Magnetic manipulation of actin orientation, polymerization, and gliding on myosin using superparamagnetic iron oxide particles

The actin cytoskeleton controls cell shape, motility, as well as intracellular molecular trafficking. The ability to remotely manipulate actin is therefore highly desirable as a tool to probe and manipulate biological processes at the molecular level. We demonstrate actin manipulation by labeling actin filaments with superparamagnetic iron oxide particles (IOPs) and applying a uniform magnetic field to affect actin orientation, polymerization and gliding on myosin. We show for the first time magnetic manipulation of magnetizable actin filaments at the molecular level while gliding on a bed of myosin molecules and during polymerization. A model for the magnetic alignment and guiding mechanism is proposed based on the torque from the induced molecular anisotropy due to interactions between neighboring IOPs distributed along magnetically labeled actin molecules.     

Graphene Oxide Nanosheets Retard Cellular Migration via Disruption of Actin Cytoskeleton

Graphene and graphene‐based nanomaterials are broadly used for various biomedical applications due to their unique physiochemical properties. However, how graphene‐based nanomaterials interact with biological systems has not been thoroughly studied. This study shows that graphene oxide (GO) nanosheets retard A549 lung carcinoma cell migration through nanosheet‐mediated disruption of intracellular actin filaments. After GO nanosheets treatment, A549 cells display slower migration and the structure of the intracellular actin filaments is dramatically changed. It is found that GO nanosheets are capable of absorbing large amount of actin and changing the secondary structures of actin monomers. Large‐scale all‐atom molecular dynamics simulations further reveal the interactions between GO nanosheets and actin filaments at molecular details. GO nanosheets can insert into the interstrand gap of actin tetramer (helical repeating unit of actin filament) and cause the separation of the tetramer which eventually leads to the disruption of actin filaments. These findings offer a novel mechanism of GO nanosheet induced biophysical responses and provide more insights into their potential for biomedical applications.     

Structure formation in active networks

Structure formation and constant reorganization of the actin cytoskeleton are key requirements for the function of living cells. Here we show that a minimal reconstituted system consisting of actin filaments, crosslinking molecules and molecular-motor filaments exhibits a generic mechanism of structure formation, characterized by a broad distribution of cluster sizes. We demonstrate that the growth of the structures depends on the intricate balance between crosslinker-induced stabilization and simultaneous destabilization by molecular motors, a mechanism analogous to nucleation and growth in passive systems. We also show that the intricate interplay between force generation, coarsening and connectivity is responsible for the highly dynamic process of structure formation in this heterogeneous active gel, and that these competing mechanisms result in anomalous transport, reminiscent of intracellular dynamics.     

Effect of pore size of self-organized honeycomb-patterned polymer films on spreading, focal adhesion, proliferation, and function of endothelial cells

The design of nano- and microstructures based on self-organization is a key area of research in the search for new materials, and it has a variety of potential applications in tissue engineering scaffolds. We have reported a honeycomb-patterned polymer film (honeycomb film) with highly regular pores that is formed by self-organization. This study describes the behavior of vascular endothelial cells (ECs) on honeycomb films with four different pore sizes (5, 9, 12, and 16 microm) as well as on a flat film. We examined the influence of the honeycomb pattern and pore size on cell behavior. The changes in cell morphologies, actin filaments, vinculin clusters, cell proliferation, and secreted extracellular matrix (ECM) (fibronectin, laminin, type IV collagen, and elastin) production profiles were observed by using optical, fluorescence, and scanning electron microscopy. The ECs that adhered to the flat film showed an elongated morphology with random orientation; the actin filaments and focal adhesions were not conspicuous. On the other hand, the ECs on the honeycomb films exhibited greater spreading and flattening; the degree of spreading of the ECs increased with an increase in the pore size. The actin filaments and focal adhesions appeared conspicuous, and the focal adhesions localized along the edge of the honeycomb pores were distributed over the entire projected cell area. The honeycomb film with a pore size of 5 microm showed the highest cell proliferation and ECM production profiles. These results suggest that the honeycomb film is a suitable material for designing a new vascular device.     

Force-velocity relationship of single actin filament interacting with immobilised myosin measured by electromagnetic technique

The effect of applying an external load to actin filaments moving in the in vitro motility assay is studied. Bead-tailed actin filaments were made by polymerising actin onto 2.8 microm diameter Dynabeads conjugated with gelsolin-G actin. These were introduced into a motility cell coated with 100 microg/ml rabbit fast skeletal myosin in the presence of ATP and 0.5% methylcellulose. The motility cell was inserted between the pole-pieces of an electromagnet and the fluorescent beads and filaments were observed. The force-current relationship of the electromagnet was determined from the velocity of free beads in viscous solution and Stokes' equation. The magnet produced up to 6 pN force on the Dynabeads at 1 A. Many bead-tailed actin filaments stuck to the surface, but the beads that did move moved at the same speed as unloaded f-actin in the same cell. Bead-tailed filaments slowed down under an increasing magnetic load, eventually stalled and then slid backward under increasing load before detaching from the surface. Single-filament force-velocity curves were constructed and a stalling force of about 0.6 pN/mm of actin filament estimated.     

Polymer physics of the cytoskeleton

The cytoskeleton is generally visualized by light or electron microscopy as a meshwork of protein filaments that spans the space between the nuclear envelope and the plasma membrane. In most cell types, this meshwork is formed by a three dimensional composite network of actin filaments, microtubules (MT), and intermediate filaments (IF) together with the host of proteins that bind to the sides or ends of these linear polymers. Cytoskeletal binding proteins regulate filament length, crosslink filaments to each other, and apply forces to the filaments. One approach to modeling the mechanical properties of the cytoskeleton and of cell in general is to consider the elements of the cytoskeleton as polymers, using experimental methods and theoretical models developed for traditional polymers but modified for the much larger, stiffer, and fragile biopolymers comprising the cytoskeleton. The presence of motor proteins that move actin filaments and microtubules also creates a new class of active materials that are out of thermodynamic equilibrium, and unconstrained by limitations of the fluctuation–dissipation theorem. These active materials create rich opportunities for experimental design and theoretical developments. The degree to which the mechanics of live cells can usefully be modeled as highly complex polymer networks is by no means certain, and this article will discuss recent progress in quantitatively measuring cytoskeletal polymer systems and relating them to the properties of the cell.     

Utilization of Myosin and Actin Bundles for the Transport of Molecular Cargo

The utilization of motor proteins for the movement and assembly of synthetic components is currently a goal of nanoengineering research. Application of the myosin actin motor system for nanotechnological uses has been hampered due to the low flexural rigidity of individual F‐actin filaments. Here it is demonstrated how actin bundling can be used to affect the translational behavior of myosin‐propelled filaments, transport molecules across a motor‐patterned surface, and that the movement of bundled actin can be regulated photonically. These data suggest that actin bundling may significantly improve the applicability of the myosin motor for future nanotechnological applications.     

Comparing guiding track requirements for myosin- and kinesin-powered molecular shuttles

The design of nanoscale transport systems utilizing motor proteins as engines has advanced rapidly. Here, actin/myosin- and microtubule/kinesin-based molecular shuttles are compared with respect to their requirements for track designs. To this end, the trajectory persistence length of actin filaments gliding on myosin-coated surfaces has been experimentally determined to be equal to 8.8 +/- 2 microm. This measurement complements an earlier determination of the trajectory persistence length of microtubules gliding on kinesin-coated surfaces and enables a comparison of the accessible track designs for kinesin and myosin motor-powered systems. Despite the 200-fold smaller stiffness of actin filaments compared to that of microtubules, the dimensions of myosin tracks for actin filaments have to be quite similar to the dimensions of kinesin tracks for microtubules (radii larger than 200 nm and widths smaller than 0.9 microm compared to 600 nm and 19 microm). The difference in gliding speed is shown to require additional consideration in the design of track modules.     

Crystallization of fluorescent quantum dots within a three-dimensional bio-organic template of actin filaments and lipid membranes

Biological molecules and molecular self-assemblies are promising templates to organize well-defined inorganic nanostructures. We demonstrate the ability of a self-assembled three-dimensional crystal template of helical actin protein filaments and lipids bilayers to generate a hierarchical self-assembly of quantum dots. Functionnalized tricystein peptidic quantum dots (QDs) are incorporated during the dynamical self-assembly of this actin/lipid template resulting in the formation of crystalline fibers. The crystal parameters, 26.5×18.9×35.5 nm3, are imposed by the membrane thickness, the diameter, and the pitch of the actin self-assembly. This process ensures the high quality of the crystal and results in unexpected fluorescence properties. This method of preparation offers opportunities to generate crystals with new symmetries and a large range of distance parameters.     

Plasmonic nanosensors for imaging intracellular biomarkers in live cells

Here we present the first intracellular molecular imaging platform using multifunctional gold nanoparticles which incorporate both cytosolic delivery and targeting moieties on the same particle. The utility of these intracellular sensors was demonstrated by monitoring actin rearrangement in live fibroblasts. We observed a strong molecular specific optical signal associated with effective targeting of actin filaments. These multifunctional nanosensors can be adapted to target various intracellular processes especially where transfection or cytotoxic labels are not feasible.     

Orthogonal (transverse) arrangements of actin in endothelia and fibroblasts.

Though actin filaments running across the cell (transverse actin) have been occasionally reported for epithelial cells in groups and for cells growing on fibres, there has been no report heretofore of transverse actin in cells grown on planar substrata. This paper describes evidence in support of this possibility derived from actin staining, polarization microscopy and force measurements. The paper introduces two new methods for detecting the orientation and activity of contractile elements in cells. The orthogonal actin is most obvious in cells grown on groove ridge structures, but can be detected in cells grown on flat surfaces.     

Measuring forces between two single actin filaments during bundle formation

Bundles of filamentous actin are dominant cytoskeletal structures, which play a crucial role in various cellular processes. As yet quantifying the fundamental interaction between two individual actin filaments forming the smallest possible bundle has not been realized. Applying holographic optical tweezers integrated with a microfluidic platform, we were able to measure the forces between two actin filaments during bundle formation. Quantitative analysis yields forces up to 0.2 pN depending on the concentration of bundling agents.     

Modelling planar polarity of epithelia: the role of signal relay in collective cell polarization

Collective cell polarization is an important characteristic of tissues. Epithelia commonly display cellular structures that are polarized within the plane of the tissue. Establishment of this planar cell polarity requires mechanisms that locally align polarized structures between neighbouring cells, as well as cues that provide global information about alignment relative to an axis of a tissue. In the Drosophila ovary, the cadherin Fat2 is required to orient actin filaments located at the basal side of follicle cells perpendicular to the long axis of the egg chamber. The mechanisms directing this orientation of actin filaments, however, remain unknown. Here we show, using genetic mosaic analysis, that fat2 is not essential for the local alignment of actin filaments between neighbouring cells. Moreover, we provide evidence that Fat2 is involved in the propagation of a cue specifying the orientation of actin filaments relative to the tissue axis. Monte Carlo simulations of actin filament orientation resemble the results of the genetic mosaic analysis, if it is assumed that a polarity signal can propagate from a signal source only through a connected chain of wild-type cells. Our results suggest that Fat2 is required for propagating global polarity information within the follicle epithelium through direct cell–cell contact. Our computational model might be more generally applicable to study collective cell polarization in tissues.     

Bridging defects in nerve continuity: influence of variations in synthetic fiber composition

Synthetic filaments introduced into a silicone tube may help to enhance axonal growth over extended defects in nerve continuity [1]. Here we test the influence of number (0, 3, 7 or 15), size (diameter 150 or 250 m) and material of filaments (polyamide or catgut) enclosed in such tubes (inner diameter 1.98 mm) on axonal growth across a 10 mm defect in rat sciatic nerve. The morphology of the tube content was analyzed four weeks post-surgery. The area of the formed tissue matrix inside the tube showed no difference between the groups. Myelinated axons were observed in the formed tissue matrix inbetween and peripheral to the filaments, however, separated from the filaments by concentric cell layers. The number of myelinated axons was less in the tubes with 15 filaments, most pronounced when catgut filaments were used. In most cases, except in tubes with 15 catgut filaments, fibers had grown into the distal nerve segment (pinch reflex test/light microscopy). We conclude that an intrinsic framework consisting of a limited number of synthetic filaments inside an extrinsic framework (silicone tube) does not disturb nerve regeneration. The formed tissue matrix was neither influenced by the presence or the numbers (if less than or equal to seven filaments), type of filaments nor the size of the filaments indicating the importance of the inserted nerve segments. © 1999 Kluwer Academic Publishers     

Actin flow and talin dynamics govern rigidity sensing in actin–integrin linkage through talin extension

At cell–substrate adhesion sites, the linkage between actin filaments and integrin is regulated by mechanical stiffness of the substrate. Of potential molecular regulators, the linker proteins talin and vinculin are of particular interest because mechanical extension of talin induces vinculin binding with talin, which reinforces the actin–integrin linkage. For understanding the molecular and biophysical mechanism of rigidity sensing at cell–substrate adhesion sites, we constructed a simple physical model to examine a role of talin extension in the stiffness-dependent regulation of actin–integrin linkage. We show that talin molecules linking between retrograding actin filaments and substrate-bound integrin are extended in a manner dependent on substrate stiffness. The model predicts that, in adhesion complexes containing ≈30 talin links, talin is extended enough for vinculin binding when the substrate is stiffer than 1 kPa. The lifetime of talin links needs to be 2–5 s to achieve an appropriate response of talin extension against substrate stiffness. Furthermore, changes in actin velocity drastically shift the range of substrate stiffness that induces talin–vinculin binding. Our results suggest that talin extension is a key step in sensing and responding to substrate stiffness at cell adhesion sites.     

Design of binary subtractor using actin quantum cellular automata

Actin is a biological protein that provides support to the cellular structure and plays a crucial role in cytoskeletal and intra‐cellular signalling events. Logic circuits can be designed with actin filaments with the help of actin quantum automata. The authors use a rule (4,27) to implement some novel designs of logic subtractor circuits on this automata to achieve the difference in two binary bits. Logic design of both half and full binary subtractors is proposed in this study. Actin‐based quantum cellular automata can be used in different combinations of input to get optimised results from the circuits. The authors focus on consolidating the designs inside single automata block to generate output in a less number of timesteps and less overheads. The designs are simulated with Simulink and this way output is verified for these different design approaches. Reliability and fault‐tolerance check is another interesting part of this study. To get a better idea of the optimisation achieved, the authors have also presented a comparative study between the proposed designs in terms of circuit size and efficiency. With all these parameters involved, this study explores opportunities for future implementation of unconventional computing in nano‐scale and cost‐effective bio‐molecular networks.Inspec keywords: cellular automata, molecular biophysics, molecular configurations, biology computing, proteins, logic circuits, biomolecular electronics, cellular biophysics, fault toleranceOther keywords: binary subtractor, actin quantum cellular automata, biological protein, cellular structure, cytoskeletal signalling events, intracellular signalling events, actin filaments, logic subtractor circuits, binary bits, logic design, half binary subtractors, full binary subtractors, optimised results, single automata block, Simulink, design approaches, reliability, fault‐tolerance check, circuit size, circuit efficiency, unconventional computing, nanoscale biomolecular networks, cost‐effective biomolecular networks     

High-quality single-walled carbon nanotubes synthesis by hot filament CVD on Ru nanoparticle catalyst

We investigated the single-walled carbon nanotubes (SWCNTs) growth on Ru nanoparticle catalyst via hot filament assisted chemical vapor deposition (HFCVD) with two independent W filaments for the carbon precursor (methane) and the hydrogen dissociation respectively. The Ru nanoparticles were obtained following a two-step strategy. At first the growth substrate is functionalized by silanisation, then a self assembly of a ruthenium porphyrin complex monolayer on pyridine-functionalized metal oxide substrates. We have studied the impact of the filaments power and we optimized the SWCNTs growth temperature. The as grown SWCNTs were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM) and Raman spectroscopy. It was found that the quality, density and the diameter of SWCNTs depends on the filament and growth temperature. Results of this study can be used to improve the understanding of the growth of SWCNTs by HFCVD.     

3D nanometer images of biological fibers by directed motion of gold nanoparticles

Using near-infrared femtosecond pulses, we move single gold nanoparticles (AuNPs) along biological fibers, such as collagen and actin filaments. While the AuNP is sliding on the fiber, its trajectory is measured in three dimensions (3D) with nanometer resolution providing a high-resolution image of the fiber. Here, we systematically moved a single AuNP along nanometer-size collagen fibers and actin filament inside chinese hamster ovary K1 living cells, mapping their 3D topography with high fidelity.     

Slow filament dynamics and viscoelasticity in entangled and active actin networks

This paper deals with correlations between the viscoelastic impedance of entangled actin networks and the slow conformational dynamics and diffusive motions of single filaments. The single filament dynamics is visualized and analysed by analysing the Brownian motion of attached colloidal beads, which enables independent measurements of characteristic viscoelastic response times such as the entanglement and reptation times. We further studied the frequency-dependent viscoelastic impedance of active actin-heavy-meromyosin II networks by magnetic-tweezers microrheometry to gain insight into the effect of such highly dynamic and force-generating crosslinkers (exhibiting bond lifetimes of less than 1 s) on the rheological properties. We show that at high frequencies (higher than 1 Hz) the viscoelastic loss modulus is slightly increased relative to the entangled network (associated with an increase in the energy dissipated during mechanical excitations), while at low frequencies the plateau of the impedance spectrum becomes more pronounced as a consequence of the cross-linking of the network and the suppression of the terminal regime. Our data provide evidence that the myosin motor protein may play a role as softener of the actin cortex, enabling the adaptive reduction of the yield stress of cells and thus facilitating cellular deformations.     

Remodeling of fibrinogen by endothelial cells in dependence on fibronectin matrix assembly. Effect of substratum wettability

The endothelization of cardiovascular implants is desirable to improve their blood compatibility. The capacity of the endothelial cells to attach, migrate, proliferate and function on the implant surface depends on the presence of matrix proteins such as fibronectin (FN) and fibrinogen (FNG). In this study, we show that the deposition of fibrinogen into extracellular matrix-like structures by human umbilical vein endothelial cells (HUVEC) is dependent on FN matrix formation. We found further that the process of organization of both adsorbed and soluble FN and FNG is dependent on the wettability of materials since it was observed only on a hydrophilic and not on a hydrophobic model surface. ₃ integrin was involved in the process of cell attachment to adsorbed FNG, while the mechanism of FNG fibrillogenesis required the activity of the ₁ integrin. Studies of EC morphology showed the predominant peripheral organization of actin filaments and the formation of distinct leading and trailing cell edges suggesting a motile phenotype of cells when they are seeded on FNG. In summary, we concluded that adsorbed fibrinogen may enhance the motility of HUVEC and that soluble FNG requires FN matrix assembly to be organized in fibrilar structures.