhibited reduced diffusion, suggesting glycan lattices can serve as nucleation sites for ordered, raft-like microdomains. Finally, we also reveal that these Galectin-3-induced changes to membrane dynamics significantly amplifies Ca²⁺ triggered scrambling of phosphatidylserine exposure. Together, these findings identify Galectin-3 as an extracellular phase organizer that translates glycan recognition into nanoscale mechanical remodeling of the plasma membrane, potentially serving as a generalizable mechanism for fine-tuning cell behavior.