Scanning STED-FCS reveals spatiotemporal heterogeneity of lipid interaction in the plasma membrane of living cells

• DOI: 10.1038/ncomms6412
• Publication Date: 2014-11-20T10:54:51Z
• Authors: Alf Honigmann; Veronika Mueller; Haisen Ta; Andreas Schoenle; Erdinc Sezgin; Stefan W. Hell; Christian Eggeling
• Keywords: Microscopy; Sphingolipids; 0303 health sciences; Cell Membrane; Membrane Proteins; Cell Line; Membrane Lipids; 03 medical and health sciences; Cholesterol; Spectrometry, Fluorescence; Potoroidae; Animals; Phospholipids

The interaction of lipids and proteins plays an important role in plasma membrane bioactivity, and much can be learned from their diffusion characteristics. Here we present the combination of super-resolution STED microscopy with scanning fluorescence correlation spectroscopy (scanning STED-FCS, sSTED-FCS) to characterize the spatial and temporal heterogeneity of lipid interactions. sSTED-FCS reveals transient molecular interaction hotspots for a fluorescent sphingolipid analogue. The interaction sites are smaller than 80 nm in diameter and lipids are transiently trapped for several milliseconds in these areas. In comparison, newly developed fluorescent phospholipid and cholesterol analogues with improved phase-partitioning properties show more homogenous diffusion, independent of the preference for liquid-ordered or disordered membrane environments. Our results do not support the presence of nanodomains based on lipid-phase separation in the basal membrane of our cultured nonstimulated cells, and show that alternative interactions are responsible for the strong local trapping of our sphingolipid analogue.