Clathrin-independent endocytosis (CIE) is usually a form of endocytosis that lacks a defined cytoplasmic machinery. fluidity and cell spreading. Furthermore, changes in membrane dynamics mediated by the galectin lattice affected macropinocytosis, an altered form of CIE, in HT1080 cells. Our results suggest that glycans play an important and nuanced role in CIE, with each cargo being affected uniquely by alterations in galectin and glycan profiles and their interactions. We conclude that galectin-driven effects exist on a continuum from stimulatory to inhibitory, with distinct CIE cargo proteins having unique response landscapes and with different cell types starting at different positions on these conceptual landscapes. of CIE and CME and the two modes of action that have been reported for glycan interactions on CIE: 1) stimulatory via promotion of entry into endocytic pits (23) and 2) inhibitory due to cell-surface sequestration of cargo (30). 0.05. Here, we investigate whether the well-characterized CIE cargo proteins major histocompatibility complex class I (MHCI) and CD59 are sensitive to glycan Rabbit Polyclonal to C1QC interactions. Further, as depicted in Fig. 1agglutinin (RCA) (which detects terminal galactose residues), and leucoagglutinin (PHA-L) (which binds to tri- and tetra-antennary branched complex agglutinin 1 (UEA1) (which binds fucose) and concanavalin A (which binds high-mannose residues) was not significantly changed by GlcNAc treatment. These results demonstrate that this increased availability of GlcNAc led to increases in glycan branching. These data also indicated that other glycan pattern characteristics were not indirectly affected. Lactose is usually a competitive inhibitor of galectinCglycan interactions (23, 26), a major focus of this study. RCA is usually a galactoside-binding lectin that binds the same epitope as galectins and hence was used to demonstrate the efficacy of lactose competitive inhibition on galectin binding. Lactose treatment during lectin binding completely inhibited RCA binding, with lactose-treated cells having fluorescent intensity histograms very similar to cells that were not subjected to lectin binding. This demonstrates the efficacy of lactose in blocking galectinCglycan interactions (Fig. PTC299 1and 0.05. We PTC299 found that GlcNAc treatment resulted in an increase in MHCI internalization (Fig. 2shows 2% lysate used as the input for immunoprecipitation, and the gel around the shows the material pulled down with the beads. The blots were probed with antibodies to MHCI (HC110) and galectin 3, and representative Western blots are shown. 0.05. GlcNAc treatment increases galectin 3 associated with MHCI To try to better understand whether the effects driven by GlcNAc treatment were direct or indirect, we immunoprecipitated MHCI from control cells and cells treated for 48 h with GlcNAc and blotted for galectin 3 to detect whether galectin 3 associated with the cargo protein (Fig. 3and and 0.05. As observed previously, increased glycan branching led to an increase in MHCI internalization, but this was not observed PTC299 in galectin 3Cdepleted cells (Fig. 4the galectin 3Cmediated portion of the galectin lattice was disrupted by knocking down galectin 3, it led to a dramatic increase in CD59 internalization. This suggests that inhibiting galectin 3 disrupts primarily the sequestration mode of action, allowing alternate galectins to drive an increase in internalization of CD59 via the endocytic pit entry mode, whereas, when galectin interactions are inhibited by lactose, both the endosomal pit entry PTC299 and the sequestration phases of the model are disrupted, resulting in an inhibition in CD59 CIE (Fig. 4CD44, EGFR, etc.) (23, 25, 26, 29). The galectin lattice affects cell-surface protein mobility and cell spreading To examine further what effect the galectin lattice has on membrane characteristics and the outcome when we disrupt these interactions, we measured the mobility of proteins in the membrane using fluorescence recovery after photobleaching (FRAP). The galectin lattice was disrupted using lactose in HeLa cells, and the FRAP of surface bound fluorescently labeled anti-CD98 antibodies (Fig. 5and square values. *, 0.05. As another means of studying the effect of altering the galectin lattice on cell behavior, we measured the ability of cells to spread on coverslips over time with various treatments that would.