Production of Lipid Constructs by Design via Three-Dimensional Nanoprinting.

Atomic force microscopy (AFM) in conjunction with microfluidic delivery was utilized to produce three-dimensional (3D) lipid structures following a custom design. While AFM is well-known for its spatial precision in imaging and 2D nanolithography, the development of AFM-based nanotechnology into 3D nanoprinting requires overcoming the technical challenges of controlling material delivery and interlayer registry. This work demonstrates the concept of 3D nanoprinting of amphiphilic molecules such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Various formulations of POPC solutions were tested to achieve point, line, and layer-by-layer material delivery. The produced structures include nanometer-thick disks, long linear spherical caps, stacking grids, and organizational chiral architectures. The POPC molecules formed stacking bilayers in these constructions, as revealed by high-resolution structural characterizations. The 3D printing reached nanometer spatial precision over a range of 0.5 mm. The outcomes reveal the promising potential of our designed technology and methodology in the production of 3D structures from nanometer to continuum, opening opportunities in biomaterial sciences and engineering, such as in the production of 3D nanodevices, chiral nanosensors, and scaffolds for tissue engineering and regeneration.

Mechanical Cues for Triggering and Regulating Cellular Movement Selectively at the Single-Cell Level.

Cell motility plays important roles in many biophysical and physiological processes ranging from in vitro biomechanics, wound healing, to cancer metastasis. This work introduces a new means to trigger and regulate motility individually using transient mechanical stimulus applied to designated cells. Using BV2 microglial cells, our investigations indicate that motility can be reproducibly and reliably initiated using mechanical compression of the cells. The location and magnitude of the applied force impact the movement of the cell. Based on observations from this investigation and current knowledge of BV2 cellular motility, new physical insights are revealed into the underlying mechanism of force-induced single cellular movement. The process involves high degrees of myosin activation to repair actin cortex breakages induced by the initial mechanical compression, which leads to focal adhesion degradation, lamellipodium detachment, and finally, cell polarization and movement. Modern technology enables accurate control over force magnitude and location of force delivery, thus bringing us closer to programming cellular movement at the single-cell level. This approach is of generic importance to other cell types beyond BV2 cells and has the intrinsic advantages of being transient, non-toxic, and non-destructive, thus exhibiting high translational potentials including mechano-based therapy.

Impact of Surface Polarity on Lipid Assembly under Spatial Confinement.

Molecular dynamics (MD) simulations in the MARTINI model are used to study the assembly of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) molecules under spatial confinement, such as during solvent evaporation from ultrasmall (femtoliter quantity) droplets. The impact of surface polarity on molecular assembly is discussed in detail. To the best of our knowledge, this work represents the first of its kind. Our results reveal that solvent evaporation gives rise to the formation of well-defined stacks of lipid bilayers in a smectic alignment. These smectic mesophases form on both polar and nonpolar surfaces but with a notable distinction. On polar surfaces, the director of the stack is oriented perpendicular to the support surface. By contrast, the stacks orient at an angle on the nonpolar surfaces. The packing of head groups on surfaces and lipid molecular mobility exhibits significant differences as surface polarity changes. The role of glycerol in the assembly and stability is also revealed. The insights revealed from the simulation have a significant impact on additive manufacturing, biomaterials, model membranes, and engineering protocells. For example, POPC assemblies via evaporation of ultrasmall droplets were produced and characterized. The trends compare well with the bilayer stack models. The surface polarity influences the local morphology and structures at the interfaces, which could be rationalized via the molecule-surface interactions observed from simulations.

Direct Observations of Silver Nanowire-Induced Frustrated Phagocytosis among NR8383 Lung Alveolar Macrophages.

The interaction of long nanowires and living cells is directly related to nanowires' nanotoxicity and health impacts. Interactions of silver nanowires (AgNWs) and macrophage cell lines (NR8383) were investigated using laser scanning confocal microscopy and single cell compression (SCC). With high-resolution imaging and mechanics measurement of individual cells, AgNW-induced frustrated phagocytosis was clearly captured in conjunction with structural and property changes of cells. While frustrated phagocytosis is known for long microwires and long carbon nanotubes, this work reports first direct observations of frustrated phagocytosis of AgNWs among living cells in situ. In the case of partial penetration of AgNWs into NR8383 cells, confocal imaging revealed actin participation at the entry sites, whose behavior differs from microwire-induced frustrated phagocytosis. The impacts of frustrated phagocytosis on the cellular membrane and cytoskeleton were also quantified by measuring the mechanical properties using SCC. Taken collectively, this study reveals the structural and property characteristics of nanowire-induced frustrated phagocytosis, which deepens our understanding of nanowire-cell interactions and nanocytotoxicity.

Direct Visualization of the Binding of Transforming Growth Factor Beta 1 with Cartilage Oligomeric Matrix Protein via High-Resolution Atomic Force Microscopy.

This work reports the first direct observations of binding and complex formation between transforming growth factor beta 1 (TGF-ß1) and cartilage oligomeric matrix protein (COMP) using high-resolution atomic force microscopy (AFM). Each COMP molecule consists of pentamers whose five identical monomeric units bundle at N-termini. From this central point, the five monomers' flexible arms extend outward with C-terminal domains at the distal ends, forming a bouquet-like structure. In commonly used buffer solutions, TGF-ß1 molecules typically form homodimers (majority), double dimers (minority), and aggregates (trace amount). Mixing TGF-ß1 and COMP leads to rapid binding and complex formation. The TGF-ß1/COMP complexes contain one to three COMP and multiple TGF-ß1 molecules. For complexes with one COMP, the structure is more compact and less flexible than that of COMP alone. For complexes with two or more COMP molecules, the conformation varies to a large degree from one complex to another. This is attributed to the presence of double dimers or aggregates of TGF-ß1 molecules, whose size and multiple binding sites enable binding to more than one COMP. The number and location of individual TGF-ß1 dimers are also clearly visible in all complexes. This molecular-level information provides a new insight into the mechanism of chondrogenesis enhancement by TGF-ß1/COMP complexes, i.e., simultaneous and multivalent presentation of growth factors. These presentations help explain the high efficacy in sustained activation of the signaling pathway to augment chondrogenesis.

Label-Free and Direct Visualization of Multivalent Binding of Bone Morphogenetic Protein-2 with Cartilage Oligomeric Matrix Protein.

This work presents the first direct evidence of multivalent binding between bone morphogenetic protein-2 (BMP-2) and cartilage oligomeric matrix protein (COMP) using high-resolution atomic force microscopy (AFM) imaging. AFM topographic images reveal the molecular morphology of COMP, a pentameric protein whose five identical monomer units bundle together at N-termini, extending out with flexible chains to C-termini. Upon addition of BMP-2, COMP molecules undergo conformational changes at the C-termini to enable binding with BMP-2 molecules. AFM enables local structural changes of COMP to be revealed upon binding various numbers, 1-5, of BMP-2 molecules. These BMP-2/COMP complexes exhibit very different morphologies from those of COMP: much more compact and thus less flexible. These molecular-level insights deepen current understanding of the mechanism of how the BMP-2/COMP complex enhances osteogenesis among osteoprogenitor cells, i.e., multivalent presentation of BMP-2 via the stable and relatively rigid BMP-2/COMP complex could form a lattice of interaction between multiple BMP-2 and BMP-2 receptors. These ligand-receptor clusters lead to fast initiation and sustained activation of the Smad signaling pathway, resulting in enhanced osteogenesis. This work is also of translational importance as the outcome may enable use of lower BMP-2 dosage for bone repair and regeneration.

Local Mechanical Perturbation Provides an Effective Means to Regulate the Growth and Assembly of Functional Peptide Fibrils.

Mucin 1 (MUC1) peptide fused with Q11 (MUC1-Q11) having 35 residues has previously been shown to form amyloid fibrils. Using time-dependent and high-resolution atomic force microscopy (AFM) imaging, it is revealed that the formation of individual MUC1-Q11 fibrils entails nucleation and extension at both ends. This process can be altered by local mechanical perturbations using AFM probes. This work reports two specific perturbations and outcomes. First, by increasing load while maintaining tip-surface contact, the fibrils are cut during the scan due to shearing. Growth of fibrils occurs at the newly exposed termini, following similar mechanism of the MUC1-Q11 nucleation growth. As a result, branched fibrils are seen on the surface whose orientation and length can be controlled by the nuclei orientation and reaction time. In contrast to the "one-time-cut", fibrils can be continuously fragmented by modulation at sufficiently high amplitude. As a result, short and highly branched fibrils accumulate and pile on surfaces. Since the fibril formation and assembly of MUC1-Q11 can be impacted by local mechanical force, this approach offers a nonchemical and label-free means to control the presentation of MUC1 epitopes, and has promising application in MUC1 fibril-based immunotherapy.

Single-Cell Mechanics Provides an Effective Means To Probe in Vivo Interactions between Alveolar Macrophages and Silver Nanoparticles.

Single-cell mechanics, derived from atomic force microscopy-based technology, provides a new and effective means to investigate nanomaterial-cell interactions upon in vivo exposure. Lung macrophages represent initial and important responses upon introducing nanoparticles into the respiratory tract, as well as particle clearance with time. Cellular mechanics has previously proven effective to probe in vitro nanomaterial-cell interactions. This study extends technology further to probe the interactions between primary alveolar macrophages (AM) and silver nanoparticles (AgNPs) upon in vivo exposure. Two types of AgNPs, 20 and 110 nm, were instilled to rat lung at 0.5 mg AgNPs/kg body weight, and allowed 24 h interaction. The consequences of these interactions were investigated by harvesting the primary AMs while maintaining their biological status. Cellular mechanics measurements revealed the diverse responses among AM cells, due to variations in AgNP uptake and oxidative dissolving into Ag(+). Three major responses are evident: zero to low uptake that does not alter cellular mechanics, intracellular accumulation of AgNPs trigger cytoskeleton rearrangement resulting in the stiffening of mechanics, and damage of cytoskeleton that softens the mechanical profile. These effects were confirmed using confocal imaging of F-actin and measurements of reactive oxygen species production. More detailed intracellular interactions will also be discussed on the basis of this study in conjunction with prior knowledge of AgNP toxicity.

High-Resolution Imaging of Polyethylene Glycol Coated Dendrimers via Combined Atomic Force and Scanning Tunneling Microscopy.

Dendrimers have shown great promise as drug delivery vehicles in recent years because they can be synthesized with designed size and functionalities for optimal transportation, targeting, and biocompatibility. One of the most well-known termini used for biocompatibility is polyethylene glycol (PEG), whose performance is affected by its actual conformation. However, the conformation of individual PEG bound to soft materials such as dendrimers has not been directly observed. Using atomic force microscopy (AFM) and scanning tunneling microscopy (STM), this work characterizes the structure adopted by PEGylated dendrimers with the highest resolution reported to date. AFM imaging enables visualization of the individual dendrimers, as well as the differentiation and characterization of the dendrimer core and PEG shell. STM provides direct imaging of the PEG extensions with high-resolution. Collectively, this investigation provides important insight into the structure of coated dendrimers, which is crucial for the design and development of better drug delivery vehicles.

Engineering amyloid fibrils from ß-solenoid proteins for biomaterials applications.

Nature provides numerous examples of self-assembly that can potentially be implemented for materials applications. Considerable attention has been given to one-dimensional cross-ß or amyloid structures that can serve as templates for wire growth or strengthen materials such as glue or cement. Here, we demonstrate controlled amyloid self-assembly based on modifications of ß-solenoid proteins. They occur naturally in several contexts (e.g., antifreeze proteins, drug resistance proteins) but do not aggregate in vivo due to capping structures or distortions at their ends. Removal of these capping structures and regularization of the ends of the spruce budworm and rye grass antifreeze proteins yield micron length amyloid fibrils with predictable heights, which can be a platform for biomaterial-based self-assembly. The design process, including all-atom molecular dynamics simulations, purification, and self-assembly procedures are described. Fibril formation with the predicted characteristics is supported by evidence from thioflavin-T fluorescence, circular dichroism, dynamic light scattering, and atomic force microscopy. Additionally, we find evidence for lateral assembly of the modified spruce budworm antifreeze fibrils with sufficient incubation time. The kinetics of polymerization are consistent with those for other amyloid formation reactions and are relatively fast due to the preformed nature of the polymerization nucleus.

Cross-species mechanical fingerprinting of cardiac myosin binding protein-C.

Cardiac myosin binding protein-C (cMyBP-C) is a member of the immunoglobulin (Ig) superfamily of proteins and consists of 8 Ig- and 3 fibronectin III (FNIII)-like domains along with a unique regulatory sequence referred to as the MyBP-C motif or M-domain. We previously used atomic force microscopy to investigate the mechanical properties of murine cMyBP-C expressed using a baculovirus/insect cell expression system. Here, we investigate whether the mechanical properties of cMyBP-C are conserved across species by using atomic force microscopy to manipulate recombinant human cMyBP-C and native cMyBP-C purified from bovine heart. Force versus extension data obtained in velocity-clamp experiments showed that the mechanical response of the human recombinant protein was remarkably similar to that of the bovine native cMyBP-C. Ig/Fn-like domain unfolding events occurred in a hierarchical fashion across a threefold range of forces starting at relatively low forces of ~50 pN and ending with the unfolding of the highest stability domains at ~180 pN. Force-extension traces were also frequently marked by the appearance of anomalous force drops suggestive of additional mechanical complexity such as structural coupling among domains. Both recombinant and native cMyBP-C exhibited a prominent segment ~100 nm-long that could be stretched by forces <50 pN before the unfolding of Ig- and FN-like domains. Combined with our previous observations of mouse cMyBP-C, these results establish that although the response of cMyBP-C to mechanical load displays a complex pattern, it is highly conserved across species.

Distinct annular oligomers captured along the assembly and disassembly pathways of transthyretin amyloid protofibrils.

BACKGROUND: Defects in protein folding may lead to severe degenerative diseases characterized by the appearance of amyloid fibril deposits. Cytotoxicity in amyloidoses has been linked to poration of the cell membrane that may involve interactions with amyloid intermediates of annular shape. Although annular oligomers have been detected in many amyloidogenic systems, their universality, function and molecular mechanisms of appearance are debated. METHODOLOGY/PRINCIPAL FINDINGS: We investigated with high-resolution in situ atomic force microscopy the assembly and disassembly of transthyretin (TTR) amyloid protofibrils formed of the native protein by pH shift. Annular oligomers were the first morphologically distinct intermediates observed in the TTR aggregation pathway. Morphological analysis suggests that they can assemble into a double-stack of octameric rings with a 16 ± 2 nm diameter, and displaying the tendency to form linear structures. According to light scattering data coupled to AFM imaging, annular oligomers appeared to undergo a collapse type of structural transition into spheroid oligomers containing 8-16 monomers. Disassembly of TTR amyloid protofibrils also resulted in the rapid appearance of annular oligomers but with a morphology quite distinct from that observed in the assembly pathway. CONCLUSIONS/SIGNIFICANCE: Our observations indicate that annular oligomers are key dynamic intermediates not only in the assembly but also in the disassembly of TTR protofibrils. The balance between annular and more compact forms of aggregation could be relevant for cytotoxicity in amyloidogenic disorders.

Mechanical unfolding of cardiac myosin binding protein-C by atomic force microscopy.

Cardiac myosin-binding protein-C (cMyBP-C) is a thick-filament-associated protein that performs regulatory and structural roles within cardiac sarcomeres. It is a member of the immunoglobulin (Ig) superfamily of proteins consisting of eight Ig- and three fibronectin (FNIII)-like domains, along with a unique regulatory sequence referred to as the M-domain, whose structure is unknown. Domains near the C-terminus of cMyBP-C bind tightly to myosin and mediate the association of cMyBP-C with thick (myosin-containing) filaments, whereas N-terminal domains, including the regulatory M-domain, bind reversibly to myosin S2 and/or actin. The ability of MyBP-C to bind to both myosin and actin raises the possibility that cMyBP-C cross-links myosin molecules within the thick filament and/or cross-links myosin and thin (actin-containing) filaments together. In either scenario, cMyBP-C could be under mechanical strain. However, the physical properties of cMyBP-C and its behavior under load are completely unknown. Here, we investigated the mechanical properties of recombinant baculovirus-expressed cMyBP-C using atomic force microscopy to assess the stability of individual cMyBP-C molecules in response to stretch. Force-extension curves showed the presence of long extensible segment(s) that became stretched before the unfolding of individual Ig and FNIII domains, which were evident as sawtooth peaks in force spectra. The forces required to unfold the Ig/FNIII domains at a stretch rate of 500 nm/s increased monotonically from ∼30 to ∼150 pN, suggesting a mechanical hierarchy among the different Ig/FNIII domains. Additional experiments using smaller recombinant proteins showed that the regulatory M-domain lacks significant secondary or tertiary structure and is likely an intrinsically disordered region of cMyBP-C. Together, these data indicate that cMyBP-C exhibits complex mechanical behavior under load and contains multiple domains with distinct mechanical properties.

Oriented epitaxial growth of amyloid fibrils of the N27C mutant beta 25-35 peptide.

Amyloid fibrils are present in the extracellular space of various tissues in neurodegenerative and protein misfolding diseases. Amyloid fibrils may be used in nanotechnology applications, because of their self-assembly properties and stability, if their growth and orientation can be controlled. Recently, we have shown that amyloid beta 25-35 (A beta 25-35) forms a highly oriented, K(+)-dependent network on mica. Here, we analyzed the properties of A beta 25-35_N27C, the cysteine residue of which may be used for subsequent chemical modifications. We find that A beta 25-35_N27C forms epitaxially growing fibrils on mica, which evolve into a trigonally oriented branched network. The binding is apparently more sensitive to cation concentration than that of the wild-type peptide. By nanomanipulating A beta 25-35_N27C fibrils with a gold-coated AFM tip, we show that the sulfhydryl of Cys27 is reactive and accessible from the solution. The oriented network of A beta 25-35_N27C fibrils can therefore be specifically labeled and may be used for constructing nanobiotechnological devices.

Stepwise dynamics of epitaxially growing single amyloid fibrils.

The assembly mechanisms of amyloid fibrils, tissue deposits in a variety of degenerative diseases, is poorly understood. With a simply modified application of the atomic force microscope, we monitored the growth, on mica surface, of individual fibrils of the amyloid beta25-35 peptide with near-subunit spatial and subsecond temporal resolution. Fibril assembly was polarized and discontinuous. Bursts of rapid (up to 300-nm(-1)) growth phases that extended the fibril by approximately 7 nm or its integer multiples were interrupted with pauses. Stepwise dynamics were also observed for amyloid beta1-42 fibrils growing on graphite, suggesting that the discontinuous assembly mechanisms may be a general feature of epitaxial amyloid growth. Amyloid assembly may thus involve fluctuation between a fast-growing and a blocked state in which the fibril is kinetically trapped because of intrinsic structural features. The used scanning-force kymography method may be adapted to analyze the assembly dynamics of a wide range of linear biopolymers.

Spatially and temporally synchronized atomic force and total internal reflection fluorescence microscopy for imaging and manipulating cells and biomolecules.

The atomic force microscope is a high-resolution scanning-probe instrument which has become an important tool for cellular and molecular biophysics in recent years but lacks the time resolution and functional specificities offered by fluorescence microscopic techniques. To exploit the advantages of both methods, here we developed a spatially and temporally synchronized total internal reflection fluorescence and atomic force microscope system. The instrument, which we hereby call STIRF-AFM, is a stage-scanning device in which the mechanical and optical axes are coaligned to achieve spatial synchrony. At each point of the scan the sample topography (atomic force microscope) and fluorescence (photon count or intensity) information are simultaneously recorded. The tool was tested and validated on various cellular (monolayer cells in which actin filaments and intermediate filaments were fluorescently labeled) and biomolecular (actin filaments and titin molecules) systems. We demonstrate that with the technique, correlated sample topography and fluorescence images can be recorded, soft biomolecular systems can be mechanically manipulated in a targeted fashion, and the fluorescence of mechanically stretched titin can be followed with high temporal resolution.

Lithium induces phosphoglucomutase activity in various tissues of rats and in bipolar patients.

Phosphoglucomutase catalyses the reversible conversion of glucose-6-P and glucose-1-P. Lithium is a potent inhibitor of phosphoglucomutase in vitro, however, it is not known if phosphoglucomutase was significantly inhibited by Li+ in Li+-treated bipolar patients. Here, we demonstrate that phosphoglucomutase inhibition by chronic Li+ treatment causes alterations of glucose-phosphate levels in various tissues of rats. Also, phosphoglucomutase inhibition results in compensatory elevation of phosphoglucomutase activity in rat tissues and in leukocytes isolated from Li+-treated bipolar patients. The increase of uninhibited phosphoglucomutase activity in leukocytes of Li+-treated bipolar patients is due to the increased expression of the PGM1 gene.

Reversible mechanical unzipping of amyloid beta-fibrils.

Amyloid fibrils are self-associating filamentous structures, the deposition of which is considered to be one of the most important factors in the pathogenesis of Alzheimer's disease and various other disorders. Here we used single molecule manipulation methods to explore the mechanics and structural dynamics of amyloid fibrils. In mechanically manipulated amyloid fibrils, formed from either amyloid beta (Abeta) peptides 1-40 or 25-35, beta-sheets behave as elastic structures that can be "unzipped" from the fibril with constant forces. The unzipping forces were different for Abeta1-40 and Abeta25-35. Unzipping was fully reversible across a wide range of stretch rates provided that coupling, via the beta-sheet, between bound and dissociated states was maintained. The rapid, cooperative zipping together of beta-sheets could be an important mechanism behind the self-assembly of amyloid fibrils. The repetitive force patterns contribute to a mechanical fingerprint that could be utilized in the characterization of different amyloid fibrils.