James K. Li

Cholesterol-dependent nanomechanical stability of phase-segregated multicomponent lipid bilayers.

Cholesterol is involved in endocytosis, exocytosis, and the assembly of sphingolipid/cholesterol-enriched domains, as has been demonstrated in both model membranes and living cells. In this work, we explored the influence of different cholesterol levels (5-40 mol%) on the morphology and nanomechanical stability of phase-segregated lipid bilayers consisting of dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/SM/Chol) by means of atomic force microscopy (AFM) imaging and force mapping. Breakthrough forces were consistently higher in the SM/Chol-enriched liquid-ordered domains (Lo) than in the DOPC-enriched fluid-disordered phase (Ld) at a series of loading rates. We also report the activation energies (DeltaEa) for the formation of an AFM-tip-induced fracture, calculated by a model for the rupture of molecular thin films. The obtained DeltaEa values agree remarkably well with reported values for fusion-related processes using other techniques. Furthermore, we observed that within the Chol range studied, the lateral organization of bilayers can be categorized into three distinct groups. The results are rationalized by fracture nanomechanics of a ternary phospholipid/sphingolipid/cholesterol mixture using correlated AFM-based imaging and force mapping, which demonstrates the influence of a wide range of cholesterol content on the morphology and nanomechanical stability of model bilayers. This provides fundamental insights into the role of cholesterol in the formation and stability of sphingolipid/cholesterol-enriched domains, as well as in membrane fusion.

Direct Correlation of Structures and Nanomechanical Properties of Multicomponent Lipid Bilayers

Exploring the fine structures and physicochemical properties of physiologically relevant membranes is crucial to understanding biological membrane functions including membrane mechanical stability. We report a direct correlation of the self-organized structures exhibited in phase-segregated supported lipid bilayers consisting of dioleoylphosphatidylcholine/egg sphingomyelin/cholesterol (DEC) in the absence and presence of ceramide (DEC-Ceramide) with their nanomechanical properties using AFM imaging and high-resolution force mapping. Direct incorporation of ceramide into phase-segregated supported lipid bilayers formed ceramide-enriched domains, where the height topography was found to be imaging setpoint dependent. In contrast, liquid ordered domains in both DEC and DEC-Ceramide presented similar heights regardless of AFM imaging settings. Owing to its capability for simultaneous determination of the topology and interaction forces, AFM-based force mapping was used in our study to directly correlate the structures and mechanical responses of different coexisting phases. The intrinsic breakthrough forces, regarded as fingerprints of bilayer stability, along with elastic moduli, adhesion forces, and indentation of the different phases in the bilayers were systematically determined on the nanometer scale, and the results were presented as two-dimensional visual maps using a self-developed code for force curves batch analysis. The mechanical stability and compactness were increased in both liquid ordered domains and fluid disordered phases of DEC-Ceramide, attributed to the influence of ceramide in the organization of the bilayer, as well as to the displacement of cholesterol as a result of the generation of ceramide-enriched domains. The use of AFM force mapping in studying phase segregation of multicomponent lipid membrane systems is a valuable complement to other biophysical techniques such as imaging and spectroscopy, as it provides unprecedented insight into lipid membrane mechanical properties and functions.

Atomic force microscopy force mapping in the study of supported lipid bilayers.

Investigating the structural and mechanical properties of lipid bilayer membrane systems is vital in elucidating their biological function. One route to directly correlate the morphology of phase-segregated membranes with their indentation and rupture mechanics is the collection of atomic force microscopy (AFM) force maps. These force maps, while containing rich mechanical information, require lengthy processing time due to the large number of force curves needed to attain a high spatial resolution. A force curve analysis toolset was created to perform data extraction, calculation and reporting specifically in studying lipid membrane morphology and mechanical stability. The procedure was automated to allow for high-throughput processing of force maps with greatly reduced processing time. The resulting program was successfully used in systematically analyzing a number of supported lipid membrane systems in the investigation of their structure and nanomechanics.

Self-Assembly of Colloidal Quantum Dots on the Scaffold of Triblock Copolymer Micelles

This paper describes the co-self-assembly of a polystyrene-poly(4-vinylpyridine)-poly(ethylene oxide) triblock copolymer with CdSe nanocrystals (quantum dots, QDs) and with a styrene compatible phenylenevinylene conjugated polymer (MEH-PPV) in mixtures of chloroform and 2-propanol. The polymer itself (PS(577)-P4VP(302)-PEO(852), where the subscripts refer to the number average degree of polymerization) forms worm-like micelles when 2-propanol is added to a solution of the polymer in CHCl(3). In the presence of increasing amounts of QDs, the structures become shorter and form only spherical hybrid micelles (with QDs bound to the surface) at 4:1 QD/polymer w/w, accompanied by free QDs. These structures retain their colloidal stability in 2-PrOH, suggesting that even the free QDs bear a surface shell of block copolymer. The presence of MEH-PPV has no affect on this self assembly. One of the most remarkable observations occurred when the samples in 2-PrOH were centrifuged to remove the free QDs accompanying the hybrid micelles. The micelles sedimented, but upon redispersion in 2-PrOH, rearranged to form colloidally stable long branched cylindrical structures including cylindrical networks.

Binding Forces of Streptococcus mutans P1 Adhesin

Streptococcus mutans is a Gram-positive oral bacterium that is a primary etiological agent associated with human dental caries. In the oral cavity, S. mutans adheres to immobilized salivary agglutinin (SAG) contained within the salivary pellicle on the tooth surface. Binding to SAG is mediated by cell surface P1, a multifunctional adhesin that is also capable of interacting with extracellular matrix proteins. This may be of particular importance outside of the oral cavity as S. mutans has been associated with infective endocarditis and detected in atherosclerotic plaque. Despite the biomedical importance of P1, its binding mechanisms are not completely understood. In this work, we use atomic force microscopy-based single-molecule and single-cell force spectroscopy to quantify the nanoscale forces driving P1-mediated adhesion. Single-molecule experiments show that full-length P1, as well as fragments containing only the P1 globular head or C-terminal region, binds to SAG with relatively weak forces (∼50 pN). In contrast, single-cell analyses reveal that adhesion of a single S. mutans cell to SAG is mediated by strong (∼500 pN) and long-range (up to 6000 nm) forces. This is likely due to the binding of multiple P1 adhesins to self-associated gp340 glycoproteins. Such a cooperative, long-range character of the S. mutans-SAG interaction would therefore dramatically increase the strength and duration of cell adhesion. We also demonstrate, at single-molecule and single-cell levels, the interaction of P1 with fibronectin and collagen, as well as with hydrophobic, but not hydrophilic, substrates. The binding mechanism (strong forces, cooperativity, broad specificity) of P1 provides a molecular basis for its multifunctional adhesion properties. Our methodology represents a valuable approach to probe the binding forces of bacterial adhesins and offers a tractable methodology to assess anti-adhesion therapy.

Quantification of the Nanomechanical Stability of Ceramide-Enriched Domains

The quantification of the mechanical stability of lipid bilayers is important in establishing composition-structure-property relations and sheds light on our understanding of the functions of biological membranes. Here, we designed an experiment to directly probe and quantify the nanomechanical stability and rigidity of the ceramide-enriched platforms that play a distinctive role in a variety of cellular processes. Our force mapping results have demonstrated that the ceramide-enriched domains require both methyl beta-cyclodextrin (MbCD) and chloroform treatments to weaken their highly ordered organization, suggesting a lipid packing that is different from that in typical gel states. Our results also show the expulsion of cholesterol from the sphingolipid/cholesterol-enriched domains as a result of ceramide incorporation. This work provides quantitative information on the nanomechanical stability and rigidity of coexisting phase-segregated lipid bilayers with the presence of ceramide-enriched platforms, indicating that that generation of ceramide in cells drastically alters the structural organization and the mechanical property of biological membranes.

Phase segregation of untethered zwitterionic model lipid bilayers observed on mercaptoundecanoic-acid-modified gold by AFM imaging and force mapping.

Planar supported lipid bilayers (SLBs) are often studied as model cell membranes because they are accessible to a variety of surface-analytic techniques. Specifically, recent studies of lipid phase coexistence in model systems suggest that membrane lateral organization is important to a range of cellular functions and diseases. We report the formation of phase-segregated dioleoylphosphatidylcholine (DOPC)/sphingomyelin/cholesterol bilayers on mercaptoundecanoic-acid-modified (111) gold by spontaneous fusion of unilamellar vesicles, without the use of charged or chemically modified headgroups. The liquid-ordered (l(o)) and liquid-disordered (l(d)) domains are observed by atomic force microscopy (AFM) height and phase imaging. Furthermore, the mechanical properties of the bilayer were characterized by force-indentation maps. Fits of force indentation to Sneddon mechanics yields average apparent Youngs moduli of the l(o) and l(d) phases of 100 +/- 2 and 59.8 +/- 0.9 MPa, respectively. The results were compared to the same lipid membrane system formed on mica with good agreement, though modulus values on mica appeared higher. Semiquantitative comparisons suggest that the mechanical properties of the l(o) phase are dominated by intermolecular van der Waals forces, while those of the fluid l(d) phase, with relatively weak van der Waals forces, are influenced appreciably by differences in surface charge density between the two substrates, which manifests as a difference in apparent Poisson ratios.

Pressure-induced restructuring of a monolayer film nanojunction produces threshold and power law conduction.

The electrical conduction of metal-molecule-metal junctions formed between Au-supported self-assembled monolayers of structurally different 1-hexanethiol, 1-decanethiol, and ferrocenyl-1-undecanethiol and a Pt-coated atomic force microscope (AFM) tip has been measured under different compression forces using conducting-probe AFM. The observed junction resistance had two distinct power law scaling changes with the compression force. Different scaling regions were assigned to the change in the contact area, tunneling distance, number of conduction pathways, and structure of the film under compression.

Mechanical stability of phase-segregated multicomponent lipid bilayers enhanced by PS-b-PEO diblock copolymers

Polymeric additives affect the mechanics of phospholipid vesicles, but little is known about the effect to supported lipid bilayers that are phase segregated on the submicron length scale. In this study, we use AFM-based force mapping, by means of breakthrough forces, to quantify the spreading pressures and line tensions of raft-forming lipid bilayers consisting of dioleoylphosphatidylcholine (DOPC), egg sphingomyelin (ESM), and cholesterol (Chol) in the presence of diblock copolymers comprised of polystyrene (PS) and poly(ethylene oxide) (PEO), PS-b-PEO. Varying molecular weights of PS-b-PEO were used in the experiments. The presence of the polymer leads to higher breakthrough forces when compared to pure DOPC/ESM/Chol bilayers. The lipid–polymer composite made with a PS block radius of gyration comparable to the bilayer thickness and a PEO block length that is the shortest exhibits the highest breakthrough forces and hence is the most stable mechanically. The breakthrough force distributions are analyzed to extract the spreading pressures and line tensions of the lipid–polymer composites. The spreading pressure is seen to increase with the addition of PS-b-PEO, and on average, increases with decreasing PEO block length. Based on the results, we propose the incorporation of the PS moiety into the bilayer core as the main mechanism of this enhanced resistance to bilayer breakthrough by the AFM tip.

Nanomechanical Stability of Phase-Segregated Multicomponent Lipid Bilayers Enhanced by Pluronics

Lipid-polymer/peptide systems have been used as platforms to understand various cellular components, functions, and processes such as the cell glycocalyx, the hydrophobic mismatch, and interaction of specific peptides with lipid bilayers. In this study, we used AFM-based force mapping to quantify by means of breakthrough forces the nanomechanical stability of raft-forming lipid bilayers consisting of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol (DEC) in the presence of diblock copolymers comprised of polystyrene(PS) and polyethylene oxide(PEO). Varying molecular weights of PS-b-PEO (Pluronics-mimics): PS(3.6)-b-PEO(25), PS(3.6)-b-PEO(16.6), PS(3.8)-b-PEO(6.5), PS(19)-b-PEO(6.4) were used in the experiments. The presence of the polymer led to a significant increase in the breakthrough forces when compared to a pure DEC bilayer. Bilayers with greater proportion of PS exhibited the highest breakthrough force hence is the most mechanically stable. Based on the results, we proposed the incorporation of the PS moiety into the bilayer core as the main mechanism of the enhanced stability. Our force mapping results provide a direct measurement of the effect of Pluronics on the stability of phase-separated multicomponent lipid bilayers that mimic biological membrane. In addition, the lipid bilayer-Pluronic-mimics systems presented in this study pose as an attractive platform for obtaining fundamental understanding on the role of Pluronics in drug delivery application as well as being a biological response modifier.