Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy

http://rrichter.net/main/download/Wolny_JBC_2010_285_p30170.pdf

Analysis of CD44-Hyaluronan Interactions in an Artificial Membrane System: INSIGHTS INTO THE DISTINCT BINDING PROPERTIES OF HIGH AND LOW MOLECULAR WEIGHT HYALURONAN*

In 2010 there has again been an increase in the number of papers published involving piezoelectric acoustic sensors, or quartz crystal microbalances (QCM), when compared to the last period reviewed 2006-2009. The average number of QCM publications per annum was 124 in the period 2001-2005, 223 in the period 2006-9, and 273 in 2010. There are trends towards increasing use of QCM in the study of protein adsorption to surfaces (93% increase), homeostasis (67% increase), protein-protein interactions (40% increase), and carbohydrates (43% increase). New commercial systems have been released that are driving the uptake of the technology for characterisation of binding specificities, affinities, kinetics and conformational changes associated with a molecular recognition event. This article highlights theoretical and practical aspects of the principals that underpin acoustic analysis, then reviews exemplary papers in key application areas involving small molecular weight ligands, carbohydrates, proteins, nucleic acids, viruses, bacteria, cells, and membrane interfaces.

A survey of the 2010 quartz crystal microbalance literature

Clathrin-mediated endocytosis (CME) involves membrane-associated scaffolds of the bin-amphiphysin-rvs (BAR) domain protein family as well as the GTPase dynamin, and is accompanied and perhaps triggered by changes in local lipid composition. How protein recruitment, scaffold assembly and membrane deformation is spatiotemporally controlled and coupled to fission is poorly understood. We show by computational modelling and super-resolution imaging that phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] synthesis within the clathrin-coated area of endocytic intermediates triggers selective recruitment of the PX-BAR domain protein SNX9, as a result of complex interactions of endocytic proteins competing for phospholipids. The specific architecture induces positioning of SNX9 at the invagination neck where its self-assembly regulates membrane constriction, thereby providing a template for dynamin fission. These data explain how lipid conversion at endocytic pits couples local membrane constriction to fission. Our work demonstrates how computational modelling and super-resolution imaging can be combined to unravel function and mechanisms of complex cellular processes.

Lipid-mediated PX-BAR domain recruitment couples local membrane constriction to endocytic vesicle fission.

We present a new method for creating patches of fluid lipid bilayers with conjugated biotin and other compounds down to 1 μm resolution using a photolithographically patterned polymer lift-off technique. The patterns are realized as the polymer is mechanically peeled away in one contiguous piece in solution. The functionality of these surfaces is verified with binding of antibodies and avidin on these uniform micron-scale platforms. The biomaterial patches, measuring 1 μm-76 μm on edge, provide a synthetic biological substrate for biochemical analysis that is ∼100× smaller in width than commercial printing technologies. 100 nm unilamellar lipid vesicles spread to form a supported fluid lipid bilayer on oxidized silicon surface as confirmed by fluorescence photobleaching recovery. Fluorescence photobleaching recovery measurements of Dil (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC 1 8 (3))) stained bilayer patches yielded an average diffusion coefficient of 7.54 ′ 1.25 μm 2 s - 1 , equal to or slightly faster than typically found in Dil stained cells. This diffusion rate is ∼3× faster than previous values for bilayers on glass. This method provides a new means to form functionalized fluid lipid bilayers as micron-scale platforms to immobilize biomaterials, capture antibodies and biotinylated reagents from solution, and form antigenic stimuli for cell stimulation.

Creating biological membranes on the micron scale: Forming patterned lipid bilayers using a polymer lift-off technique

Protein organization on biomembranes and their dynamics are essential for cellular function It is not clear, however, how protein binding may influence the assembly of underlying lipids or how the membrane structure leads to functional protein organization Toward this goal, we investigated the effects of annexin a5 binding to biomimetic membranes using fluorescence imaging and correlation spectroscopy Annexin a5 (anx a5), a peripheral intracellular protein that plays a membrane remodeling role in addition to other functions, binds specifically and tightly to anionic (eg, phosphatidylserine)-containing membranes in the presence of calcium ion Our fluorescence microscopy reveals that annexin likely forms assemblies, along with a more dispersed population, upon binding to anionic biomembranes in the presence of calcium ion, which is reflected in its two-component Brownian motion To investigate the effects of annexin binding on the underlying lipids, we used specific acyl chain labeled phospholipid analogues, NBD-phosphatidylcholine (NBD-PC) and NBD-phosphatidylserine (NBD-PS) We find that both NBD-labeled lipids cluster under anx a5 assemblies, as compared with when they are found under the dispersed annexin population, and NBD-PS exhibits two-component lateral diffusion under the annexin assemblies In contrast, NBD-PC diffusion is slower by an order of magnitude under the annexin assemblies in contrast to its diffusion when not localized under anx a5 assemblies Our results indicate that, upon binding to membranes, the peripheral protein annexin organizes the underlying lipids into domains, which may have functional implications in vivo

/pdf/peripheral-protein-organization-and-its-influence-on-lipid-592tma3l4x.pdf

Kanika Vats

Papers

Peripheral protein organization and its influence on lipid diffusion in biomimetic membranes.

Characterizing the chemical complexity of patterned biomimetic membranes.