Galectins are a class of proteins that bind to β-galactose–containing glycoconjugates and play critical roles in developmental, homeostatic, and pathological contexts (1, 2). Expressed across animal tissues, galectins are synthesized in the cytoplasm and trafficked to the extracellular milieu via the unconventional secretion pathway, although they may also be localized within the cytoplasm and nuclei of cells (3). Secreted galectins bind to cell surface glycoconjugates and modulate adhesion of cells to each other and to the extracellular matrix, as well as exert influence on intracellular signaling (4, 5). All galectins possess one or two evolutionarily conserved carbohydrate recognition domains (CRDs) that are responsible for binding their cognate sugars (6). Shaped like a jelly roll, the structure of a CRD consists of two opposing β-sheets: a five-stranded F-face and a six-stranded sugar-binding S-face. Among galectins, only galectin-3 (Gal-3) belongs to the chimera type: It is characterized by a long and intrinsically disordered proline-rich N-terminal tail (NT)(7). Binding to cell surface glycoconjugates induces secreted Gal-3 to oligomerize through mechanisms that are as yet incompletely understood; quantitative precipitation studies indicate that Gal-3 may pentamerize in the presence of multivalent glycan ligands and form heterogeneous disorganized complexes with the latter (8). The multimerization of Gal-3 has been proposed to occur both by selfassociation through its C-terminal CRDs as well as through self-association of its NTs (9). In addition, the NT has been shown to form intramolecular links with the F-face of Gal-3 CRD (which implies a potential unification of the self-association models)(10). However, the exact role of NT in the specific cellular functions of Gal-3 is still unclear. Equally enigmatic is the presence of multiple prolines in the NT of Gal-3. In PNAS, Zhao et al.(11) address these important questions. By creating a series of Gal-3 mutants that have their individual prolines mutated to alanines (and in one case to histidine in accordance with a P64H polymorphism that is associated with pathologies such as breast and thyroid cancer), the authors show that the removal of individual NT prolines impairs the ability of Gal-3 to induce human endothelial cell migration, T cell activation, and erythrocyte agglutination [all classically described functions of Gal-3 (12–14)]. The extent of impairment depends on the identity of the mutated proline; moreover, different, if overlapping, sets of prolines seem to contribute to each of the above cellular functions. Gal-3 has earlier been shown to mediate the formation of structures known as clathrin-independent carriers responsible for endocytosis of cell surface proteins such as CD44 and integrins (15). A set of NT prolines (different from the set inducing migration in the same cells) are also found to be necessary for the successful dispensation of this function of Gal-3.

Employing a series of biophysical techniques, the authors show that in the presence of glycoprotein ligands, Gal-3 may aggregate through a phenomenon akin to liquid–liquid phase separation (LLPS). The latter pertains to the sequestration of chemical reactions and the molecules mediating them in a membraneindependent manner through phase separation from the surrounding liquid milieu (16, 17). Zhao et al. show that this aggregation is dependent on the concentrations of Gal-3 and its ligands. By testing droplet formation using covalently delinked Gal-3 CRDs and NTs, they demonstrate that intermolecular interactions between the NTs and F-faces of the CRDs may drive LLPS-like behavior. This separation is impaired by attenuating the binding of the S …