Outer membrane monolayer domains from two-dimensional surface scanning resistance measurements

Cellular plasma membranes have domains that are defined, in most cases, by cytoskeletal elements. The outer half of the bilayer may also contain domai ns that organize glycosylphosphatidylinositol (GPI)-linked proteins. To def ine outer membrane barriers, we measured the resistive force on membrane bo und beads as they were scanned across the plasma membrane of HEPA-OVA cells with optical laser tweezers. Beads were bound by antibodies to fluorescein -phosphatidylethanolamine (FI-PE) or to the class I major histocompatibilit y complex (MHC class I) Qa-2 (a GPI-anchored protein). Two-dimensional scan s of resistive force showed both occasional, resistive barriers and a veloc ity-dependent, continuous resistance. At the lowest antibody concentration, which gave specific binding, the continuous friction coefficient of Qa-2 w as consistent with that observed by single-particle tracking (SPT) of small gold particles. At high antibody concentrations, the friction coefficient was significantly higher but decreased with increasing temperature, additio n of deoxycholic acid, or treatment with heparinase I. Barriers to lateral movement (>3 times the continuous resistance) were consistently observed. E lastic barriers (with elastic constants from 1 to 20 pN/mu m and sensitive to cytochalasin D) and small nonelastic barriers (<100 nm) were specificall y observed with beads bound to the GPI-linked Qa-2. We suggest that GPI-lin ked proteins interact with transmembrane proteins when aggregated by antibo dy-coated beads and the transmembrane proteins encounter cytoplasmic barrie rs to lateral movement. The barriers to lateral movement are dynamic, disco ntinuous, and low in density.