ABSTRACT
The kinetics of annexin A1 binding to solid-supported lipid bilayers consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS; 4:1) has been investigated as a function of the calcium ion concentration in the bulk phase. Quartz crystal microbalance measurements in conjunction with scanning force microscopy, fluorescence microscopy, and computer simulations indicate that at a given Ca2+ concentration annexin A1 adsorbs irreversibly on membrane domains enriched in POPS. By contrast, annexin A1 adsorbs reversibly on the POPC-enriched phase, which is composed of single POPS molecules embedded within a POPC matrix. The overall area occupied by the POPS-enriched phase is controlled by the CaCl2 concentration. Monte Carlo simulations suggest that the area of the POPS-enriched phase increases by a factor of 7 when the Ca2+ concentration is changed from 0.01 to 1 mM.
Subject(s)
Annexin A1/chemistry , Biosensing Techniques/methods , Lipid Bilayers/chemistry , Membranes, Artificial , Phosphatidylcholines/chemistry , Phosphatidylserines/chemistry , Adsorption , Biosensing Techniques/instrumentation , Computer Simulation , Microscopy, Atomic Force , Microscopy, Fluorescence/methods , Monte Carlo Method , Particle Size , Stress, Mechanical , Surface Properties , Time FactorsABSTRACT
By means of scanning force and fluorescence microscopy of artificial membranes immobilized on mica surfaces, the lateral organization of the annexin A2/S100A10 heterotetramer (annexin A2t) and its influence on the lateral organization of the lipids within the membrane have been elucidated. Planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) were prepared on atomically flat mica surfaces by the spreading of unilamellar vesicles. Fluorescence images of fluorescently labeled annexin A2t and scanning force microscopy images of nonlabeled protein bound to POPC/POPS bilayers show the formation of micrometer-sized lateral protein domains in the presence of 1 mM CaCl2. By means of scanning force microscopy, not only protein domains became discernible but also small membrane domains, which were attributed to POPS-enriched areas. A depletion of these POPS domains was observed in the vicinity of annexin A2t protein domains. These results indicate that annexin A2t is a peripheral membrane-binding complex capable of inducing lipid segregation.
Subject(s)
Annexin A2/chemistry , Lipid Bilayers/chemistry , Phosphatidylserines/chemistry , Protein Structure, Tertiary , S100 Proteins/chemistry , Microscopy, Atomic Force , Microscopy, Fluorescence , Phosphatidylcholines/chemistry , Protein Structure, QuaternaryABSTRACT
Phase separation of glycolipids in lipid mono- and bilayers is of great interest for the understanding of membrane function. The distribution of the ganglioside GM1 in sphingomyelin (SM)/1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), SM/1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DOPC) and SM/cholesterol/POPC Langmuir-Blodgett (LB) monolayers transferred at 36 mN/m has been studied by scanning force microscopy. Besides lateral organization of the glycolipid in LB monolayers as deduced from topography, material properties have been investigated by phase imaging, pulsed force mode and force modulation microscopy. It was shown that GM1 preferentially clusters in an ordered lipid matrix, i.e. the SM phase in the case of the SM/POPC and SM/DOPC mixture or in the ordered phase of POPC/SM/cholesterol monolayers. At higher local concentrations, three-dimensional protrusions enriched in GM1 occur, which may represent a precursor for the formation of micelles budding into the aqueous subphase. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00249-002-0232-4.
