Using fluorescence correlation spectroscopy, we show that the diffusive movements of catalase enzyme molecules increase in the presence of the substrate, hydrogen peroxide, in a concentration-dependent manner. Employing a microfluidic device to generate a substrate concentration gradient, we show that both catalase and urease enzyme molecules spread toward areas of higher substrate concentration, a form of chemotaxis at the molecular scale. Using glucose oxidase and glucose to generate a hydrogen peroxide gradient, we induce the migration of catalase toward glucose oxidase, thereby showing that chemically interconnected enzymes can be drawn together.

We show that diffusion of single urease enzyme molecules increases in the presence of urea in a concentration-dependent manner and calculate the force responsible for this increase. Urease diffusion measured using fluorescence correlation spectroscopy increased by 16-28% over buffer controls at urea concentrations ranging from 0.001 to 1 M. This increase was significantly attenuated when urease was inhibited with pyrocatechol, demonstrating that the increase in diffusion was the result of enzyme catalysis of urea. Local molecular pH changes as measured using the pH-dependent fluorescence lifetime of SNARF-1 conjugated to urease were not sufficient to explain the increase in diffusion. Thus, a force generated by self-electrophoresis remains the most plausible explanation. This force, evaluated using Brownian dynamics simulations, was 12 pN per reaction turnover. These measurements demonstrate force generation by a single enzyme molecule and lay the foundation for a further understanding of biological force generation and the development of enzyme-driven nanomotors.

Encapsulation of imaging agents and drugs in calcium phosphate nanoparticles (CPNPs) has potential as a nontoxic, bioresorbable vehicle for drug delivery to cells and tumors. The objectives of this study were to develop a calcium phosphate nanoparticle encapsulation system for organic dyes and therapeutic drugs so that advanced fluoresence methods could be used to assess the efficiency of drug delivery and possible mechanisms of nanoparticle bioabsorption. Highly concentrated CPNPs encapsulating a variety of organic fluorophores were successfully synthesized. Well-dispersed CPNPs encapsulating Cy3 amidite exhibited nearly a 5-fold increase in fluorescence quantum yield when compared to the free dye in PBS. FCS diffusion data and cell staining were used to show pH-dependent dissolution of the particles and cellular uptake, respectively. Furthermore, an experimental hydrophobic cell growth inhibitor, ceramide, was successfully delivered in vitro to human vascular smooth muscle cells via encapsulation in CPNPs. These studies demonstrate that CPNPs are effective carriers of dyes and drugs for bioimaging and, potentially, for therapeutic intervention.

Bioinspired artificial water channels aim to combine the high permeability and selectivity of biological aquaporin (AQP) water channels with chemical stability. Here, we carefully characterized a class of artificial water channels, peptide-appended pillar [5]arenes (PAPs). The average single-channel osmotic water permeability for PAPs is 1.0(±0.3) × 10 −14 cm 3 /s or 3.5(±1.0) × 10 8 water molecules per s, which is in the range of AQPs (3.4∼40.3 × 10 8 water molecules per s) and their current synthetic analogs, carbon nanotubes (CNTs, 9.0 × 10 8 water molecules per s). This permeability is an order of magnitude higher than first-generation artificial water channels (20 to ∼10 7 water molecules per s). Furthermore, within lipid bilayers, PAP channels can self-assemble into 2D arrays. Relevant to permeable membrane design, the pore density of PAP channel arrays (∼2.6 × 10 5 pores per μm 2 ) is two orders of magnitude higher than that of CNT membranes (0.1∼2.5 × 10 3 pores per μm 2 ). PAP channels thus combine the advantages of biological channels and CNTs and improve upon them through their relatively simple synthesis, chemical stability, and propensity to form arrays.artificial aquaporins | artificial water channels | peptide-appended pillar [5]arene | single-channel water permeability | two-dimensional arrays T he discovery of the high water and gas permeability of aquaporins (AQPs) and the development of artificial analogs, carbon nanotubes (CNTs), have led to an explosion in studies aimed at incorporating such channels into materials and devices for applications that use their unique transport properties (1-9). Areas of application include liquid and gas separations (10-13), drug delivery and screening (14), DNA recognition (15), and sensors (16). CNTs are promising channels because they conduct water and gas three to four orders of magnitude faster than predicted by conventional Hagen-Poiseuille flow theory (11). However, their use in large-scale applications has been hampered by difficulties in producing CNTs with subnanometer pore diameters and fabricating membranes in which the CNTs are vertically aligned (4). AQPs also efficiently conduct water across membranes (∼3 billion molecules per second) (17) and are therefore being studied intensively for their use in biomimetic membranes for water purification and other applications (1, 2, 18). The largescale applications of AQPs is complicated by the high cost of membrane protein production, their low stability, and challenges in membrane fabrication (1).Artificial water channels, bioinspired analogs of AQPs created using synthetic chemistry (19), ideally have a structure that forms a water-permeable channel in the center and an outer surface that is compatible with a lipid membrane environment (1). Interest in artificial water channels has grown in recent years, following decades of research and focus on synthetic ion channels (19). However, two fundamental questions remain: (i) Can artificial channels approach the permeability and selectivity of AQP water chan...

T he migration of vascular endothelial cells (ECs) plays an important role in angiogenesis and postangioplasty wound healing. Cell migration is a coordinated process consisting of adhesion at the leading edge and detachment at the rear (1, 2). The focal adhesions (FAs), cytoskeleton, and signaling pathways that mediate cell migration need to respond to diverse extracellular signals and translate them into precisely regulated intracellular responses. There have been many studies on EC migration in response to gradients of soluble chemicals (chemotaxis) and immobilized extracellular matrix (haptotaxis; refs. 3-6). However, the effect of mechanical environment on EC migration is not well understood.ECs are constantly subjected to shear stress, the tangential component of hemodynamic force caused by blood flow. It has been shown that shear stress induces EC monolayer remodeling, e.g., increase of stress fibers and alterations in gene expression (7,8). Shear stress can modulate EC migration in wounding area and vascular stent surface (9-12), but the kinetics and molecular mechanism of EC migration in response to shear stress remain to be determined.Integrins are transmembrane adhesion receptors that link the extracellular matrix to cytoskeletal proteins and signaling molecules at FAs (13-15). Integrin-matrix binding activates the signaling cascade at FAs to modulate cell migration (13,14). Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that colocalizes with integrins at FAs. FAK mediates the FA dynamics and signaling in response to growth factors and integrin-ligand binding (16,17). Phosphorylation of FAK at Tyr-397 [p-FAK(Y397)] upon cell adhesion allows FAK to associate with Src, which triggers downstream signaling events such as phosphorylation of mitogen-activated kinases, p130 cas , and paxillin to mediate cell adhesion and migration (18)(19)(20)(21)(22)(23)(24). Recent studies show that FAK is required for mechanosensing and persistent migration of fibroblasts (25, 26). We and others have shown that shear stress induces a transient activation of FAK in EC monolayer (27)(28)(29). These previous studies focused on the analysis of the global activity of FAK by using traditional biochemical assays; the subcellular distribution and dynamics of FAK at FAs and the role of this spatial dynamics in cell migration in response to mechanical and chemical stimuli remain to be determined.Here, we defined the kinetics of shear stress-induced directional migration of ECs. By expressing green fluorescence protein (GFP)-tagged FAK, we demonstrated the molecular dynamics of FAK at FAs in migrating ECs in response to shear stress and serum. The results showed that p-FAK(Y397) was correlated with FAK dynamics at FAs. Our findings indicate that the spatial dynamics of signaling at FAs is critical in directional migration, and that mechanotaxis is an important mechanism controlling EC migration. Materials and MethodsCell Culture. Cell culture reagents were obtained from GIBCO͞ BRL. Bovine aortic ECs (BAECs) before passa...