Actin filaments attachment at the plasma membrane in live cells cause the formation of ordered lipid domains

The relationship between ordered plasma membrane nanodomains, known as lipid rafts, and actin filaments is the focus of this study. Plasma membrane order was followed in live cells at 37 degrees C using laurdan and di-4-ANEPPDHQ to report on lipid packing. Disrupting actin polymerisation decreased the fraction of ordered domains, which strongly argue that unstimulated cells have a basal level of ordered domains. Stabilising actin filaments had the opposite effect and increased the proportion of ordered domains. Decreasing the plasma membrane level of 4-phosphate-inositides lowers the number of attachment points for actin filaments and reduced the proportion of ordered domains. Aggregation of plasma membrane molecules, both lipid raft and non-lipid raft markers, lead to the formation of ordered domains. The increase in ordered domains was correlated with an increase in actin filaments just beneath the plasma membrane. In live cell plasma membrane blebs, which are detached from the underlying actin filaments, the fraction of ordered domains was low and GM1 could not be patched to form ordered domains. We conclude that ordered domains form when actin filaments attach to the plasma membrane. This downplays lipid-lipid interactions as the main driving force behind the formation of ordered membrane domains in vivo, giving greater prominence to membrane-intracellular filament interactions. (C) 2012 Elsevier B.V. All rights reserved.