Publications

Lateral segregation of cell membranes is accepted as a primary mechanism for cells to regulate a diversity of cellular functions. In this context, lipid rafts have been conceptualized as organizing principle of biological membranes where underlying cholesterol-mediated selective connectivity must exist even at the resting state. However, such a level of nanoscale compositional connectivity has been challenging to prove. Here we used single-molecule near-field scanning optical microscopy to visualize the nanolandscape of raft ganglioside GM1 after tightening by its ligand cholera toxin (CTxB) on intact cell membranes. We show that CTxB tightening of GM1 is sufficient to initiate a minimal raft coalescence unit, resulting in the formation of cholesterol-dependent GM1 nanodomains <120 nm in size. This particular arrangement appeared independent of cell type and GM1 expression level on the membrane. Simultaneous dual color high-resolution images revealed that GPI anchored and certain transmembrane proteins were recruited to regions proximal (<150 nm) to CTxB-GM1 nanodomains without physical intermixing. Together with in silico experiments, our high-resolution data conclusively demonstrate the existence of raft-based interconnectivity at the nanoscale. Such a linked state on resting cell membranes constitutes thus an obligatory step toward the hierarchical evolution of large-scale raft coalescence upon cell activation.

JTD Keywords: Cholera toxin, Membrane heterogeneity, Near-field scanning optical microscopy, Raft ganglioside GM1, Single-molecule detection

Interleukin 2 and interleukin 15 (IL2 and IL15, respectively) provide quite distinct contributions to T-cell-mediated immunity, despite having similar receptor composition and signaling machinery. As most of the proposed mechanisms underlying this apparent paradox attribute key significance to the individual {alpha}-chains of IL2 and IL15 receptors, we investigated the spatial organization of the receptors IL2R{alpha} and IL15R{alpha} at the nanometer scale expressed on a human CD4+ leukemia T cell line using single-molecule-sensitive near-field scanning optical microscopy (NSOM). In agreement with previous findings, we here confirm clustering of IL2R{alpha} and IL15R{alpha} at the submicron scale. In addition to clustering, our single-molecule data reveal that a non-negligible percentage of the receptors are organized as monomers. Only a minor fraction of IL2R{alpha} molecules reside outside the clustered domains, whereas [~]30% of IL15R{alpha} molecules organize as monomers or small clusters, excluded from the main domain regions. Interestingly, we also found that the packing densities per unit area of both IL2R{alpha} and IL15R{alpha} domains remained constant, suggesting a `building block' type of assembly involving repeated structures and composition. Finally, dual-color NSOM demonstrated co-clustering of the two {alpha}-chains. Our results should aid understanding the action of the IL2R-IL15R system in T cell function and also might contribute to the more rationale design of IL2R- or IL15R-targeted immunotherapy agents for treating human leukemia.

JTD Keywords: Near-field scanning optical microscopy (NSOM), Interleukin receptors IL2R, IL15R, Single-molecule detection, Nanometer-scale membrane organization

We present a single-molecule study on femtosecond dynamics in multichromophoric systems, combining fs pump-probe, emission-spectra and fluorescence-lifetime analysis. The ultrafast fs approach gives direct information on the initial exciton dynamics after excitation. The lifetime data show superradiance, a direct measure for the extent of the coherent coupling and static disorder. The spectra finally reveal the role of exciton-phonon coupling. At the single-molecule level a wide range of exciton delocalization lengths and energy redistribution times is revealed.

JTD Keywords: Single-molecule detection, Pump-probe, Exciton delocalization, Superradiance, Exciton-phonon coupling