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.     

Simultaneous Observation of the Lever Arm and Head Explains Myosin VI Dual Function

Myosin VI is an adenosine triphosphate (ATP)‐driven dimeric molecular motor that has dual function as a vesicle transporter and a cytoskeletal anchor. Recently, it was reported that myosin VI generates three types of steps by taking either a distant binding or adjacent binding state (noncanonical hand‐over‐hand step pathway). The adjacent binding state, in which both heads bind to an actin filament near one another, is unique to myosin VI and therefore may help explain its distinct features. However, detailed information of the adjacent binding state remains unclear. Here simultaneous observations of the head and tail domain during stepping are presented. These observations show that the lever arms tilt forward in the adjacent binding state. Furthermore, it is revealed that either head could take the subsequent step with equal probability from this state. Together with previous results, a comprehensive stepping scheme is proposed; it includes the tail domain motion to explain how myosin VI achieves its dual function.     

Processive behaviour of kinesin observed using micro-fabricated cantilevers

The mechanical characterization of biomolecular motors requires force sensors with sub-piconewton resolution. The coupling of a nanoscale motor to this type of microscale sensors introduces structural deformations in the motor according to the thermally activated degrees of freedom of the sensor. At present, no simple solution is available to reduce these effects. Here, we exploit the advantages of micro-fabricated cantilevers to produce a force sensor with essentially one degree of freedom and a spring constant of 0.03 pN nm(-1) for the study of the molecular motor protein kinesin-1. During processive runs, the cantilever constrains the movement of the cargo binding domain of kinesin in a straight line, parallel to the microtubule track, and excludes specific reaction coordinates such as cargo rotation. In these conditions, we measured a step size of 8.0 ± 0.4 nm and a maximal unloaded velocity of 820 ± 80 nm s(-1) at saturated adenosine triphosphate (ATP) concentration. We concluded that the motor does not need to rotate its tail as it moves through consecutive stepping cycles.     

Cytoplasmic streaming in plant cells: the role of wall slip

We present a computer simulation study, via lattice Boltzmann simulations, of a microscopic model for cytoplasmic streaming in algal cells such as those of Chara corallina. We modelled myosin motors tracking along actin lanes as spheres undergoing directed motion along fixed lines. The sphere dimension takes into account the fact that motors drag vesicles or other organelles, and, unlike previous work, we model the boundary close to which the motors move as walls with a finite slip layer. By using realistic parameter values for actin lane and myosin density, as well as for endoplasmic and vacuole viscosity and the slip layer close to the wall, we find that this simplified view, which does not rely on any coupling between motors, cytoplasm and vacuole other than that provided by viscous Stokes flow, is enough to account for the observed magnitude of streaming velocities in intracellular fluid in living plant cells.     

Dynamin II involves in cell migration and actin formation of NIH3T3 cells

It was previously reported that in Ras transformed NIH3T3 cells, dynamin II acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration. However, these results do not provide sufficient evidence of a relationship between dynamin II and the Ras signal transduction pathway leading to membrane ruffling and cell migration. The results showed that a dynamin II association with myosin II as a signaling molecule is involved in NIH3T3 cell migration through the Ras/PI3K signaling pathway, and is associated with the p85 subunit of PI3K. Confocal microscopy also revealed co-localization between dynamin II and paxillin after PDGF stimulation. In addition, immunofluorescence results showed that dynamin II was colocalized with the actin filament. After stimulating the NIH3T3 cells with PDGF and treating them with an actin inhibitor, such as Cytochalasin D, it was observed that dynamin II with the myosin II complex inhibited binding to the actin. Therefore, dynamin II is localized in focal adhesion when cell migration is triggered and binds to the actin filament component, suggesting that it is a good candidate nanomolecule to regulate the cell attachment and migration to the materials such as implants etc.     

Rear actomyosin contractility-driven directional cell migration in three-dimensional matrices: a mechano-chemical coupling mechanism

Cell migration is of vital importance in many biological processes, including organismal development, immune response and development of vascular diseases. For instance, migration of vascular smooth muscle cells from the media to intima is an essential part of the development of atherosclerosis and restenosis after stent deployment. While it is well characterized that cells use actin polymerization at the leading edge to propel themselves to move on two-dimensional substrates, the migration modes of cells in three-dimensional matrices relevant to in vivo environments remain unclear. Intracellular tension, which is created by myosin II activity, fulfils a vital role in regulating cell migration. We note that there is compelling evidence from theoretical and experimental work that myosin II accumulates at the cell rear, either isoform-dependent or -independent, leading to three-dimensional migration modes driven by posterior myosin II tension. The scenario is not limited to amoeboid migration, and it is also seen in mesenchymal migration in which a two-dimensional-like migration mode based on front protrusions is often expected, suggesting that there may exist universal underlying mechanisms. In this review, we aim to shed some light on how anisotropic myosin II localization induces cell motility in three-dimensional environments from a biomechanical view. We demonstrate an interesting mechanism where an interplay between mechanical myosin II recruitment and biochemical myosin II activation triggers directional migration in three-dimensional matrices. In the case of amoeboid three-dimensional migration, myosin II first accumulates at the cell rear to induce a slight polarization displayed as a uropod-like structure under the action of a tension-dependent mechanism. Subsequent biochemical signalling pathways initiate actomyosin contractility, producing traction forces on the adhesion system or creating prominent motile forces through blebbing activity, to drive cells to move. In mesenchymal three-dimensional migration, cells can also take advantage of the elastic properties of three-dimensional matrices to move. A minor myosin isoform, myosin IIB, is retained by relatively stiff three-dimensional matrices at the posterior side, then activated by signalling cascades, facilitating prominent cell polarization by establishing front–back polarity and creating cell rear. Myosin IIB initiates cell polarization and coordinates with the major isoform myosin IIA-assembled stress fibres, to power the directional migration of cells in the three-dimensional matrix.     

Control of a biomolecular motor-powered nanodevice with an engineered chemical switch

The biophysical and biochemical properties of motor proteins have been well-studied, but these motors also show promise as mechanical components in hybrid nano-engineered systems. The cytoplasmic F(1) fragment of the adenosine triphosphate synthase (F1-ATPase) can function as an ATP-fuelled rotary motor and has been integrated into self-assembled nanomechanical systems as a mechanical actuator. Here we present the rational design, construction and analysis of a mutant F1-ATPase motor containing a metal-binding site that functions as a zinc-dependent, reversible on/off switch. Repeated cycles of zinc addition and removal by chelation result in inhibition and restoration, respectively, of both ATP hydrolysis and motor rotation of the mutant, but not of the wild-type F1 fragment. These results demonstrate the ability to engineer chemical regulation into a biomolecular motor and represent a critical step towards controlling integrated nanomechanical devices at the single-molecule level.     

Nanometre resolution tracking of myosin-1b motility

The movement produced by a small number of myosin molecular motors was measured with nanometre precision using single-molecule fluorescence localisation methods. The positional precision of the measurements was sufficient to reveal fluctuations in sliding velocity due to stochastic interactions between individual myosin motors and the actin filament. Dependence of sliding velocity upon filament length was measured and fluctuations in velocity were quantified by autocorrelation analysis. Optical tweezers-based nanometry was used to measure the myosin-1b step-size directly. The 10 nm power-stroke and its duty cycle ratio were consistent with values derived from in vitro sliding assays.     

Cell-Mimic Directional Cargo Transportation in a Visible-Light-Activated Colloidal Motor/Lipid Tube System

Active tether and transportation of cargoes on cytoskeletal highway enabled by molecular motors is key for accurate delivery of vesicles and organelles in the complex intracellular environment. Here, a hybrid system composed of colloidal motors and self-assembled lipid tubes is designed to mimic the subcellular traffic system in living cells. The colloidal motors, composed of gold-coated hematite, display light-activated self-propulsion tunable by the light intensity and the concentration of hydrogen peroxide fuel. Importantly, the motors show light-switchable binding with lipid cargoes and attachment to the lipid tubes, whereby the latter act as the motor highways. Upon assembly, the colloidal motor/lipid tube system demonstrates directional delivery of lipid vesicles, emulating intracellular transportation. The assembly and function of the hybrid system are rationalized by a cooperative action of light-triggered electrophoretic and hydrodynamic effects, supported by finite element analysis. A synthetic analog of the biological protein motor/cytoskeletal filament system is realized for the manipulation and delivery of different matter at the microscale, which is expected to be a promising platform for various applications in materials science, nanotechnology, microfluidics, and synthetic biology.     

Engineering controllable bidirectional molecular motors based on myosin

Cytoskeletal motors drive the transport of organelles and molecular cargoes within cells and have potential applications in molecular detection and diagnostic devices. Engineering molecular motors with controllable properties will allow selective perturbation of mechanical processes in living cells and provide optimized device components for tasks such as molecular sorting and directed assembly. Biological motors have previously been modified by introducing activation/deactivation switches that respond to metal ions and other signals. Here, we show that myosin motors can be engineered to reversibly change their direction of motion in response to a calcium signal. Building on previous protein engineering studies and guided by a structural model for the redirected power stroke of myosin VI, we have constructed bidirectional myosins through the rigid recombination of structural modules. The performance of the motors was confirmed using gliding filament assays and single fluorophore tracking. Our strategy, in which external signals trigger changes in the geometry and mechanics of myosin lever arms, should make it possible to achieve spatiotemporal control over a range of motor properties including processivity, stride size and branchpoint turning.     

Elucidation of the separate roles of myosins IIA and IIB during neurite outgrowth, adhesion and retraction

The functions of nonmuscle myosin isoforms are key to an understanding of process outgrowth from nerve cells during animal development. Despite considerable structural similarity, myosin IIA and myosin IIB play distinct and complementary roles during the actin-based mechanisms of nerve process extension. An overview is given of evidence that implicates myosin IIB as the motor essential for nerve process outgrowth and myosin IIA both as the motor required to maintain cell adhesion to the substrate as well as the motor required to power retraction of the nerve cell process. These actions are placed in context within a model for nerve process extension that is consistent with many observations in the literature and provides testable hypotheses regarding possible roles for these nonmuscle myosin motors. The relevance of a fundamental understanding of the mechanisms underpinning nerve cell process extension to the application of nanotechnology in this area is also discussed.     

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.     

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.     

Bubble‐Enabled Underwater Motion of a Light‐Driven Motor

This work reports the photothermally driven horizontal motion of a motor as well as the suspending and vertical movements underwater. A motor is designed by attaching two polydimethylsiloxane‐coated oxidized copper foams (POCF) to the two opposite sides of an oxidized copper foam (OCF). When the hydrophobic POCF is immersed in water, it serves as both an air bubble trapper and a light‐to‐heat conversion center. As bubbles grow under photothermal heating, they provide lifting force and result in the revolving motion of the motor. With removal of light illumination, bubbles are cooled by the surrounding water and shrink, and the buoyance is lowered. The resultant force of gravitational force, buoyance, and fluid resistance drives the motor to move forward horizontally. Furthermore, the motors are utilized as oil collectors and oil/water separation is achieved successfully. To effectively control the suspending motion, a polydimethylsiloxane foam doped with carbon black (C‐foam) is designed under the photothermal principle. It is maintained at a certain position underwater by controlling the on/off of light. The vertical motion is also studied and utilized to generate electricity. It is expected that different types of underwater motion will open up new opportunities for various applications including drug delivery, collection of heavy oil underwater, and electricity generation.     

Modelling muscle motor conformations using low-angle X-ray diffraction

New results on myosin head organization using analysis of low-angle X-ray diffraction patterns from relaxed insect flight muscle (IFM) from a giant waterbug, building on previous studies of myosin filaments in bony fish skeletal muscle (BFM), show that the information content of such low-angle diffraction patterns is very high despite the 'crystallographically low' resolution limit (65 A) of the spacings of the Bragg diffraction peaks being used. This high information content and high structural sensitivity arises because: (i) the atomic structures of the domains of the myosin head are known from protein crystallography; and (ii) myosin head action appears to consist mainly of pivoting between domains which themselves stay rather constant in structure, thus (iii) the intensity distribution among diffraction peaks in even the low resolution diffraction pattern is highly determined by the high-resolution distribution of atomically modelled domain mass. A single model was selected among 5000+ computer-generated variations as giving the best fit for the 65 reflections recorded within the selected resolution limit of 65 A. Clear evidence for a change in shape of the insect flight muscle myosin motor between the resting (probably like the pre-powerstroke) state and the rigor state (considered to mimic the end-of-powerstroke conformation) has been obtained. This illustrates the power of the low-angle X-ray diffraction method. The implications of these new results about myosin motor action during muscle contraction are discussed.     

Dual‐Hole Excitons Activated Photoelectrolysis in Neutral Solution

II–VI semiconductors exhibit unique behaviors that can generate dual‐holes (“heavy and light”), but the application in photocatalysis is still missing. Herein, an empirical utilization of light/heavy holes in a hybrid metal cluster‐2D semiconductor nanoplatelets is reported. This hybrid material can boost the hole‐transfer at the surface and suppress the recombination. Different roles are enacted by light‐holes and heavy‐holes, in which the light‐holes with higher energy and mobility can facilitate the slow kinetics of water oxidation and further reduce the onset voltage, while the massive heavy‐holes can increase the resulting photocurrent by about five times, achieving a photocurrent of 2 mA cm^?2 at 1.23 V versus RHE under AM 1.5 G illumination in nonsacrificial neutral solution. These strategies can be the solutions for photoelectrolysis and be beneficial for sustainable development in solar conversion.     

Molecular motors: nature's nanomachines

Molecular motors are protein-based machines that convert chemical potential energy into mechanical work. This paper aims to introduce the non-specialist reader to molecular motors, in particular, acto-myosin, the prototype system for motor protein studies. These motors produce their driving force from changes in chemical potential arising directly from chemical reactions and are responsible for muscle contraction and a variety of other cell motilities.     

Computational modelling identifies the impact of subtle anatomical variations between amphibian and mammalian skeletal muscle on spatiotemporal calcium dynamics

The physical sites of calcium entry and exit in the skeletal muscle cell are distinct and highly organised in space. It was investigated whether the highly structured spatial organisation of sites of Ca² release, uptake and action in skeletal muscle cells substantially impacts the dynamics of cytosolic Ca² handling and thereby the physiology of the cell. Hereto, the spatiotemporal dynamics of the free calcium distribution in a fast-twitch (FT) muscle sarcomere was studied using a reaction?diffusion computational model for two genotypes with known anatomical differences. A computational model of a murine FT muscle sarcomere is developed, de novo including a closed calcium mass balance to simulate spatiotemporal high stimulation frequency calcium dynamics at 358°C. Literature data on high-frequency calcium dye measurements were used as a first step towards model validation. The murine and amphibian sarcomere models were phenotypically distinct to capture known differences in positions of troponin C, actin?myosin overlap and calcium release within the sarcomere between frog and mouse. The models predicted large calcium gradients throughout the myoplasm as well as differences in calcium concentrations near the mitochondria of frog and mouse. Furthermore, the predicted Ca² concentration was high at positions where Ca² has a regulatory function, close to the mitochondria and troponin C.