Parkinson's disease is characterized by the presence of intracellular aggregates composed primarily of the neuronal protein α-synuclein (αS). Interactions between αS and various cellular membranes are thought to be important to its native function as well as relevant to its role in disease. We use fluorescence correlation spectroscopy to investigate binding of αS to lipid vesicles as a function of the lipid composition and membrane curvature. We determine how these parameters affect the molar partition coefficient of αS, providing a quantitative measure of the binding energy, and calculate the number of lipids required to bind a single protein. Specific anionic lipids have a large effect on the free energy of binding. Lipid chain saturation influences the binding interaction to a lesser extent, with larger partition coefficients measured for gel-phase vesicles than for fluid-phase vesicles, even in the absence of anionic lipid components. Although we observe variability in the binding of the mutant proteins, differences in the free energies of partitioning are less dramatic than with varied lipid compositions. Vesicle curvature has a strong effect on the binding affinity, with a>15-fold increase in affinity for small unilamellar vesicles over large unilamellar vesicles, suggesting that αS may be a curvature-sensing protein. Our findings provide insight into how physical properties of the membrane may modulate interactions of αS with cellular membranes.

Mycobacterium abscessus is an increasingly important cause of human disease; however, virulence determinants are largely uncharacterized. Previously, it was demonstrated that a rough, wild-type human clinical isolate (390R) causes persistent, invasive infection, while a smooth isogenic mutant (390S) has lost this capability. During serial passage of 390S, a spontaneous rough revertant was obtained, which was named 390V. This revertant regained the ability to cause persistent, invasive infection in human monocytes and the lungs of mice. Glycopeptidolipid (GPL), which plays a role in environmental colonization, was present in abundance in the cell wall of 390S, and was associated with sliding motility and biofilm formation. In contrast, a marked reduction in the amount of GPL in the cell wall of 390R and 390V was correlated with cord formation, a property associated with mycobacterial virulence. These results indicate that the ability to switch between smooth and rough morphologies may allow M. abscessus to transition between a colonizing phenotype and a more virulent, invasive form.

Alpha-synuclein (alphaS) is a soluble synaptic protein that is the major proteinaceous component of insoluble fibrillar Lewy body deposits that are the hallmark of Parkinson's disease. The interaction of alphaS with synaptic vesicles is thought to be critical both to its normal function as well as to its pathological role in Parkinson's disease. We demonstrate the use of fluorescence correlation spectroscopy as a tool for rapid and quantitative analysis of the binding of alphaS to large unilamellar vesicles of various lipid compositions. We find that alphaS binds preferentially to vesicles containing acidic lipids, and that this interaction can be blocked by increasing the concentration of NaCl in solution. Negative charge is not the only factor determining binding, as we clearly observe binding to vesicles composed entirely of zwitterionic lipids. Additionally, we find enhanced binding to lipids with less bulky headgroups. Quantification of the protein-to-lipid ratio required for binding to different lipid compositions, combined with other data in the literature, yields an upper bound estimate for the number of lipid molecules required to bind each individual molecule of alphaS. Our results demonstrate that fluorescence correlation spectroscopy provides a powerful tool for the quantitative characterization of alphaS-lipid interactions.

Analysis of infected macrophages revealed that lipidcontaining moieties of the mycobacterial cell wall are actively trafficked out of the mycobacterial vacuole. To facilitate the analysis of vesicular trafficking from mycobacteria-containing phagosomes, surface-exposed carbohydrates were labeled with hydrazide-tagged markers. The distribution of labeled carbohydrate/lipid moieties and subsequent interaction with cellular compartments were analyzed by immunoelectron microscopy and by fluorescence microscopy of live cells. The released mycobacterial constituents were associated with several intracellular organelles and were enriched strikingly in tubular endocytic compartments. Subcellular fractionation of infected macrophages by density gradient electrophoresis showed temporal movement of labeled bacterial constituents through early and late endosomes. Thin layer chromatography analysis of these subcellular fractions confirmed their lipid nature and revealed five dominant bacteria-derived species. These mycobacterial lipids were also found in extracellular vesicles isolated from the medium and could be observed in un-infected 'bystander' cells. Their transfer to bystander cells could expand the bacteria's sphere of influence beyond the immediate confines of the host cell.

Recent theoretical work suggests that protein folding involves an ensemble of pathways on a rugged energy landscape. We provide direct evidence for heterogeneous folding pathways from single-molecule studies, facilitated by a recently developed immobilization technique. Individual fluorophore-labeled molecules of the protein adenylate kinase were trapped within surface-tethered lipid vesicles, thereby allowing spatial restriction without inducing any spurious interactions with the environment, which often occur when using direct surface-linking techniques. The conformational fluctuations of these protein molecules, prepared at the thermodynamic midtransition point, were studied by using fluorescence resonance energy transfer between two specifically attached labels. Folding and unfolding transitions appeared in experimental time traces as correlated steps in donor and acceptor fluorescence intensity. The size of the steps, in fluorescence resonance energy transfer efficiency units, shows a very broad distribution. This distribution peaks at a relatively low value, indicating a preference for small-step motion on the energy landscape. The time scale of the transitions is also distributed, and although many transitions are too fast to be time-resolved here, the slowest ones may take >1 sec to complete. These extremely slow changes during the folding of single molecules highlight the possible importance of correlated, non-Markovian conformational dynamics.

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Interactions between the synaptic protein α-Synuclein and cellular membranes may be relevant both to its native function as well as its role in Parkinson's disease. We use single molecule Förster resonance energy transfer to probe the structure of α-Synuclein bound to detergent micelles and lipid vesicles. We find evidence that it forms a bent-helix when bound to highly curved detergent micelles, whereas it binds more physiological 100 nm diameter lipid vesicles as an elongated helix. Our results highlight the influence of membrane curvature in determining α-Synuclein conformation, which may be important for both its normal and disease-associated functions.α-Synuclein (AS) is the primary protein constituent of cytoplasmic Lewy bodies and Lewy neurites that are the pathological hallmark of Parkinson's Disease (PD)(1 , 2). Although it is strongly implicated in disease progression (3) the precise role of AS in PD is unclear. The native function of AS is also poorly understood, although evidence suggests that it may play a role both in maintaining neuronal plasticity and in the regulation of synaptic vesicle recycling (4 , 5).AS is disordered in solution(6) but undergoes a conformational change to an α-helical structure upon association with negatively charged membranes (7 , 8). A number of in vitro studies have characterized the interactions of AS both with detergent micelles and lipid membranes (reviewed in (9)). However, there is conflicting evidence as to whether AS demonstrates preferential affinities for specific phospholipids(7 -12) as well as if association with lipids inhibits or promotes AS aggregation or oligomerization (12 -16). A further matter of debate is the configuration of micelle or vesicle bound AS, with contrasting models proposing either an extended, continuous helix (17 -20) or two anti-parallel, non-interacting helices(21 -24), with an unstructured loop region between residues ~40-45 (Figure 1). Characterizing these conformations is of great interest, as membrane-bound structures may be pertinent both to native and disease-associated functions.Here we use single molecule Förster resonance energy transfer (FRET) to probe the helical structure of AS bound to SDS micelles and large unilamellar vesicles (LUVs). In FRET, the energy transfer efficiency (ET eff ) is dependent upon the distance between donor and acceptor fluorophores to the sixth power (25). In single molecule studies of protein conformations, each protein is labeled with a donor and an acceptor fluorophore. Photon bursts from the labeled † This work was supported by a grant from the Ellison Medical Foundation.. *To whom correspondence should be addressed: 266 Whitney Avenue, Box 208114, New Haven, CT 06520-8114; Telephone: 203-432-5342; Fax: 203-432-5175 proteins are collected as they diffuse through a diffraction-limited excitation volume. ET eff is calculated as: ET eff = I a°/ (I a°+ γI d°) , where I a° and I d° are the photon counts on the acceptor and donor channels, respectively, corrected for background and signal b...

Development of rapid processes combining hierarchical self-assembly with mesoscopic shape control has remained a challenge. This is particularly true for high-surface-area porous materials essential for applications including separation and detection, catalysis, and energy conversion and storage. We introduce a simple and rapid laser writing method compatible with semiconductor processing technology to control three-dimensionally continuous hierarchically porous polymer network structures and shapes. Combining self-assembly of mixtures of block copolymers and resols with spatially localized transient laser heating enables pore size and pore size distribution control in all-organic and highly conducting inorganic carbon films with variable thickness. The method provides all-laser-controlled pathways to complex high-surface-area structures, including fabrication of microfluidic devices with high-surface-area channels and complex porous crystalline semiconductor nanostructures.