Molecular Dynamics and Concentration in Raft and Boundary Domains in Actin-Depleted Plasma Membrane Vesicles as Revealed by Single-Molecule Imaging

Abstract

Compartmentalization of the cellular plasma membrane has been considered a critical mechanism for regulating molecular interactions occurring in the plane of the membrane, by varying local molecular concentration and dynamics within and across the compartments. Among various compartments, raft domains, enriched in sterols, sphingolipids, and proteins anchored by saturated aliphatic lipid tails, have been drawing extensive attention but remained elusive due to their nano-meso-scale sizes. Recently developed plasma membrane vesicles (PMVs), which are largely depleted of the actin filaments (actin-based membrane skeleton) but contain virtually the full complement of lipids and proteins of native membranes, provide a unique platform for investigating raft domains because, by lowering the temperature, micron-sized raft-like, liquid-ordered-phase (Lo)-like domains can be induced. Here, using these PMVs with coexisting domains and single-molecule imaging-tracking methods, we examined molecular dynamics and concentration of various molecules in raft and boundary domains. One of the most interesting findings is that GPI-APs, although they preferentially partition into the Lo-like raft domains, continually move back and forth between Lo-like domains and the bulk domain, showing very dynamic partitioning, without any particular concentration in the boundary domain. Their diffusion coefficient within the boundary region has been measured for the first time: it was in the middle of the values for the Lo-like domain and the bulk domain. Other observations using molecules with various levels of raft affinity will be reported and discussed in the context of signal transduction.