Membrane phase separation drives responsive assembly of receptor signaling domains.

Plasma membrane heterogeneity has been tied to a litany of cellular functions and is often explained by analogy to membrane phase separation; however, models based on phase separation alone fall short of describing the rich organization available within cell membranes. Here we present comprehensive experimental evidence motivating an updated model of plasma membrane heterogeneity in which membrane domains assemble in response to protein scaffolds. Quantitative super-resolution nanoscopy measurements in live B lymphocytes detect membrane domains that emerge upon clustering B cell receptors (BCRs). These domains enrich and retain membrane proteins based on their preference for the liquid-ordered phase. Unlike phase-separated membranes that consist of binary phases with defined compositions, membrane composition at BCR clusters is modulated through the protein constituents in clusters and the composition of the membrane overall. This tunable domain structure is detected through the variable sorting of membrane probes and impacts the magnitude of BCR activation.

Chemical potential measurements constrain models of cholesterol-phosphatidylcholine interactions.

Bilayer membranes composed of cholesterol and phospholipids exhibit diverse forms of nonideal mixing. In particular, many previous studies document macroscopic liquid-liquid phase separation as well as nanometer-scale heterogeneity in membranes of phosphatidylcholine (PC) lipids and cholesterol. Here, we present experimental measurements of cholesterol chemical potential (µc) in binary membranes containing dioleoyl PC (DOPC), 1-palmitoyl-2-oleoyl PC (POPC), or dipalmitoyl PC (DPPC), and in ternary membranes of DOPC and DPPC, referenced to crystalline cholesterol. µc is the thermodynamic quantity that dictates the availability of cholesterol to bind other factors, and notably must be equal between coexisting phases of a phase separated mixture. It is simply related to concentration under conditions of ideal mixing, but is far from ideal for the majority of lipid mixtures investigated here. Measurements of µc can vary with phospholipid composition by 1.5 kBT at constant cholesterol mole fraction implying a more than fivefold change in its availability for binding receptors and other reactions. Experimental measurements are fit to thermodynamic models including cholesterol-DPPC complexes or pairwise interactions between lipid species to provide intuition about the magnitude of interactions. These findings reinforce that µc depends on membrane composition overall, suggesting avenues for cells to alter the availability of cholesterol without varying cholesterol concentration.

Single-molecule imaging reveals aß42:aß40 ratio-dependent oligomer growth on neuronal processes.

Soluble oligomers of the amyloid-ß peptide have been implicated as proximal neurotoxins in Alzheimer's disease. However, the identity of the neurotoxic aggregate(s) and the mechanisms by which these species induce neuronal dysfunction remain uncertain. Physiologically relevant experimentation is hindered by the low endogenous concentrations of the peptide, the metastability of Aß oligomers, and the wide range of observed interactions between Aß and biological membranes. Single-molecule microscopy represents one avenue for overcoming these challenges. Using this technique, we find that Aß binds to primary rat hippocampal neurons at physiological concentrations. Although amyloid-ß(1-40) as well as amyloid-ß(1-42) initially form larger oligomers on neurites than on glass slides, a 1:1 mix of the two peptides result in smaller neurite-bound oligomers than those detected on-slide or for either peptide alone. With 1 nM peptide in solution, Aß40 oligomers do not grow over the course of 48 h, Aß42 oligomers grow slightly, and oligomers of a 1:1 mix grow substantially. Evidently, small Aß oligomers are capable of binding to neurons at physiological concentrations and grow at rates dependent on local Aß42:Aß40 ratios. These results are intriguing in light of the increased Aß42:Aß40 ratios shown to correlate with familial Alzheimer's disease mutations.

Direct observation of single amyloid-ß(1-40) oligomers on live cells: binding and growth at physiological concentrations.

Understanding how amyloid-ß peptide interacts with living cells on a molecular level is critical to development of targeted treatments for Alzheimer's disease. Evidence that oligomeric Aß interacts with neuronal cell membranes has been provided, but the mechanism by which membrane binding occurs and the exact stoichiometry of the neurotoxic aggregates remain elusive. Physiologically relevant experimentation is hindered by the high Aß concentrations required for most biochemical analyses, the metastable nature of Aß aggregates, and the complex variety of Aß species present under physiological conditions. Here we use single molecule microscopy to overcome these challenges, presenting direct optical evidence that small Aß(1-40) oligomers bind to living neuroblastoma cells at physiological Aß concentrations. Single particle fluorescence intensity measurements indicate that cell-bound Aß species range in size from monomers to hexamers and greater, with the majority of bound oligomers falling in the dimer-to-tetramer range. Furthermore, while low-molecular weight oligomeric species do form in solution, the membrane-bound oligomer size distribution is shifted towards larger aggregates, indicating either that bound Aß oligomers can rapidly increase in size or that these oligomers cluster at specific sites on the membrane. Calcium indicator studies demonstrate that small oligomer binding at physiological concentrations induces only mild, sporadic calcium leakage. These findings support the hypothesis that small oligomers are the primary Aß species that interact with neurons at physiological concentrations.

Simultaneous single-molecule fluorescence and conductivity studies reveal distinct classes of Abeta species on lipid bilayers.

The extracellular senile plaques prevalent in brain tissue in Alzheimer's disease (AD) are composed of amyloid fibrils formed by the Abeta peptide. These fibrils have been traditionally believed to be featured in neurotoxicity; however, numerous recent studies provide evidence that cytotoxicity in AD may be associated with low-molecular weight oligomers of Abeta that associate with neuronal membranes and may lead to membrane permeabilization and disruption of the ion balance in the cell. The underlying mechanism leading to disruption of the membrane is the subject of many recent studies. Here we report the application of single-molecule optical detection, using fluorescently labeled human Alphabeta40, combined with membrane conductivity measurements, to monitor the interaction of single-oligomeric peptide structures with model planar black lipid membranes (BLMs). In a qualitative study, we show that the binding of Alphabeta to the membrane can be described by three distinctly different behaviors, depending on the Alphabeta monomer concentration. For concentrations much below 10 nM, there is uniform binding of monomers over the surface of the membrane with no evidence of oligomer formation or membrane permeabilization. Between 10 nM and a few hundred nanomolar, the uniform monomer binding is accompanied by the presence of peptide species ranging from dimers to small oligomers. The dimers are not found to permeabilize the membrane, but the larger oligomers lead to permeabilization with individual oligomers producing ion conductances of <10 pS/pore. At higher concentrations, perhaps beyond physiologically relevant concentrations, larger extended and dynamic structures are found with large conductances (hundreds of picosiemens), suggesting a major disruption of the membrane.

Amyloid-beta membrane binding and permeabilization are distinct processes influenced separately by membrane charge and fluidity.

The 40 and 42 residue amyloid-beta (Abeta) peptides are major components of the proteinaceous plaques prevalent in the Alzheimer's disease-afflicted brain and have been shown to have an important role in instigating neuronal degeneration. Whereas it was previously thought that Abeta becomes cytotoxic upon forming large fibrillar aggregates, recent studies suggest that soluble intermediate-sized oligomeric species cause cell death through membrane permeabilization. The present study examines the interactions between Abeta40 and lipid membranes using liposomes as a model system to determine how changes in membrane composition influence the conversion of Abeta into these toxic species. Abeta40 membrane binding was monitored using fluorescence-based assays with a tryptophan-substituted peptide (Abeta40 [Y10W]). We extend previous observations that Abeta40 interacts preferentially with negatively charged membranes, and show that binding of nonfibrillar, low molecular mass oligomers of Abeta40 to anionic, but not neutral, membranes involves insertion of the peptide into the bilayer, as well as sequential conformational changes corresponding to the degree of oligomerization induced. Significantly, while anionic membranes in the gel, liquid crystalline, and liquid ordered phases induce these conformational changes equally, membrane permeabilization is reduced dramatically as the fluidity of the membrane is decreased. These findings demonstrate that binding alone is not sufficient for membrane permeabilization, and that the latter is also highly dependent on the fluidity and phase of the membrane. We conclude that binding and pore formation are two distinct steps. The differences in Abeta behavior induced by membrane composition may have significant implications on the development and progression of AD as neuronal membrane composition is altered with age.

Mapping tissue-specific genes correlated with age-dependent changes in protein stability and function.

Biophysical measurements indicative of protein stability and function were performed on crude extracts from liver, muscle, and lens of a genetically heterogeneous mouse population. Genetic information was used to search for quantitative trait loci (QTL) that influenced the biophysical traits, with emphasis on phenotypes that previously have been shown to be altered in aged animals. Spectroscopic and enzymatic assays of crude liver and muscle tissue extracts from approximately 600 18-month-old mice, the progeny of (BALB/cJxC57BL/6J)F1 females and (C3H/HeJxDBA/2J)F1 males, were used to measure the susceptibility of a ubiquitous glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), to thermal denaturation. The rate constant for thermal inactivation of GAPDH correlated with markers on chromosome 5 (D5Mit79 and D5Mit251) for muscle lysates and chromosome 15 (D15Mit63 and D15Mit100) for liver tissue. The degree of variability of inactivation rate constants, a measure of the heterogeneity of muscle GAPDH in tissue extracts, was also associated with markers on chromosome 5 (D5Mit79 and D5Mit205). In addition, spectroscopic characteristics of extracted eye lens proteins were evaluated for their susceptibility to photooxidative stress. Absorbance and fluorescence emission characteristics of the lens proteins were mapped to QTL on chromosomes 5 and 15 (D5Mit25 and D15Mit171) while the degree of heterogeneity in photochemical oxidation kinetics was associated with a marker on the chromosome 8 (D8Mit42). Recent work has shown that GAPDH possesses a number of non-glycolytic functions including DNA/RNA binding and regulation of protein expression. Tissue specific differences in GAPDH stability may have significant consequences to these alternate functions during aging.