Motility of Enzyme-Powered Vesicles.

Autonomous nanovehicles powered by energy derived from chemical catalysis have potential applications as active delivery agents. For in vivo applications, it is necessary that the engine and its fuel, as well as the chassis itself, be biocompatible. Enzyme molecules have been shown to display enhanced motility through substrate turnover and are attractive candidates as engines; phospholipid vesicles are biocompatible and can serve as cargo containers. Herein, we describe the autonomous movement of vesicles with membrane-bound enzymes in the presence of the substrate. We find that the motility of the vesicles increases with increasing enzymatic turnover rate. The enhanced diffusion of these enzyme-powered systems was further substantiated in real time by tracking the motion of the vesicles using optical microscopy. The membrane-bound protocells that move by transducing chemical energy into mechanical motion serve as models for motile living cells and are key to the elucidation of the fundamental mechanisms governing active membrane dynamics and cellular movement.

Light-Driven Chloride Transport Kinetics of Halorhodopsin.

Despite growing interest in light-driven ion pumps for use in optogenetics, current estimates of their transport rates span two orders of magnitude due to challenges in measuring slow transport processes and determining protein concentration and/or orientation in membranes in vitro. In this study, we report, to our knowledge, the first direct quantitative measurement of light-driven Cl- transport rates of the anion pump halorohodopsin from Natronomonas pharaonis (NpHR). We used light-interfaced voltage clamp measurements on NpHR-expressing oocytes to obtain a transport rate of 219 (± 98) Cl-/protein/s for a photon flux of 630 photons/protein/s. The measurement is consistent with the literature-reported quantum efficiency of ∼30% for NpHR, i.e., 0.3 isomerizations per photon absorbed. To reconcile our measurements with an earlier-reported 20 ms rate-limiting step, or 35 turnovers/protein/s, we conducted, to our knowledge, novel consecutive single-turnover flash experiments that demonstrate that under continuous illumination, NpHR bypasses this step in the photocycle.

Highly permeable artificial water channels that can self-assemble into two-dimensional arrays.

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.

Exothermicity Is Not a Necessary Condition for Enhanced Diffusion of Enzymes.

Recent experiments have revealed that the diffusivity of exothermic and fast enzymes is enhanced when they are catalytically active, and different physical mechanisms have been explored and quantified to account for this observation. We perform measurements on the endothermic and relatively slow enzyme aldolase, which also shows substrate-induced enhanced diffusion. We propose a new physical paradigm, which reveals that the diffusion coefficient of a model enzyme hydrodynamically coupled to its environment increases significantly when undergoing changes in conformational fluctuations in a substrate concentration dependent manner, and is independent of the overall turnover rate of the underlying enzymatic reaction. Our results show that substrate-induced enhanced diffusion of enzyme molecules can be explained within an equilibrium picture and that the exothermicity of the catalyzed reaction is not a necessary condition for the observation of this phenomenon.

Enhanced Diffusion of Passive Tracers in Active Enzyme Solutions.

Colloidal suspensions containing microscopic swimmers have been the focus of recent studies aimed at understanding the principles of energy transfer in fluidic media at low Reynolds number conditions. Going down in scale, active enzymes have been shown to be force-generating, nonequilibrium systems, thus offering opportunity to examine energy transfer at the ultralow Reynolds number regime. By monitoring the change of diffusion of inert tracers dispersed in active enzyme solutions, we demonstrate that the nature of energy transfer in these systems is similar to that reported for larger microscopic active systems, despite the large differences in scale, modes of energy transduction, and propulsion. Additionally, even an enzyme that catalyzes an endothermic reaction behaves analogously, suggesting that heat generation is not the primary factor for the observed enhanced tracer diffusion. Our results provide new insights into the mechanism of energy transfer at the molecular level.

ß1-Integrin-Mediated Adhesion Is Lipid-Bilayer Dependent.

Integrin-mediated adhesion is a central feature of cellular adhesion, locomotion, and endothelial cell mechanobiology. Although integrins are known to be transmembrane proteins, little is known about the role of membrane biophysics and dynamics in integrin adhesion. We treated human aortic endothelial cells with exogenous amphiphiles, shown previously in model membranes, and computationally, to affect bilayer thickness and lipid phase separation, and subsequently measured single-integrin-molecule adhesion kinetics using an optical trap, and diffusion using fluorescence correlation spectroscopy. Benzyl alcohol (BA) partitions to liquid-disordered (Ld) domains, thins them, and causes the greatest increase in hydrophobic mismatch between liquid-ordered (Lo) and Ld domains among the three amphiphiles, leading to domain separation. In human aortic endothelial cells, BA increased ß1-integrin-Arg-Gly-Asp-peptide affinity by 18% with a transition from single to double valency, consistent with a doubling of the molecular brightness of mCherry-tagged ß1-integrins measured using fluorescence correlation spectroscopy. Accordingly, BA caused an increase in the size of focal-adhesion-kinase/paxillin-positive peripheral adhesions and reduced migration speeds as measured using wound-healing assays. Vitamin E, which thickens Lo domains and disperses them by lowering edge energy on domain boundaries, left integrin affinity unchanged but reduced binding probability, leading to smaller focal adhesions and equivalent migration speed relative to untreated cells. Vitamin E reversed the BA-induced decrease in migration speed. Triton X-100 also thickens Lo domains, but partitions to both lipid phases and left unchanged binding kinetics, focal adhesion sizes, and migration speed. These results demonstrate that only the amphiphile that thinned Ld lipid domains increased ß1-integrin-Arg-Gly-Asp-peptide affinity and valency, thus implicating Ld domains in modulation of integrin adhesion, nascent adhesion formation, and cell migration.

Effective encapsulation and biological activity of phosphorylated chemotherapeutics in calcium phosphosilicate nanoparticles for the treatment of pancreatic cancer.

Drug resistant cancers like pancreatic ductal adenocarcinoma (PDAC) are difficult to treat, and nanoparticle drug delivery systems can overcome some of the limitations of conventional systemic chemotherapy. In this study, we demonstrate that FdUMP and dFdCMP, the bioactive, phosphorylated metabolites of the chemotherapy drugs 5-FU and gemcitabine, can be encapsulated into calcium phosphosilicate nanoparticles (CPSNPs). The non-phosphorylated drug analogs were not well encapsulated by CPSNPs, suggesting the phosphate modification is essential for effective encapsulation. In vitro proliferation assays, cell cycle analyses and/or thymidylate synthase inhibition assays verified that CPSNP-encapsulated phospho-drugs retained biological activity. Analysis of orthotopic tumors from mice treated systemically with tumor-targeted FdUMP-CPSNPs confirmed the in vivo up take of these particles by PDAC tumor cells and release of active drug cargos intracellularly. These findings demonstrate a novel methodology to efficiently encapsulate chemotherapeutic agents into the CPSNPs and to effectively deliver them to pancreatic tumor cells.

Acoustofluidic chemical waveform generator and switch.

Eliciting a cellular response to a changing chemical microenvironment is central to many biological processes including gene expression, cell migration, differentiation, apoptosis, and intercellular signaling. The nature and scope of the response is highly dependent upon the spatiotemporal characteristics of the stimulus. To date, studies that investigate this phenomenon have been limited to digital (or step) chemical stimulation with little control over the temporal counterparts. Here, we demonstrate an acoustofluidic (i.e., fusion of acoustics and microfluidics) approach for generating programmable chemical waveforms that permits continuous modulation of the signal characteristics including the amplitude (i.e., sample concentration), shape, frequency, and duty cycle, with frequencies reaching up to 30 Hz. Furthermore, we show fast switching between multiple distinct stimuli, wherein the waveform of each stimulus is independently controlled. Using our device, we characterized the frequency-dependent activation and internalization of the ß2-adrenergic receptor (ß2-AR), a prototypic G-protein coupled receptor (GPCR), using epinephrine. The acoustofluidic-based programmable chemical waveform generation and switching method presented herein is expected to be a powerful tool for the investigation and characterization of the kinetics and other dynamic properties of many biological and biochemical processes.

Substrate stiffness regulates cellular uptake of nanoparticles.

Nanoparticle (NP)-bioconjugates hold great promise for more sensitive disease diagnosis and more effective anticancer drug delivery compared with existing approaches. A critical aspect in both applications is cellular internalization of NPs, which is influenced by NP properties and cell surface mechanics. Despite considerable progress in optimization of the NP-bioconjugates for improved targeting, the role of substrate stiffness on cellular uptake has not been investigated. Using polyacrylamide (PA) hydrogels as model substrates with tunable stiffness, we quantified the relationship between substrate stiffness and cellular uptake of fluorescent NPs by bovine aortic endothelial cells (BAECs). We found that a stiffer substrate results in a higher total cellular uptake on a per cell basis, but a lower uptake per unit membrane area. To obtain a mechanistic understanding of the cellular uptake behavior, we developed a thermodynamic model that predicts that membrane spreading area and cell membrane tension are two key factors controlling cellular uptake of NPs, both of which are modulated by substrate stiffness. Our experimental and modeling results not only open up new avenues for engineering NP-based cancer cell targets for more effective in vivo delivery but also contribute an example of how the physical environment dictates cellular behavior and function.

Enzyme molecules as nanomotors.

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.

Tuning membrane phase separation using nonlipid amphiphiles.

Lipid phase separation may be a mechanism by which lipids participate in sorting membrane proteins and facilitate membrane-mediated biochemical signaling in cells. To provide new tools for membrane lipid phase manipulation that avoid direct effects on protein activity and lipid composition, we studied phase separation in binary and ternary lipid mixtures under the influence of three nonlipid amphiphiles, vitamin E (VE), Triton-X (TX)-100, and benzyl alcohol (BA). Mechanisms of additive-induced phase separation were elucidated using coarse-grained molecular dynamics simulations of these additives in a liquid bilayer made from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dilinoleoyl-sn-glycero-3-phosphocholine [corrected]. From simulations, the additive's partitioning preference, changes in membrane thickness, and alterations in lipid order were quantified. Simulations showed that VE favored the DPPC phase but partitioned predominantly to the domain boundaries and lowered the tendency for domain formation, and therefore acted as a linactant. This simulated behavior was consistent with experimental observations in which VE promoted lipid mixing and dispersed domains in both gel/liquid and liquid-ordered/liquid-disordered systems. From simulation, BA partitioned predominantly to the DUPC phase, decreased lipid order there, and thinned the membrane. These actions explain why, experimentally, BA promoted phase separation in both binary and ternary lipid mixtures. In contrast, TX, a popular detergent used to isolate raft membranes in cells, exhibited equal preference for both phases, as demonstrated by simulations, but nonetheless, was a strong domain promoter in all lipid mixtures. Further analysis showed that TX increased membrane thickness of the DPPC phase to a greater extent than the DUPC phase and thus increased hydrophobic mismatch, which may explain experimental observation of phase separation in the presence of TX. In summary, these nonlipid amphiphiles provide new tools to tune domain formation in model vesicle systems and could provide the means to form or disperse membrane lipid domains in cells, in addition to the well-known methods involving cholesterol enrichment and sequestration.

Atomistic simulation of lipid and DiI dynamics in membrane bilayers under tension.

Membrane tension modulates cellular processes by initiating changes in the dynamics of its molecular constituents. To quantify the precise relationship between tension, structural properties of the membrane, and the dynamics of lipids and a lipophilic reporter dye, we performed atomistic molecular dynamics (MD) simulations of DiI-labeled dipalmitoylphosphatidylcholine (DPPC) lipid bilayers under physiological lateral tensions ranging from -2.6 mN m(-1) to 15.9 mN m(-1). Simulations showed that the bilayer thickness decreased linearly with tension consistent with volume-incompressibility, and this thinning was facilitated by a significant increase in acyl chain interdigitation at the bilayer midplane and spreading of the acyl chains. Tension caused a significant drop in the bilayer's peak electrostatic potential, which correlated with the strong reordering of water and lipid dipoles. For the low tension regime, the DPPC lateral diffusion coefficient increased with increasing tension in accordance with free-area theory. For larger tensions, free area theory broke down due to tension-induced changes in molecular shape and friction. Simulated DiI rotational and lateral diffusion coefficients were lower than those of DPPC but increased with tension in a manner similar to DPPC. Direct correlation of membrane order and viscosity near the DiI chromophore, which was just under the DPPC headgroup, indicated that measured DiI fluorescence lifetime, which is reported to decrease with decreasing lipid order, is likely to be a good reporter of tension-induced decreases in lipid headgroup viscosity. Together, these results offer new molecular-level insights into membrane tension-related mechanotransduction and into the utility of DiI in characterizing tension-induced changes in lipid packing.

Substrate catalysis enhances single-enzyme diffusion.

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.

Photophysics of Cy3-encapsulated calcium phosphate nanoparticles.

Progress toward clinical application of biodegradable fluorescent calcium phosphate (CP) nanoparticles as a bioimaging agent requires detailed knowledge of chromophore interaction with CP. As readouts of this cargo-matrix interaction, we determined the principle photophysical properties of Cy3 encapsulated in CP nanparticles (CPNPs) using steady-state and time-resolved fluorescence spectroscopy. Fluorescence correlation spectroscopy (FCS)-determined diffusion coefficients and associated hydrodynamic radii confirmed the presence of highly monodisperse CPNPs with radii ranging from 7 to 10 nm. Single CP nanoparticles were 20 times brighter than free dye molecules because of a CP-induced 5-fold increase in quantum efficiency and encapsulation of four dye molecules per particle. Solvatochromic shifts resulting from hydrogen bonding between free dye and solvent or restricted intramolecular mobility by solvent viscosity were absent when Cy3 was encapsulated in CP. Encapsulation-mediated increases in radiative decay rates and decreases in nonradiative decay rates resulting in longer fluorescence lifetimes of Cy3 were attributed to solvent and CP-related local refractive indices and restricted flexibility of dye by rigid CP. Enhanced brightness of CPNPs enabled imaging of single nanoparticles under epifluorescence using both standard and total internal reflection fluorescence (TIRF) modes with camera exposure times on the order of tens of milliseconds. These enhanced photophysical properties together with excellent biocompatibility make CPNPs ideal for bioimaging applications ranging from single-molecule tracking to in vivo tumor detection and offer the possibility of timed codelivery of drugs to control cell function.

Mechanobiology of dynamic enzyme systems.

This Perspective paper advances a hypothesis of mechanosensation by endothelial cells in which the cell is a dynamic crowded system, driven by continuous enzyme activity, that can be shifted from one non-equilibrium state to another by external force. The nature of the shift will depend on the direction, rate of change, and magnitude of the force. Whether force induces a pathophysiological or physiological change in cell biology will be determined by whether the dynamics of a cellular system can accommodate the dynamics and magnitude of the force application. The complex interplay of non-static cytoskeletal structures governs internal cellular rheology, dynamic spatial reorganization, and chemical kinetics of proteins such as integrins, and a flaccid membrane that is dynamically supported; each may constitute the necessary dynamic properties able to sense external fluid shear stress and reorganize in two and three dimensions. The resulting reorganization of enzyme systems in the cell membrane and cytoplasm may drive the cell to a new physiological state. This review focuses on endothelial cell mechanotransduction of shear stress, but may lead to new avenues of investigation of mechanobiology in general requiring new tools for interrogation of mechanobiological systems, tools that will enable the synthesis of large amounts of spatial and temporal data at the molecular, cellular, and system levels.

Every sponge its own name: removing Porifera homonyms.

The occurrence of different sponge species bearing the same Linnean binomial name combination, i.e. homonyms, is to be avoided for obvious reasons. In a review of sponge taxon names of the World Porifera Database, we detected 121 homonymic cases (115 species-group names, 6 genus-group names), involving a total of 272 nominal taxa. It is the object of the present study to remove their occurrence by proposing new names for the junior homonyms following the rules of the International Commission of Zoological Nomenclature as laid down in the Code (ICZN, 1999) and the on-line edition http://iczn.org/iczn/index.jsp . Homonym cases are discussed and, where applicable, junior homonyms are either replaced by nomina nova or reassigned to their earliest available synonyms. The order in which the homonyms are treated is alphabetical on original species name, with genus names separately treated at the end. A summary table with all proposed name changes is also presented to allow quick access to the junior homonyms and their proposed new names. A total of 116 nomina nova are proposed, including five new genus names.

Mechanobiology of the abluminal glycocalyx.

BACKGROUND: Endothelial cells (ECs) sense the forces from blood flow through the glycocalyx, a carbohydrate rich luminal surface layer decorating most cells, and through forces transmitted through focal adhesions (FAs) on the abluminal side of the cell. OBJECTIVES: This perspective paper explores a complementary hypothesis, that glycocalyx molecules on the abluminal side of the EC between the basement membrane and the EC membrane, occupying the space outside of FAs, work in concert with FAs to sense blood flow-induced shear stress applied to the luminal surface. RESULTS: First, we summarize recent studies suggesting that the glycocalyx repels the plasma membrane away from the basement membrane, while integrin molecules attach to extracellular matrix (ECM) ligands. This coordinated attraction and repulsion results in the focal nature of integrin-mediated adhesion making the abluminal glycocalyx a participant in mechanotransduction. Further, the glycocalyx mechanically links the plasma membrane to the basement membrane providing a mechanism of force transduction when the cell deforms in the peri-FA space. To determine if the membrane might deform against a restoring force of an elastic abluminal glycocalyx in the peri-FA space we present some analysis from a multicomponent elastic finite element model of a sheared and focally adhered endothelial cell whose abluminal topography was assessed using quantitative total internal reflection fluorescence microscopy with an assumption that glycocalyx fills the space between the membrane and extracellular matrix. CONCLUSIONS: While requiring experimental verification, this analysis supports the hypothesis that shear on the luminal surface can be transmitted to the abluminal surface and deform the cell in the vicinity of the focal adhesions, with the magnitude of deformation depending on the abluminal glycocalyx modulus.

Nonuniform Crowding Enhances Transport.

The cellular cytoplasm is crowded with macromolecules and other species that occupy up to 40% of the available volume. Previous studies have reported that for high crowder molecule concentrations, colloidal tracer particles have a dampened diffusion due to the higher solution viscosity. However, these studies employed uniform distributions of crowder molecules. We report a scenario, previously unexplored experimentally, of increased tracer transport driven by a nonuniform concentration of crowder macromolecules. In gradients of a polymeric crowder, tracer particles undergo transport several times higher than that of their bulk diffusion rate. The direction of the transport is toward regions of lower crowder concentration. Mechanistically, hard-sphere interactions and the resulting volume exclusion between the tracer and crowder increase the effective diffusion by inducing a convective motion of tracers, which we explain through modeling. Strikingly, soft deformable particles show even greater enhancement in transport in crowder gradients compared to similarly sized hard particles. Overall, this demonstration of enhanced transport in nonuniform distributions of crowders is anticipated to clarify aspects of multicomponent intracellular transport.

Tumor-promoting effects of pancreatic cancer cell exosomes on THP-1-derived macrophages.

Pancreatic ductal adenocarcinoma (PDAC) tumor growth is enhanced by tumor-associated macrophages (TAMs), yet the mechanisms by which tumor cells and TAMs communicate are not fully understood. Here we show that exosomes secreted by PDAC cell lines differed in their surface proteins, lipid composition, and efficiency of fusing with THP-1-derived macrophages in vitro. Exosomes from AsPC-1, an ascites-derived human PDAC cell line, were enriched in ICAM-1, which mediated their docking to macrophages through interactions with surface-exposed CD11c on macrophages. AsPC-1 exosomes also contained much higher levels of arachidonic acid (AA), and they fused at a higher rate with THP-1-derived macrophages than did exosomes from other PDAC cell lines or from an immortalized normal pancreatic ductal epithelial cell line (HPDE) H6c7. Phospholipase A2 enzymatic cleavage of arachidonic acid from AsPC-1 exosomes reduced fusion efficiency. PGE2 secretion was elevated in macrophages treated with AsPC-1 exosomes but not in macrophages treated with exosomes from other cell lines, suggesting a functional role for the AsPC-1 exosome-delivered arachidonic acid in macrophages. Non-polarized (M0) macrophages treated with AsPC-1 exosomes had increased levels of surface markers indicative of polarization to an immunosuppressive M2-like phenotype (CD14hi CD163hi CD206hi). Furthermore, macrophages treated with AsPC-1 exosomes had significantly increased secretion of pro-tumoral, bioactive molecules including VEGF, MCP-1, IL-6, IL-1ß, MMP-9, and TNFα. Together, these results demonstrate that compared to exosomes from other primary tumor-derived PDAC cell lines, AsPC-1 exosomes alter THP-1-derived macrophage phenotype and function. AsPC-1 exosomes mediate communication between tumor cells and TAMs that contributes to tumor progression.

Substrate-driven chemotactic assembly in an enzyme cascade.

Enzymatic catalysis is essential to cell survival. In many instances, enzymes that participate in reaction cascades have been shown to assemble into metabolons in response to the presence of the substrate for the first enzyme. However, what triggers metabolon formation has remained an open question. Through a combination of theory and experiments, we show that enzymes in a cascade can assemble via chemotaxis. We apply microfluidic and fluorescent spectroscopy techniques to study the coordinated movement of the first four enzymes of the glycolysis cascade: hexokinase, phosphoglucose isomerase, phosphofructokinase and aldolase. We show that each enzyme independently follows its own specific substrate gradient, which in turn is produced by the preceding enzymatic reaction. Furthermore, we find that the chemotactic assembly of enzymes occurs even under cytosolic crowding conditions.