Nanomaterials in biological environment: a review of computer modelling studies

Nanotechnology is set to impact a vast range of fields, including computer science, materials technology, engineering/manufacturing and medicine. As nanotechnology grows so does exposure to nanostructured materials, thus investigation of the effects of nanomaterials on biological systems is paramount. Computational techniques can allow investigation of these systems at the nanoscale, providing insight into otherwise unexaminable properties, related to both the intentional and unintentional effects of nanomaterials. Herein, we review the current literature involving computational modelling of nanoparticles and biological systems. This literature has highlighted the common modes in which nanostructured materials interact with biological molecules such as membranes, peptides/proteins and DNA. Hydrophobic interactions are the most favoured, with π-stacking of the aromatic side-chains common when binding to a carbonaceous nanoparticle or surface. van der Waals forces are found to dominate in the insertion process of DNA molecules into carbon nanotubes. Generally, nanoparticles have been observed to disrupt the tertiary structure of proteins due to the curvature and atomic arrangement of the particle surface. Many hydrophobic nanoparticles are found to be able to transverse a lipid membrane, with some nanoparticles even causing mechanical damage to the membrane, thus potentially leading to cytotoxic effects. Current computational techniques have revealed how some nanoparticles interact with biological systems. However, further research is required to determine both useful applications and possible cytotoxic effects that nanoparticles may have on DNA, protein and membrane structure and function within biosystems.     

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis¹, atherogenesis² and wound healing³. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance^4-7.Single molecule techniques such as optical trapping⁸, atomic force microscopy⁹, and magnetic tweezers^10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level¹². Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.     

Effect of fuel concentration and force on collective transport by a team of dynein motors

Motor proteins are essential components of intracellular transport inside eukaryotic cells. These protein molecules use chemical energy obtained from hydrolysis of ATP to produce mechanical forces required for transporting cargos inside cells, from one location to another, in a directed manner. Of these motors, cytoplasmic dynein is structurally more complex than other motor proteins involved in intracellular transport, as it shows force and fuel (ATP) concentration dependent step‐size. Cytoplasmic dynein motors are known to work in a team during cargo transport and force generation. Here, we use a complete Monte‐Carlo model of single dynein constrained by in vitro experiments, which includes the effect of both force and ATP on stepping as well as detachment of motors under force. We then use our complete Monte‐Carlo model of single dynein motor to understand collective cargo transport by a team of dynein motors, such as dependence of cargo travel distance and velocity on applied force and fuel concentration. In our model, cargos pulled by a team of dynein motors do not detach rapidly under higher forces, confirming the experimental observation of longer persistence time of dynein team on microtubule under higher forces.     

The effects of mechanical forces on intestinal physiology and pathology

The epithelial and non-epithelial cells of the intestinal wall experience a myriad of physical forces including strain, shear, and villous motility during normal gut function. Pathologic conditions alter these forces, leading to changes in the biology of these cells. The responses of intestinal epithelial cells to forces vary with both the applied force and the extracellular matrix proteins with which the cells interact, with differing effects on proliferation, differentiation, and motility, and the regulation of these effects involves similar but distinctly different signal transduction mechanisms. Although normal epithelial cells respond to mechanical forces, malignant gastrointestinal epithelial cells also respond to forces, most notably by increased cell adhesion, a critical step in tumor metastasis. This review will focus on the phenomenon of mechanical forces influencing cell biology and the mechanisms by which the gut responds these forces in both the normal as well as pathophysiologic states when forces are altered. Although more is known about epithelial responses to force, information regarding mechanosensitivity of vascular, neural, and endocrine cells within the gut wall will also be discussed, as will, the mechanism by which forces can regulate epithelial tumor cell adhesion.     

Resolving structure and mechanical properties at the nanoscale of viruses with frequency modulation atomic force microscopy

Structural Biology (SB) techniques are particularly successful in solving virus structures. Taking advantage of the symmetries, a heavy averaging on the data of a large number of specimens, results in an accurate determination of the structure of the sample. However, these techniques do not provide true single molecule information of viruses in physiological conditions. To answer many fundamental questions about the quickly expanding physical virology it is important to develop techniques with the capability to reach nanometer scale resolution on both structure and physical properties of individual molecules in physiological conditions. Atomic force microscopy (AFM) fulfills these requirements providing images of individual virus particles under physiological conditions, along with the characterization of a variety of properties including local adhesion and elasticity. Using conventional AFM modes is easy to obtain molecular resolved images on flat samples, such as the purple membrane, or large viruses as the Giant Mimivirus. On the contrary, small virus particles (25-50 nm) cannot be easily imaged. In this work we present Frequency Modulation atomic force microscopy (FM-AFM) working in physiological conditions as an accurate and powerful technique to study virus particles. Our interpretation of the so called "dissipation channel" in terms of mechanical properties allows us to provide maps where the local stiffness of the virus particles are resolved with nanometer resolution. FM-AFM can be considered as a non invasive technique since, as we demonstrate in our experiments, we are able to sense forces down to 20 pN. The methodology reported here is of general interest since it can be applied to a large number of biological samples. In particular, the importance of mechanical interactions is a hot topic in different aspects of biotechnology ranging from protein folding to stem cells differentiation where conventional AFM modes are already being used.     

Optical tweezers in biology and medicine

Optical trapping techniques provide unique means to manipulate biological particles such as virus, living cells and subcellular organelles. Another area of interest is the measurement of mechanical (elastic) properties of cell membranes, long strands of single DNA molecule, and filamentous proteins. One of the most attractive applications is the study of single motor molecules. With optical tweezers traps, one can measure the forces generated by single motor molecules such as kinesin and myosin, in the piconewton range and, for the first time, resolve their detailed stepping motion.     

Nuclear mechanotransduction in stem cells

In development and in homeostatic maintenance of tissues, stem cells and progenitor cells are constantly subjected to forces. These forces can lead to significant changes in gene expression and function of stem cells, mediating self-renewal, lineage specification, and even loss of function. One of the ways that has been proposed to mediate these functional changes in stem cells is nuclear mechanotransduction — the process by which forces are converted to signals in the nucleus. The purpose of this review is to discuss the means by which mechanical signals are transduced into the nucleus, through the linker of nucleoskeleton and cytoskeleton (LINC) complex and other nuclear envelope transmembrane (NET) proteins, which connect the cytoskeleton to the nucleus. We discuss how LINC/NETs confers tissue-specific mechanosensitivity to cells and further elucidate how LINC/NETs acts as a control center for nuclear mechanical signals, regulating both gene expression and chromatin organization. Throughout, we primarily focus on stem cell–specific examples, notwithstanding that this is a nascent field. We conclude by highlighting open questions and pointing the way to enhanced research efforts to understand the role nuclear mechanotransduction plays in cell fate choice.     

Biophysical approaches for studying the integrity and function of tight junctions

Cell-cell adhesion is an extremely important phenomenon as it influences several biologically important processes such as inflammation, cell migration, proliferation, differentiation and even cancer metastasis. Furthermore, proteins involved in cell-cell adhesion are also important from the perspective of facilitating better drug delivery across epithelia. The adhesion forces imparted by proteins involved in cell-cell adhesion have been the focus of research for sometime. However, with the advent of nanotechnological techniques such as the atomic force microscopy (AFM), we can now quantitatively probe these adhesion forces not only at the cellular but also molecular level. Here, we review the structure and function of tight junction proteins, highlighting some mechanistic studies performed to quantify the adhesion occurring between these proteins and where possible their association with human diseases. In particular, we will highlight two important experimental techniques, namely the micropipette step pressure technique and the AFM that allow us to quantify these adhesion forces at both the cellular and molecular levels, respectively.     

Musculoskeletal mechanobiology: Interpretation by external force and engineered substratum

Mechanobiology aims to discover how the mechanical environment affects the biological activity of cells and how cells’ ability to sense these mechanical cues is converted into elicited cellular responses. Musculoskeletal mechanobiology is of particular interest given the high mechanical loads that musculoskeletal tissues experience on a daily basis. How do cells within these mechanically active tissues interpret external loads imposed on their extracellular environment, and, how are cell–substrate interactions converted into biochemical signals? This review outlines many of the main mechanotransduction mechanisms known to date, and describes recent literature examining effects of both external forces and cell–substrate interactions on musculoskeletal cells. Whether via application of external forces and/or cell–substrate interactions, our understanding and regulation of musculoskeletal mechanobiology can benefit by expanding upon traditional models, and shedding new light through novel investigative approaches. Current and future work in this field is focused on identifying specific forces, stresses, and strains at the cellular and tissue level through both experimental and computational approaches, and analyzing the role of specific proteins through fluorescence-based investigations and knockdown models.     

Changes in surface topologies of chondrocytes subjected to mechanical forces: an AFM analysis

The cartilage is composed of chondrocytes embedded in a matrix of collagen fibrils interspersed within a network of proteoglycans and is constantly exposed to biomechanical forces during normal joint movement. Characterization of the surface morphology, cytoskeletal structure, adherance and elastic properties of these mechanosensitive cells are crucial in understanding the effects of mechanical forces around a cell and how a cell responds to changes in its physical environment. In this work, we employed the atomic force microscope (AFM) to image cultured chondrocytes before and after subjecting them to mechanical forces in the presence or absence of interleukin-1β to mimic inflammatory conditions. Nanoscale imaging and quantitative measurements from AFM data revealed that there are distinct changes in cell-surface topology and cytoskeleton arrangement in the cells following treatment with mechanical forces, IL-1β or both. Our findings for the first time demonstrate that cultured chondrocytes are amenable to high-resolution AFM imaging and dynamic tensile forces may help overcome the effect of inflammatory factors on chondrocyte response.     

Integrins as mechanochemical transducers

A recent resurgence of interest in mechanical forces and cell shape as biological regulators has revealed extracellular matrix as the site at which forces are transmitted both to and from cells. at the same time, great advances have been made in terms of defining cell-surface integrin receptors as transmembrane molecules that mediate cell attachment and physically interlink extracellular matrix with the intracellular cytoskeleton. Convergence of these two lines of research has begun to elucidate the molecular mechanism by which cells sense physical forces and transduce mechanical signals into a biochemical response.     

Reversible mechanical unfolding of single ubiquitin molecules

Single-molecule manipulation techniques have enabled the characterization of the unfolding and refolding process of individual protein molecules, using mechanical forces to initiate the unfolding transition. Experimental and computational results following this approach have shed new light on the mechanisms of the mechanical functions of proteins involved in several cellular processes, as well as revealed new information on the protein folding/unfolding free-energy landscapes. To investigate how protein molecules of different folds respond to a stretching force, and to elucidate the effects of solution conditions on the mechanical stability of a protein, we synthesized polymers of the protein ubiquitin and characterized the force-induced unfolding and refolding of individual ubiquitin molecules using an atomic-force-microscope-based single-molecule manipulation technique. The ubiquitin molecule was highly resistant to a stretching force, and the mechanical unfolding process was reversible. A model calculation based on the hydrogen-bonding pattern in the native structure was performed to explain the origin of this high mechanical stability. Furthermore, pH effects were studied and it was found that the forces required to unfold the protein remained constant within a pH range around the neutral value, and forces decreased as the solution pH was lowered to more acidic values.     

Theoretical study of two-photon absorption properties and up-conversion efficiency of new symmetric organic π-conjugated molecules for photovoltaic devices

Organic material with high intensity of two-photon absorption (TPA) induced fluorescence can be used as the frequency up-converter materials for improving efficiency of the solar cells. In this work, the organic molecular structures were designed by symmetrically grafting two elongated conjugated linkers on a conjugated core and then adding donor groups at both terminals. Fluorene derivatives as a core acceptor, phenylethynyl as the conjugated linker and diphenylamino or methyl-9H-carbozole as the donor end groups were selected. Quantum mechanical modeling techniques were applied to investigate the molecular electronic structure and properties. Absorption properties of these novel π-conjugated organic molecules were studied. The TPA cross-sections of these derivatives were calculated using few-states models, respectively. The effects of donor and acceptor groups on the TPA behaviors of these designed molecules were investigated. The up-conversion efficiency of designed molecules was also calculated.     

Force-induced adsorption and anisotropic growth of focal adhesions

Focal adhesions are micrometer-sized protein aggregates that connect actin stress fibers to the extracellular matrix, a network of macromolecules surrounding tissue cells. The actin fibers are under tension due to actin-myosin contractility. Recent measurements have shown that as the actin force is increased, these adhesions grow in size and in the direction of the force. This is in contrast to the growth of condensed domains of surface-adsorbed molecules in which the dynamics are isotropic. We predict these force-sensitive, anisotropic dynamics of focal adhesions from a model for the adsorption of proteins from the cytoplasm to the adhesion site. Our theory couples the mechanical forces and elasticity to the adsorption dynamics via force-induced conformational changes of molecular-sized mechanosensors located in the focal adhesion. We predict the velocity of both the front and back of the adhesion as a function of the applied force. In addition, our results show that the relative motion of the front and back of the adhesion is asymmetric and in different ranges of forces, the adhesion can either shrink or grow in the direction of the force.     

Imaging and manipulating the structural machinery of living cells on the micro- and nanoscale

The structure, physiology, and fate of living cells are all highly sensitive to mechanical forces in the cellular microenvironment, including stresses and strains that originate from encounters with the extracellular matrix (ECM), blood and other flowing materials, and neighbouring cells. This relationship between context and physiology bears tremendous implications for the design of cellular micro-or nanotechnologies, since any attempt to control cell behavior in a device must provide the appropriate physical microenvironment for the desired cell behavior. Cells sense, process, and respond to biophysical cues in their environment through a set of integrated, multi-scale structural complexes that span length scales from single molecules to tens of microns, including small clusters of force-sensing molecules at the cell surface, micron-sized cell-ECM focal adhesion complexes, and the cytoskeleton that permeates and defines the entire cell. This review focuses on several key technologies that have recently been developed or adapted for the study of the dynamics of structural micro-and nanosystems in living cells and how these systems contribute to spatially-and temporally-controlled changes in cellular structure and mechanics. We begin by discussing subcellular laser ablation, which permits the precise incision of nanoscale structural elements in living cells in order to discern their mechanical properties and contributions to cell structure. We then discuss fluorescence recovery after photobleaching and fluorescent speckle microscopy, two live-cell fluorescence imaging methods that enable quantitative measurement of the binding and transport properties of specific proteins in the cell. Finally, we discuss methods to manipulate cellular structural networks by engineering the extracellular environment, including microfabrication of ECM distributions of defined geometry and microdevices designed to measure cellular traction forces at micron-scale resolution. Together, these methods form a powerful arsenal that is already adding significantly to our understanding of the nanoscale architecture and mechanics of living cells and may contribute to the rational design of new cellular micro-and nanotechnologies.     

Shape-dependent control of cell growth, differentiation, and apoptosis: switching between attractors in cell regulatory networks

Development of characteristic tissue patterns requires that individual cells be switched locally between different phenotypes or "fates;" while one cell may proliferate, its neighbors may differentiate or die. Recent studies have revealed that local switching between these different gene programs is controlled through interplay between soluble growth factors, insoluble extracellular matrix molecules, and mechanical forces which produce cell shape distortion. Although the precise molecular basis remains unknown, shape-dependent control of cell growth and function appears to be mediated by tension-dependent changes in the actin cytoskeleton. However, the question remains: how can a generalized physical stimulus, such as cell distortion, activate the same set of genes and signaling proteins that are triggered by molecules which bind to specific cell surface receptors. In this article, we use computer simulations based on dynamic Boolean networks to show that the different cell fates that a particular cell can exhibit may represent a preprogrammed set of common end programs or "attractors" which self-organize within the cell's regulatory networks. In this type of dynamic network model of information processing, generalized stimuli (e.g., mechanical forces) and specific molecular cues elicit signals which follow different trajectories, but eventually converge onto one of a small set of common end programs (growth, quiescence, differentiation, apoptosis, etc.). In other words, if cells use this type of information processing system, then control of cell function would involve selection of preexisting (latent) behavioral modes of the cell, rather than instruction by specific binding molecules. Importantly, the results of the computer simulation closely mimic experimental data obtained with living endothelial cells. The major implication of this finding is that current methods used for analysis of cell function that rely on characterization of linear signaling pathways or clusters of genes with common activity profiles may overlook the most critical features of cellular information processing which normally determine how signal specificity is established and maintained in living cells.     

Protein nanomechanics in biological context

How proteins respond to pulling forces, or protein nanomechanics, is a key contributor to the form and function of biological systems. Indeed, the conventional view that proteins are able to diffuse in solution does not apply to the many polypeptides that are anchored to rigid supramolecular structures. These tethered proteins typically have important mechanical roles that enable cells to generate, sense, and transduce mechanical forces. To fully comprehend the interplay between mechanical forces and biology, we must understand how protein nanomechanics emerge in living matter. This endeavor is definitely challenging and only recently has it started to appear tractable. Here, I introduce the main in vitro single-molecule biophysics methods that have been instrumental to investigate protein nanomechanics over the last 2 decades. Then, I present the contemporary view on how mechanical force shapes the free energy of tethered proteins, as well as the effect of biological factors such as post-translational modifications and mutations. To illustrate the contribution of protein nanomechanics to biological function, I review current knowledge on the mechanobiology of selected muscle and cell adhesion proteins including titin, talin, and bacterial pilins. Finally, I discuss emerging methods to modulate protein nanomechanics in living matter, for instance by inducing specific mechanical loss-of-function (mLOF). By interrogating biological systems in a causative manner, these new tools can contribute to further place protein nanomechanics in a biological context.     

Protein interactions and misfolding analyzed by AFM force spectroscopy

Protein misfolding is conformational transition dramatically facilitating the assembly of protein molecules into aggregates of various morphologies. Spontaneous formation of specific aggregates, mostly amyloid fibrils, was initially believed to be limited to proteins involved in the development of amyloidoses. However, recent studies show that, depending on conditions, the majority of proteins undergo structural transitions leading to the appearance of amyloidogenic intermediates followed by aggregate formation. Various techniques have been used to characterize the protein misfolding facilitating the aggregation process, but no direct evidence as to how such a conformational transition increases the intermolecular interactions has been obtained as of yet. We have applied atomic force microscopy (AFM) to follow the interaction between protein molecules as a function of pH. These studies were performed for three unrelated and structurally distinctive proteins, alpha-synuclein, amyloid beta-peptide (Abeta) and lysozyme. It was shown that the attractive force between homologous protein molecules is minimal at physiological pH and increases dramatically at acidic pH. Moreover, the dependence of the pulling forces is sharp, suggesting a pH-dependent conformational transition within the protein. Parallel circular dichroism (CD) measurements performed for alpha-synuclein and Abeta revealed that the decrease in pH is accompanied by a sharp conformational transition from a random coil at neutral pH to the more ordered, predominantly beta-sheet, structure at low pH. Importantly, the pH ranges for these conformational transitions coincide with those of pulling forces changes detected by AFM. In addition, protein self-assembly into filamentous aggregates studied by AFM imaging was shown to be facilitated at pH values corresponding to the maximum of pulling forces. Overall, these results indicate that proteins at acidic pH undergo structural transition into conformations responsible for the dramatic increase in interprotein interaction and promoting the formation of protein aggregates.     

Intercellular mechanotransduction: cellular circuits that coordinate tissue responses to mechanical loading

Physical forces play an important role in modulating cell function and shaping tissue structure. Mechanotransduction, the process by which cells transduce physical force-induced signals into biochemical responses, is critical for mediating adaptations to mechanical loading in connective tissues. While much is known about mechanotransduction in cells involving forces delivered through extracellular matrix proteins and integrins, there is limited understanding of how mechanical signals are propagated through the interconnected cellular networks found in tissues and organs. We propose that intercellular mechanotransduction is a critical component for achieving coordinated remodeling responses to force application in connective tissues. We examine here recent evidence on different pathways of intercellular mechanotransduction and suggest a general model for how multicellular structures respond to mechanical loading as an integrated unit.     

Asymmetric spindle positioning

When a spindle is positioned asymmetrically in a dividing cell, the resulting daughter cells are unequal in size. Asymmetric spindle positioning is driven by regulated forces that can pull or push a spindle. The physical and molecular mechanisms that can position spindles asymmetrically have been studied in several systems, and some themes have begun to emerge from recent research. Recent work in budding yeast has presented a model for how cytoskeletal motors and cortical capture molecules can function in orienting and positioning a spindle. The temporal regulation of microtubule-based pulling forces that move a spindle has been examined in one animal system. Although the spindle positioning force generators have not been identified in most animal systems, the forces have been found to be regulated by both PAR polarity proteins and G-protein signaling pathways in more than one animal system.