Methods for making and observing model lipid droplets.

We present methods for making and testing the membrane biophysics of model lipid droplets (LDs). Methods are described for imaging LDs ranging in size from 0.1 to 40 µm in diameter with high-resolution microscopy and spectroscopy. With known LD compositions, membrane binding, sorting, diffusion, and tension were measured via fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), atomic force microscopy (AFM), and imaging flow cytometry. Additionally, a custom, small-volume pendant droplet tensiometer is described and used to measure the association of phospholipids to the LD surface. These complementary, cross-validating methods of measuring LD membrane behavior reveal the interplay of biophysical processes on lipid droplet monolayers.

Diffusion Dynamics of Water and Ethanol in Graphene Oxide.

We utilized the momentum transfer (Q)-dependence of quasi-elastic neutron scattering (QENS) to measure the dynamics of water and ethanol confined in graphene oxide (GO) powder or membranes at different temperatures and in different orientations. We found reduced diffusivities (up to 30% in the case of water) and a depression of dynamic transition temperatures. While water showed near Arrhenius behavior with an almost bulk-like activation barrier in a temperature range of 280-310 K, the diffusivity of ethanol showed little temperature dependence. For both water and ethanol, we found evidence for immobile and mobile fractions of the confined liquid. The mobile fraction exhibited jump diffusion, with a jump length consistent with the expected average spacing of hydroxide groups in the GO surfaces. From anisotropy measurements, we found weak anisotropy in the diffusivity of the mobile species and in the fraction and geometry of immobile species.

Methods for making and observing model lipid droplets.

The mechanisms by which the lipid droplet (LD) membrane is remodeled in concert with the activation of lipolysis incorporate a complex interplay of proteins, phospholipids, and neutral lipids. Model LDs (mLDs) provide an isolated, purified system for testing the mechanisms by which the droplet composition, size, shape, and tension affects triglyceride metabolism. Described here are methods of making and testing mLDs ranging from 0.1 to 40 µm diameter with known composition. Methods are described for imaging mLDs with high-resolution microscopy during buffer exchanges for the measurement of membrane binding, diffusion, and tension via fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), atomic force microscopy (AFM), pendant droplet tensiometry, and imaging flow cytometry. These complementary, cross-validating methods of measuring LD membrane behavior reveal the interplay of biophysical processes in triglyceride metabolism.

Live cell measurements of interaction forces and binding kinetics between Discoidin Domain Receptor 1 (DDR1) and collagen I with atomic force microscopy.

BACKGROUND: Discoidin Domain Receptors (DDRs) are membrane-tethered proteins of the receptor tyrosine kinase family, which signal in response to collagen. DDR expression is associated with human diseases, including fibrosis and cancer. The role of DDRs in human pathogenesis is mediated by dysregulated receptor function in response to the collagenous milieu. Thus, understanding DDR-collagen interactions is important for developing novel therapeutic strategies against DDRs. METHODS: We developed a biophysical method to isolate and measure specific interactions between DDR1 and collagen in live cells at the single molecule level using atomic force microscopy. This new method is capable of providing density and kinetics of membrane receptors in live cells. RESULTS: We isolated DDR1-collagen interactions and quantified the association and dissociation rates of the DDR1-collagen I complex. We estimated separate binding probabilities of collagen I to DDR and integrin, and by combining kinetic and binding probability data, we were able to estimate the density of receptors in two cancer cell types. We also tested the viability of a DDR1 blocking antibody and determined its efficacy in suppressing DDR1-collagen binding. CONCLUSIONS: The new method shows promise in quantifying receptor-ligand kinetics and receptor density on live cells. GENERAL SIGNIFICANCE: The new approach is applicable to other receptor-ligand systems and allows the determination of critical parameters at the single cell/single molecule level - in particular, the direct determination of kinetic and density differences of receptors in different cell types. This capability should prove to be useful in cancer research and drug design.

NaCl-Dependent Ordering and Dynamic Mechanical Response in Nanoconfined Water.

Understanding the dynamics of water under nanoscale confinement is important for biology, geology, tribology, and nanotechnology. In many naturally occurring situations, ions are present in water at various concentrations. Here we report on how the addition of sodium ions alters the squeeze-out behavior of water nanoconfined between a mica surface and silicon oxide tip. We find that Na+ ions enhance molecular ordering and lead to longer mechanical relaxation times. We also observed a critical ion concentration, above which the confined water switches from a viscous to an elastic (solid-like) response at very slow, quasistatic compression speeds.

How molecular motors extract order from chaos (a key issues review).

Molecular motors are the workhorses of living cells. Seemingly by 'magic', these molecules are able to complete purposeful tasks while being immersed in a sea of thermal chaos. Here, we review the current understanding of how these machines work, present simple models based on thermal ratchets, discuss implications for statistical physics, and provide an overview of ongoing research in this important and fascinating field of study.

Squeeze-out dynamics of nanoconfined water: A detailed nanomechanical study.

In this study, we present a detailed analysis of the squeeze-out dynamics of nanoconfined water confined between two hydrophilic surfaces measured by small-amplitude dynamic atomic force microscopy. Explicitly considering the instantaneous tip-surface separation during squeeze-out, we confirm the existence of an adsorbed molecular water layer on mica and at least two hydration layers. We also confirm the previous observation of a sharp transition in the viscoelastic response of the nanoconfined water as the compression rate is increased beyond a critical value (previously determined to be about 0.8 nm/s). We find that below the critical value, the tip passes smoothly through the molecular layers of the film, while above the critical speed, the tip encounters "pinning" at separations where the film is able to temporarily order. Preordering of the film is accompanied by increased force fluctuations, which lead to increased damping preceding a peak in the film stiffness once ordering is completed. We analyze the data using both Kelvin-Voigt and Maxwell viscoelastic models. This provides a complementary picture of the viscoelastic response of the confined water film.

Dissociation kinetics of an enzyme-inhibitor system using single-molecule force measurements.

We report on an improved method to interpret single molecule dissociation measurements using atomic force microscopy. We describe an easy to use methodology to reject nonspecific binding events, as well as estimating the number of multiple binding events. The method takes nonlinearities in the force profiles into account that result from the deformation of the used polymeric linkers. This new method is applied to a relevant enzyme-inhibitor system, latent matrix metalloprotease 9 (ProMMP-9, a gelatinase), and its inhibitor, tissue inhibitor of metalloproteases 1 (TIMP 1), which are important players in cancer metastasis. Our method provides a measured kinetic off-rate of 0.010 ± 0.003 s(-1) for the dissociation of ProMMP9 and TIMP1, which is consistent with values measured by ensemble methods.

Dynamic solidification in nanoconfined water films.

Mechanical properties of nanoconfined water layers are still poorly understood and continue to create controversy, despite their importance for biology and nanotechnology. We report on dynamic nanomechanical measurements of water films compressed to a few single molecular layers. We show that the mechanical properties of nanoconfined water layers change significantly with their dynamic state. In particular, we observed a sharp transition from viscous to elastic response even at extremely slow compression rates, indicating that mechanical relaxation times increase dramatically once water is compressed to less than 3-4 molecular layers.

Improving a high-resolution fiber-optic interferometer through deposition of a TiO2 reflective coating by simple dip-coating.

Fiber-optic based interferometers are used to detect small displacements, down to the subnanometer range. Coating the end of the optical fiber with a partially reflecting thin film greatly improves the resolution of interferometers by increasing the multiple reflections between the fiber end and the measured object. In this work, we present a quick and easy thin film deposition technique to coat the end of a single optical fiber by dip-coating a metal-organic precursor, which is then decomposed in a propane flame. The coated fiber was tested for morphology and usefulness for interferometric application. We found that this coating technique is much faster and easier than conventional thin coating techniques, and yields results that are comparable or better than can be achieved with sputtering or thermal evaporation.

Simultaneous normal and shear measurements of nanoconfined liquids in a fiber-based atomic force microscope.

We have developed an atomic force microscopy (AFM) technique that can perform simultaneous normal and shear stiffness measurements of nanoconfined liquids with angstrom-range amplitudes. The AFM technique is based on a fiber-interferometric, small-amplitude, off-resonance AFM. This AFM is capable of providing linear quasistatic measurements of the local mechanical properties of confined liquid layers while only minimally disturbing the layers themselves. A detailed analysis of the measurement geometry reveals that shear stiffness measurements are extremely challenging, as even small deviations from perfect orthogonality can lead to data that is very difficult to interpret. We will show ways out of this dilemma and present results that show simultaneous measurement of the shear and normal stiffness of confined liquid layers.

Solid or liquid? Solidification of a nanoconfined liquid under nonequilibrium conditions.

There has been a long-standing debate about the physical state and possible phase transformations of confined liquids. In this report, we show that a model-confined liquid can behave both as a Newtonian liquid with very little change in its dynamics and as a pseudosolid, depending solely on the rate of approach of the confining surfaces. Thus, the confined liquid does not exhibit any confinement-induced solidification in thermodynamic equilibrium. Instead, solidification is induced kinetically when the two confining surfaces are approached with a minimum critical rate. This critical rate is surprisingly slow (on the order of 6 A/s), explaining the frequent observation of confinement-induced solidification.