Conformational Changes in Surface-Immobilized Proteins Measured Using Combined Atomic Force and Fluorescence Microscopy.

Biological organisms rely on proteins to perform the majority of their functions. Most protein functions are based on their physical motions (conformational changes), which can be described as transitions between different conformational states in a multidimensional free-energy landscape. A comprehensive understanding of this free-energy landscape is therefore of paramount importance for understanding the biological functions of proteins. Protein dynamics includes both equilibrium and nonequilibrium motions, which typically exhibit a wide range of characteristic length and time scales. The relative probabilities of various conformational states in the energy landscape, the energy barriers between them, their dependence on external parameters such as force and temperature, and their connection to the protein function remain largely unknown for most proteins. In this paper, we present a multimolecule approach in which the proteins are immobilized at well-defined locations on Au substrates using an atomic force microscope (AFM)-based patterning method called nanografting. This method enables precise control over the protein location and orientation on the substrate, as well as the creation of biologically active protein ensembles that self-assemble into well-defined nanoscale regions (protein patches) on the gold substrate. We performed AFM-force compression and fluorescence experiments on these protein patches and measured the fundamental dynamical parameters such as protein stiffness, elastic modulus, and transition energies between distinct conformational states. Our results provide new insights into the processes that govern protein dynamics and its connection to protein function.

Feedback-controlled dynamics of neuronal cells on directional surfaces.

The formation of neuronal networks is a complex phenomenon of fundamental importance for understanding the development of the nervous system. The basic process underlying the network formation is axonal growth, a process involving the extension of axons from the cell body and axonal navigation toward target neurons. Axonal growth is guided by the interactions between the tip of the axon (growth cone) and its extracellular environmental cues, which include intercellular interactions, the biochemical landscape around the neuron, and the mechanical and geometrical features of the growth substrate. Here, we present a comprehensive experimental and theoretical analysis of axonal growth for neurons cultured on micropatterned polydimethylsiloxane (PDMS) surfaces. We demonstrate that closed-loop feedback is an essential component of axonal dynamics on these surfaces: the growth cone continuously measures environmental cues and adjusts its motion in response to external geometrical features. We show that this model captures all the characteristics of axonal dynamics on PDMS surfaces for both untreated and chemically modified neurons. We combine experimental data with theoretical analysis to measure key parameters that describe axonal dynamics: diffusion (cell motility) coefficients, speed and angular distributions, and cell-substrate interactions. The experiments performed on neurons treated with Taxol (inhibitor of microtubule dynamics) and Y-27632 (disruptor of actin filaments) indicate that the internal dynamics of microtubules and actin filaments plays a critical role for the proper function of the feedback mechanism. Our results demonstrate that axons follow geometrical patterns through a contact-guidance mechanism, in which high-curvature geometrical features impart high traction forces to the growth cone. These results have important implications for our fundamental understanding of axonal growth as well as for bioengineering novel substrate to guide neuronal growth and promote nerve repair.

Quantitative characterization of dielectric properties of polymer fibers and polymer composites using electrostatic force microscopy.

We use a new method based on electrostatic force microscopy (EFM) to perform quantitative measurements of the dielectric constants of individual electrospun nanofibers of poly(L-lactic acid) (PLLA), as well as composite fibers of PLLA with embedded multiwall carbon nanotubes (MWCNT-PLLA). The EFM data record the oscillation phase of an atomic force microscope (AFM) cantilever as a function of the AFM tip position. In our experiments the relative dielectric constants ϵ of the sample are measured from the EFM phase shifts vs. the tip-surface separation, according to a simple analytical model describing the tip-surface interactions. We perform a comprehensive study of how the dielectric constant depends on the fiber diameter for both electrospun PLLA and MWCNT/PLLA fiber composites. Our measurements show that EFM can distinguish between dielectric properties of PLLA fibers and fiber composites with different diameters. Dielectric constants of both PLLA and MWCNT-PLLA composite fibers decrease with increasing fiber diameter. In the limit of large fiber diameters (D > 100 nm), we measure dielectric constants in the range: ϵ = 3.4-3.8, similar to the values obtained for unoriented PLLA films: ϵfilm = 2.4-3.8. Moreover, the dielectric constants of the small diameter MWCNT-PLLA composites are significantly larger than the corresponding values obtained for PLLA fibers. For MWCNT-PLLA nanofiber composites of small diameters (D < 50 nm), ϵ approaches the values measured for neat MWCNT: ϵCN = 12 ± 2. These results are consistent with a simple fiber structural model that shows higher polarizability of thinner fibers, and composites that contain MWCNTs. The experimental method has a high-resolution for measuring the dielectric constant of soft materials, and is simple to implement on standard atomic force microscopes. This non-invasive technique can be applied to measure the electrical properties of polymers, interphases, and polymer nanocomposites.

Variations of Elastic Modulus and Cell Volume with Temperature for Cortical Neurons.

Neurons change their growth dynamics and mechanical properties in response to external stimuli such as stiffness of the local microenvironment, ambient temperature, and biochemical or geometrical guidance cues. Here we use combined atomic force microscopy (AFM) and fluorescence microscopy experiments to investigate the relationship between external temperature, soma volume, and elastic modulus for cortical neurons. We measure how changes in ambient temperature affect the volume and the mechanical properties of neuronal cells at both the bulk (elastic modulus) and local (elasticity maps) levels. The experimental data demonstrate that both the volume and the elastic modulus of the neuron soma vary with changes in temperature. Our results show a decrease by a factor of 2 in the soma elastic modulus as the ambient temperature increases from room (25 °C) to physiological (37 °C) temperature, while the volume of the soma increases by a factor of 1.3 during the same temperature sweep. Using high-resolution AFM force mapping, we measure the temperature-induced variations within different regions of the elasticity maps (low and high values of elastic modulus) and correlate these variations with the dynamics of cytoskeleton components and molecular motors. We quantify the change in soma volume with temperature and propose a simple theoretical model that relates this change with variations in soma elastic modulus. These results have significant implications for understanding neuronal development and functions, as ambient temperature, cytoskeletal dynamics, and cellular volume may change with variations in physiological conditions, for example, during tissue compression and infections in vivo as well as during cell manipulation and tissue regeneration ex vivo.

Neuron dynamics on directional surfaces.

Geometrical features play a very important role in neuronal growth and the formation of functional connections between neuronal cells. Here, we analyze the dynamics of axonal growth for neuronal cells cultured on micro-patterned polydimethylsiloxane surfaces. We utilize fluorescence microscopy to image axons, quantify their dynamics, and demonstrate that periodic geometrical patterns impart strong directional bias to neuronal growth. We quantify axonal alignment and present a general stochastic approach that quantitatively describes the dynamics of the growth cones. Neuronal growth is described by a general phenomenological model, based on a simple automatic controller with a closed-loop feedback system. We demonstrate that axonal alignment on these substrates is determined by the surface geometry, and it is quantified by the deterministic part of the stochastic (Langevin and Fokker-Planck) equations. We also show that the axonal alignment with the surface patterns is greatly suppressed by the neuron treatment with Blebbistatin, a chemical compound that inhibits the activity of myosin II. These results give new insight into the role played by the molecular motors and external geometrical cues in guiding axonal growth, and could lead to novel approaches for bioengineering neuronal regeneration platforms.

Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies.

We present a programmable bioengineered 3-dimensional silk-based bone marrow niche tissue system that successfully mimics the physiology of human bone marrow environment allowing us to manufacture functional human platelets ex vivo. Using stem/progenitor cells, megakaryocyte function and platelet generation were recorded in response to variations in extracellular matrix components, surface topography, stiffness, coculture with endothelial cells, and shear forces. Millions of human platelets were produced and showed to be functional based on multiple activation tests. Using adult hematopoietic progenitor cells our system demonstrated the ability to reproduce key steps of thrombopoiesis, including alterations observed in diseased states. A critical feature of the system is the use of natural silk protein biomaterial allowing us to leverage its biocompatibility, nonthrombogenic features, programmable mechanical properties, and surface binding of cytokines, extracellular matrix components, and endothelial-derived proteins. This in turn offers new opportunities for the study of blood component production ex vivo and provides a superior tissue system for the study of pathologic mechanisms of human platelet production.

A new path to platelet production through matrix sensing.

Megakaryocytes (MK) in the bone marrow (BM) are immersed in a network of extracellular matrix components that regulates platelet release into the circulation. Combining biological and bioengineering approaches, we found that the activation of transient receptor potential cation channel subfamily V member 4 (TRPV4), a mechano-sensitive ion channel, is induced upon MK adhesion on softer matrices. This response promoted platelet production by triggering a cascade of events that lead to calcium influx, ß1 integrin activation and internalization, and Akt phosphorylation, responses not found on stiffer matrices. Lysyl oxidase (LOX) is a physiological modulator of BM matrix stiffness via collagen crosslinking. In vivo inhibition of LOX and consequent matrix softening lead to TRPV4 activation cascade and increased platelet levels. At the same time, in vitro proplatelet formation was reduced on a recombinant enzyme-mediated stiffer collagen. These results suggest a novel mechanism by which MKs, through TRPV4, sense extracellular matrix environmental rigidity and release platelets accordingly.

Load Rate and Temperature Dependent Mechanical Properties of the Cortical Neuron and Its Pericellular Layer Measured by Atomic Force Microscopy.

When studying the mechanical properties of cells by an indentation technique, it is important to take into account the nontrivial pericellular interface (or pericellular "brush") which includes a pericellular coating and corrugation of the pericellular membrane (microvilli and microridges). Here we use atomic force microscopy (AFM) to study the mechanics of cortical neurons taking into account the presence of the above pericellular brush surrounding cell soma. We perform a systematic study of the mechanical properties of both the brush layer and the underlying neuron soma and demonstrate that the brush layer is likely responsible for the low elastic modulus (<1 kPa) typically reported for cortical neurons. When the contribution of the pericellular brush is excluded, the average elastic modulus of the cortical neuron soma is found to be 3-4 times larger than previously reported values measured under similar physiological conditions. We also demonstrate that the underlying soma behaves as a nonviscous elastic material over the indentation rates studied (1-10 µm/s). As a result, it seems that the brush layer is responsible for the previously reported viscoelastic response measured for the neuronal cell body as a whole, within these indentation rates. Due to of the similarities between the macroscopic brain mechanics and the effective modulus of the pericellular brush, we speculate that the pericellular brush layer might play an important role in defining the macroscopic mechanical properties of the brain.

Quantitative analysis of mechanical and electrostatic properties of poly(lactic) acid fibers and poly(lactic) acid-carbon nanotube composites using atomic force microscopy.

We use atomic force microscopy (AFM) to perform a systematic quantitative characterization of the elastic modulus and dielectric constant of poly(L-lactic acid) electrospun nanofibers (PLLA), as well as composites of PLLA fibers with 1.0 wt% embedded multiwall carbon nanotubes (MWCNTs-PLLA). The elastic moduli are measured in the fiber skin region via AFM nanoindentation, and the dielectric constants are determined by measuring the phase shifts obtained via electrostatic force microscopy (EFM). We find that the average value for the elastic modulus for PLLA fibers is (9.8 ± 0.9) GPa, which is a factor of 2 larger than the measured average elastic modulus for MWCNT-PLLA composites (4.1 ± 0.7) GPa. We also use EFM to measure dielectric constants for both types of fibers. These measurements show that the dielectric constants of the MWCNT-PLLA fibers are significantly larger than the corresponding values obtained for PLLA fiber. This result is consistent with the higher polarizability of the MWCNT-PLLA composites. The measurement methods presented are general, and can be applied to determine the mechanical and electrical properties of other polymers and polymer nanocomposites.

Effect of sequence features on assembly of spider silk block copolymers.

Bioengineered spider silk block copolymers were studied to understand the effect of protein chain length and sequence chemistry on the formation of secondary structure and materials assembly. Using a combination of in vitro protein design and assembly studies, we demonstrate that silk block copolymers possessing multiple repetitive units self-assemble into lamellar microstructures. Additionally, the study provides insights into the assembly behavior of spider silk block copolymers in concentrated salt solutions.

Nonlinear Growth Dynamics of Neuronal Cells Cultured on Directional Surfaces.

During the development of the nervous system, neuronal cells extend axons and dendrites that form complex neuronal networks, which are essential for transmitting and processing information. Understanding the physical processes that underlie the formation of neuronal networks is essential for gaining a deeper insight into higher-order brain functions such as sensory processing, learning, and memory. In the process of creating networks, axons travel towards other recipient neurons, directed by a combination of internal and external cues that include genetic instructions, biochemical signals, as well as external mechanical and geometrical stimuli. Although there have been significant recent advances, the basic principles governing axonal growth, collective dynamics, and the development of neuronal networks remain poorly understood. In this paper, we present a detailed analysis of nonlinear dynamics for axonal growth on surfaces with periodic geometrical patterns. We show that axonal growth on these surfaces is described by nonlinear Langevin equations with speed-dependent deterministic terms and gaussian stochastic noise. This theoretical model yields a comprehensive description of axonal growth at both intermediate and long time scales (tens of hours after cell plating), and predicts key dynamical parameters, such as speed and angular correlation functions, axonal mean squared lengths, and diffusion (cell motility) coefficients. We use this model to perform simulations of axonal trajectories on the growth surfaces, in turn demonstrating very good agreement between simulated growth and the experimental results. These results provide important insights into the current understanding of the dynamical behavior of neurons, the self-wiring of the nervous system, as well as for designing innovative biomimetic neural network models.

Impact of Silk-Ionomer Encapsulation on Immune Cell Mechanical Properties and Viability.

Encapsulation of single cells is a powerful technique used in various fields, such as regenerative medicine, drug delivery, tissue regeneration, cell-based therapies, and biotechnology. It offers a method to protect cells by providing cytocompatible coatings to strengthen cells against mechanical and environmental perturbations. Silk fibroin, derived from the silkworm Bombyx mori, is a promising protein biomaterial for cell encapsulation due to the cytocompatibility and capacity to maintain cell functionality. Here, THP-1 cells, a human leukemia monocytic cell line, were encapsulated with chemically modified silk polyelectrolytes through electrostatic layer-by-layer deposition. The effectiveness of the silk nanocoating was assessed using scanning electron microscopy (SEM) and confocal microscopy and on cell viability and proliferation by Alamar Blue assay and live/dead staining. An analysis of the mechanical properties of the encapsulated cells was conducted using atomic force microscopy nanoindentation to measure elasticity maps and cellular stiffness. After the cells were encapsulated in silk, an increase in their stiffness was observed. Based on this observation, we developed a mechanical predictive model to estimate the variations in stiffness in relation to the thickness of the coating. By tuning the cellular assembly and biomechanics, these encapsulations promote systems that protect cells during biomaterial deposition or processing in general.

Temperature response of the neuronal cytoskeleton mapped via atomic force and fluorescence microscopy.

Neuronal cells change their growth properties in response to external physical stimuli such as variations in external temperature, stiffness of the growth substrate, or topographical guidance cues. Detailed knowledge of the mechanisms that control these biomechanical responses is necessary for understanding the basic principles that underlie neuronal growth and regeneration. Here, we present elasticity maps of living cortical neurons (embryonic rat) as a function of temperature, and correlate these maps to the locations of internal structural components of the cytoskeleton. Neurons display a significant increase in the average elastic modulus upon a decrease in ambient temperature from 37 to 25 °C. We demonstrate that the dominant mechanism by which the elasticity of the neurons changes in response to temperature is the stiffening of the actin components of the cytoskeleton induced by myosin II. We also report a reversible shift in the location and composition of the high-stiffness areas of the neuron cytoskeleton with temperature. At 37 °C the areas of the cell displaying high elastic modulus overlap with the tubulin-dense regions, while at 25 °C these high-stiffness areas correspond to the actin-dense regions of the cytoskeleton. These results demonstrate the importance of considering temperature effects when investigating cytoskeletal dynamics in cells.

Extracellular matrix structure and nano-mechanics determine megakaryocyte function.

Cell interactions with matrices via specific receptors control many functions, with chemistry, physics, and membrane elasticity as fundamental elements of the processes involved. Little is known about how biochemical and biophysical processes integrate to generate force and, ultimately, to regulate hemopoiesis into the bone marrow-matrix environment. To address this hypothesis, in this work we focus on the regulation of MK development by type I collagen. By atomic force microscopy analysis, we demonstrate that the tensile strength of fibrils in type I collagen structure is a fundamental requirement to regulate cytoskeleton contractility of human MKs through the activation of integrin-α2ß1-dependent Rho-ROCK pathway and MLC-2 phosphorylation. Most importantly, this mechanism seemed to mediate MK migration, fibronectin assembly, and platelet formation. On the contrary, a decrease in mechanical tension caused by N-acetylation of lysine side chains in type I collagen completely reverted these processes by preventing fibrillogenesis.

Kelvin probe microscopy and electronic transport measurements in reduced graphene oxide chemical sensors.

Reduced graphene oxide (RGO) is an electronically hybrid material that displays remarkable chemical sensing properties. Here, we present a quantitative analysis of the chemical gating effects in RGO-based chemical sensors. The gas sensing devices are patterned in a field-effect transistor geometry, by dielectrophoretic assembly of RGO platelets between gold electrodes deposited on SiO2/Si substrates. We show that these sensors display highly selective and reversible responses to the measured analytes, as well as fast response and recovery times (tens of seconds). We use combined electronic transport/Kelvin probe microscopy measurements to quantify the amount of charge transferred to RGO due to chemical doping when the device is exposed to electron-acceptor (acetone) and electron-donor (ammonia) analytes. We demonstrate that this method allows us to obtain high-resolution maps of the surface potential and local charge distribution both before and after chemical doping, to identify local gate-susceptible areas on the RGO surface, and to directly extract the contact resistance between the RGO and the metallic electrodes. The method presented is general, suggesting that these results have important implications for building graphene and other nanomaterial-based chemical sensors.

Neuron biomechanics probed by atomic force microscopy.

Mechanical interactions play a key role in many processes associated with neuronal growth and development. Over the last few years there has been significant progress in our understanding of the role played by the substrate stiffness in neuronal growth, of the cell-substrate adhesion forces, of the generation of traction forces during axonal elongation, and of the relationships between the neuron soma elastic properties and its health. The particular capabilities of the Atomic Force Microscope (AFM), such as high spatial resolution, high degree of control over the magnitude and orientation of the applied forces, minimal sample damage, and the ability to image and interact with cells in physiologically relevant conditions make this technique particularly suitable for measuring mechanical properties of living neuronal cells. This article reviews recent advances on using the AFM for studying neuronal biomechanics, provides an overview about the state-of-the-art measurements, and suggests directions for future applications.

Biased Random Walk Model of Neuronal Dynamics on Substrates with Periodic Geometrical Patterns.

Neuronal networks are complex systems of interconnected neurons responsible for transmitting and processing information throughout the nervous system. The building blocks of neuronal networks consist of individual neurons, specialized cells that receive, process, and transmit electrical and chemical signals throughout the body. The formation of neuronal networks in the developing nervous system is a process of fundamental importance for understanding brain activity, including perception, memory, and cognition. To form networks, neuronal cells extend long processes called axons, which navigate toward other target neurons guided by both intrinsic and extrinsic factors, including genetic programming, chemical signaling, intercellular interactions, and mechanical and geometrical cues. Despite important recent advances, the basic mechanisms underlying collective neuron behavior and the formation of functional neuronal networks are not entirely understood. In this paper, we present a combined experimental and theoretical analysis of neuronal growth on surfaces with micropatterned periodic geometrical features. We demonstrate that the extension of axons on these surfaces is described by a biased random walk model, in which the surface geometry imparts a constant drift term to the axon, and the stochastic cues produce a random walk around the average growth direction. We show that the model predicts key parameters that describe axonal dynamics: diffusion (cell motility) coefficient, average growth velocity, and axonal mean squared length, and we compare these parameters with the results of experimental measurements. Our findings indicate that neuronal growth is governed by a contact-guidance mechanism, in which the axons respond to external geometrical cues by aligning their motion along the surface micropatterns. These results have a significant impact on developing novel neural network models, as well as biomimetic substrates, to stimulate nerve regeneration and repair after injury.

Elasticity maps of living neurons measured by combined fluorescence and atomic force microscopy.

Detailed knowledge of mechanical parameters such as cell elasticity, stiffness of the growth substrate, or traction stresses generated during axonal extensions is essential for understanding the mechanisms that control neuronal growth. Here, we combine atomic force microscopy-based force spectroscopy with fluorescence microscopy to produce systematic, high-resolution elasticity maps for three different types of live neuronal cells: cortical (embryonic rat), embryonic chick dorsal root ganglion, and P-19 (mouse embryonic carcinoma stem cells) neurons. We measure how the stiffness of neurons changes both during neurite outgrowth and upon disruption of microtubules of the cell. We find reversible local stiffening of the cell during growth, and show that the increase in local elastic modulus is primarily due to the formation of microtubules. We also report that cortical and P-19 neurons have similar elasticity maps, with elastic moduli in the range 0.1-2 kPa, with typical average values of 0.4 kPa (P-19) and 0.2 kPa (cortical). In contrast, dorsal root ganglion neurons are stiffer than P-19 and cortical cells, yielding elastic moduli in the range 0.1-8 kPa, with typical average values of 0.9 kPa. Finally, we report no measurable influence of substrate protein coating on cell body elasticity for the three types of neurons.

Combined Traction Force-Atomic Force Microscopy Measurements of Neuronal Cells.

In the course of the development of the nervous system, neuronal cells extend (grow) axons, which navigate over distances of the order of many cell diameters to reach target dendrites from other neurons and establish neuronal circuits. Some of the central challenges in biophysics today are to develop a quantitative model of axonal growth, which includes the interactions between the neurons and their growth environment, and to describe the complex architecture of neuronal networks in terms of a small number of physical variables. To address these challenges, researchers need new experimental techniques for measuring biomechanical interactions with very high force and spatiotemporal resolutions. Here we report a unique experimental approach that integrates three different high-resolution techniques on the same platform-traction force microscopy (TFM), atomic force microscopy (AFM) and fluorescence microscopy (FM)-to measure biomechanical properties of cortical neurons. To our knowledge, this is the first literature report of combined TFM/AFM/FM measurements performed for any type of cell. Using this combination of powerful experimental techniques, we perform high-resolution measurements of the elastic modulus for cortical neurons and relate these values with traction forces exerted by the cells on the growth substrate (poly acrylamide hydrogels, or PAA, coated with poly D-lysine). We obtain values for the traction stresses exerted by the cortical neurons in the range 30-70 Pa, and traction forces in the range 5-11 nN. Our results demonstrate that neuronal cells stiffen when axons exert forces on the PAA substrate, and that neuronal growth is governed by a contact guidance mechanism, in which axons are guided by external mechanical cues. This work provides new insights for bioengineering novel biomimetic platforms that closely model neuronal growth in vivo, and it has significant impact for creating neuroprosthetic interfaces and devices for neuronal growth and regeneration.

Cytoprotection of Human Progenitor and Stem Cells through Encapsulation in Alginate Templated, Dual Crosslinked Silk and Silk-Gelatin Composite Hydrogel Microbeads.

Susceptibility of mammalian cells against harsh processing conditions limit their use in cell transplantation and tissue engineering applications. Besides modulation of the cell microenvironment, encapsulation of mammalian cells within hydrogel microbeads attract attention for cytoprotection through physical isolation of the encapsulated cells. The hydrogel formulations used for cell microencapsulation are largely dominated by ionically crosslinked alginate (Alg), which suffer from low structural stability under physiological culture conditions and poor cell-matrix interactions. Here the fabrication of Alg templated silk and silk/gelatin composite hydrogel microspheres with permanent or on-demand cleavable enzymatic crosslinks using simple and cost-effective centrifugation-based droplet processing are demonstrated. The composite microbeads display structural stability under ion exchange conditions with improved mechanical properties compared to ionically crosslinked Alg microspheres. Human mesenchymal stem and neural progenitor cells are successfully encapsulated in the composite beads and protected against environmental factors, including exposure to polycations, extracellular acidosis, apoptotic cytokines, ultraviolet (UV) irradiation, anoikis, immune recognition, and particularly mechanical stress. The microbeads preserve viability, growth, and differentiation of encapsulated stem and progenitor cells after extrusion in viscous polyethylene oxide solution through a 27-gauge fine needle, suggesting potential applications in injection-based delivery and three-dimensional bioprinting of mammalian cells with higher success rates.