Darryl Y. Sasaki

Membrane bending by protein–protein crowding

Curved membranes are an essential feature of dynamic cellular structures, including endocytic pits, filopodia protrusions and most organelles. It has been proposed that specialized proteins induce curvature by binding to membranes through two primary mechanisms: membrane scaffolding by curved proteins or complexes; and insertion of wedge-like amphipathic helices into the membrane. Recent computational studies have raised questions about the efficiency of the helix-insertion mechanism, predicting that proteins must cover nearly 100% of the membrane surface to generate high curvature, an improbable physiological situation. Thus, at present, we lack a sufficient physical explanation of how protein attachment bends membranes efficiently. On the basis of studies of epsin1 and AP180, proteins involved in clathrin-mediated endocytosis, we propose a third general mechanism for bending fluid cellular membranes: protein–protein crowding. By correlating membrane tubulation with measurements of protein densities on membrane surfaces, we demonstrate that lateral pressure generated by collisions between bound proteins drives bending. Whether proteins attach by inserting a helix or by binding lipid heads with an engineered tag, protein coverage above ~20% is sufficient to bend membranes. Consistent with this crowding mechanism, we find that even proteins unrelated to membrane curvature, such as green fluorescent protein (GFP), can bend membranes when sufficiently concentrated. These findings demonstrate a highly efficient mechanism by which the crowded protein environment on the surface of cellular membranes can contribute to membrane shape change.

Polydiacetylene films: a review of recent investigations into chromogenic transitions and nanomechanical properties

Polydiacetylenes (PDAs) form a unique class of polymeric materials that couple highly aligned and conjugated backbones with tailorable pendant sidegroups and terminal functionalities. They can be structured in the form of bulk materials, multilayer and monolayer films, polymerized vesicles, and even incorporated into inorganic host matrices to form nanocomposites. The resulting materials exhibit an array of spectacular properties, beginning most notably with dramatic chromogenic transitions that can be activated optically, thermally, chemically, and mechanically. Recent studies have shown that these transitions can even be controlled and observed at the nanometre scale. These transitions have been harnessed for the purpose of chemical and biomolecular sensors, and on a more fundamental level have led to new insights regarding chromogenic phenomena in polymers. Other recent studies have explored how the strong structural anisotropy that thes em aterials possess leads to anisotropic nanomechanical behaviour. These recen ta dvances suggest that PDAs could be considered as a potential component in nanostructured devices due to the large number of tunable properties. In this paper, we provide a succinct review of the latest insights and applications involving PDA. We then focus in more detail on our work concerning ultrathin films, specifically structural properties, mechanochromism, thermochromism, and in-plane mechanical anisotropy of PDA monolayers. Atomic force microscopy (AFM) and fluorescence microscopy confirm that films 1–3 monolayers thick can be organized into highly ordered domains,with the conjugated backbones parallel to the substrate. The number of stable layers is controlled by the head-group functionality. Local mechanical stress applied by AFM an dn ear-field optical probes induces the chromogenic transition in the film at the nanometre scale. The transition

Steric confinement of proteins on lipid membranes can drive curvature and tubulation

Deformation of lipid membranes into curved structures such as buds and tubules is essential to many cellular structures including endocytic pits and filopodia. Binding of specific proteins to lipid membranes has been shown to promote membrane bending during endocytosis and transport vesicle formation. Additionally, specific lipid species are found to colocalize with many curved membrane structures, inspiring ongoing exploration of a variety of roles for lipid domains in membrane bending. However, the specific mechanisms by which lipids and proteins collaborate to induce curvature remain unknown. Here we demonstrate a new mechanism for induction and amplification of lipid membrane curvature that relies on steric confinement of protein binding on membrane surfaces. Using giant lipid vesicles that contain domains with high affinity for his-tagged proteins, we show that protein crowding on lipid domain surfaces creates a protein layer that buckles outward, spontaneously bending the domain into stable buds and tubules. In contrast to previously described bending mechanisms relying on local steric interactions between proteins and lipids (i.e. helix insertion into membranes), this mechanism produces tubules whose dimensions are defined by global parameters: domain size and membrane tension. Our results suggest the intriguing possibility that confining structures, such as lipid domains and protein lattices, can amplify membrane bending by concentrating the steric interactions between bound proteins. This observation highlights a fundamental physical mechanism for initiation and control of membrane bending that may help explain how lipids and proteins collaborate to create the highly curved structures observed in vivo.

Biologically functional cationic phospholipid-gold nanoplasmonic carriers of RNA

Biologically functional cationic phospholipid-gold nanoplasmonic carriers have been designed to simultaneously exhibit carrier capabilities, demonstrate improved colloidal stability, and show no cytotoxicity under physiological conditions. Cargo, such as RNA, DNA, proteins, or drugs, can be adsorbed onto or incorporated into the cationic phospholipid bilayer membrane. These carriers are able to retain their unique nanoscale optical properties under physiological conditions, making them particularly useful in a wide range of imaging, therapeutic, and gene delivery applications that utilize selective nanoplasmonic properties.

Large friction anisotropy of a polydiacetylene monolayer

Friction force microscopy measurements of a polydiacetylene monolayer film reveal a 300% friction anisotropy that is correlated with the film structure. The film consists of a monolayer of the red form of N‐(2‐ethanol)‐10,12‐pentacosadiynamide, prepared on a Langmuir trough and deposited on a mica substrate. As confirmed by atomic force microscopy and fluorescence microscopy, the monolayer consists of domains of linearly oriented conjugated backbones with pendant hydrocarbon side chains above and below the backbones. Maximum friction occurs when the sliding direction is perpendicular to the backbones. We propose that this effect is due to anisotropic film stiffness, which is a result of anisotropic side chain packing and/or anisotropic stiffness of the backbone itself. Friction anisotropy is therefore a sensitive, optically‐independent indicator of polymer backbone direction and monolayer structural properties.

Microfabricated silicon gas chromatographic micro-channels: fabrication and performance

Using both wet and plasma etching, we have fabricated micro- channels in silicon substrates suitable for use as gas chromatography (GC) columns. Micro-channel dimensions range from 10 to 80 micrometer wide, 200 to 400 micrometer deep, and 10 cm to 100 cm long. Micro-channels 100 cm long take up as little as 1 cm2 on the substrate when fabricated with a high aspect ratio silicon etch (HARSE) process. Channels are sealed by anodically bonding Pyrex lids to the Si substrates. We have studied micro-channel flow characteristics to establish model parameters for system optimization. We have also coated these micro-channels with stationary phases and demonstrated GC separations. We believe separation performance can be improved by increasing stationary phase coating uniformity through micro-channel surface treatment prior to stationary phase deposition. To this end, we have developed microfabrication techniques to etch through silicon wafers using the HARSE process. Etching completely through the Si substrate facilitates the treatment and characterization of the micro-channel sidewalls, which dominate the GC physico- chemical interaction. With this approach, we separately treat the Pyrex lid surfaces that form the top and bottom surfaces of the GC flow channel.

Polymerization-Induced Epitaxy: Scanning Tunneling Microscopy of a Hydrogen-Bonded Sheet of Polyamide on Graphite

A molecularly thin film of a two-dimensional polymer network formed by hydrogen bonding was synthesized and investigated with scanning tunneling microscopy. Poly(∈-caprolactam) (nylon 6) was epitaxially grown on the basal plane of graphite and an ultrathin film of the polymer was obtained after the bulk materials had been washed away with solvents. The polymer chain has a planar, all-trans conformation and adjacent chains run in the antiparallel direction. This produces complete pairing of hydrogen bonding groups, with each amide group lying on a straight line perpendicular to the polymer backbone. This hydrogen-bonded sheet is oriented so that each polymer backbone lies in the (1010) direction on the graphite hexagonal lattice, as opposed to the (1120) direction taken by other paraffinic molecules studied so far. This experiment shows that hydrogen bonding can be used to control the orientation of macromolecules in two dimensions.

Directed formation of lipid membrane microdomains as high affinity sites for His-tagged proteins.

Lipid membranes composed of an iminodiacetic acid functionalized lipid, DSIDA, in a POPC matrix exhibited switchable properties via Cu(2+) recognition to rapidly assemble microdomains that act as high affinity sites for His-tagged proteins. The microdomains demonstrated an order of magnitude enhanced affinity for the proteins compared to homogeneously functionalized POPC membranes with Ni(2+)-NTA DOGS or Cu(2+)-DOIDA, while a rapid release and restoration of the original membrane was accomplished with micromolar concentrations of EDTA.

Control of membrane structure and organization through chemical recognition

Cell membranes consist of a fluidic medium of lipids and proteins that organize into specific submicron scale structures for signaling and molecular trafficking processes. These organized molecular assemblies form as a result of the structure and chemistry of the membrane components as well as the interactions of those components with analytes from solution. Although considerable research has focused on the structure and chemistry of membrane components and their ability to form organized assemblies, less attention has been paid toward the influence that chemical recognition has upon membrane reorganization. This review focuses on the recognition and binding of metal ions, small molecules, polyelectrolytes, and proteins on model membrane systems to assess the effects of long- and short-range interactions up on the molecular organization of the membrane. Chemical recognition can induce dramatic changes on the membranes phase transition temperature and the clustering or dispersion of membrane components.

Lipid membrane reorganization induced by chemical recognition.

Nanoscale structural reorganization of a lipid bilayer membrane induced by a chemical recognition event has been imaged using in situ atomic force microscopy (AFM). Supported lipid bilayers, composed of distearylphosphatidylcholine (DSPC) and a synthetic lipid functionalized with a Cu(2+) receptor, phase-separate into nanoscale domains that are distinguishable by the 9 A height difference between the two molecules. Upon binding of Cu(2+) the electrostatic nature of the receptor changes, causing a dispersion of the receptor molecules and subsequent shrinking of the structural features defined by the receptors in the membrane. Complete reversibility of the process was demonstrated through the removal of metal ions with EDTA.