Visualizing Single V-ATPase Rotation Using Janus Nanoparticles.

Understanding the function of rotary molecular motors, such as the rotary ATPases, relies on our ability to visualize the single-molecule rotation. Traditional imaging methods often involve tagging those motors with nanoparticles (NPs) and inferring their rotation from translational motion of NPs. Here, we report an approach using "two-faced" Janus NPs to directly image the rotation of single V-ATPase from Enterococcus hirae, an ATP-driven rotary ion pump. By employing a 500-nm silica/gold Janus NP, we exploit its asymmetric optical contrast - silica core with a gold cap on one hemisphere - to achieve precise imaging of the unidirectional counter-clockwise rotation of single V-ATPase motors immobilized on surfaces. Despite the added viscous load from the relatively large Janus NP probe, our approach provides accurate torque measurements of single V-ATPase. This study underscores the advantages of Janus NPs over conventional probes, establishing them as powerful tools for single-molecule analysis of rotary molecular motors.

A very versatile molecular machine.

In this issue (J Bacteriol. 206: e0014024, https://doi.org/10.1128/jb.00140-24), Ridone and Baker describe hybrids between two 5:2 heteroheptameric ion-powered motors. Chimeras were constructed between stator units of a bacterial flagellum and ExbBD of the Ton outer-membrane transport system. Only one of the 14 hybrids supported swimming in Escherichia coli. Three additional residue changes at sites distant from the hybrid region enhanced motility. This work suggests that flagellar stator units and ExbBD share an ancestor that diverged during evolution to perform different tasks.

Constraint-based reasoning in cell biology: on the explanatory role of context.

Cell biologists, including those seeking molecular mechanistic explanations of cellular phenomena, frequently rely on experimental strategies focused on identifying the cellular context relevant to their investigations. We suggest that such practices can be understood as a guided decomposition strategy, where molecular explanations of phenomena are defined in relation to natural contextual (cell) boundaries. This "top-down" strategy contrasts with "bottom-up" reductionist approaches where well-defined molecular structures and activities are orphaned by their displacement from actual biological functions. We focus on the central role of microscopic imaging in cell biology to uncover possible constraints on the system. We show how identified constraints are used heuristically to limit possible mechanistic explanations to those that are biologically meaningful. Historical examples of this process described here include discovery of the mechanism of oxidative phosphorylation in mitochondria, molecular explanation of the first steps in protein secretion, and identification of molecular motors. We suggest that these instances are examples of a form of downward causation or, more specifically, constraining relations, where higher-level structures and variables delimit and enable lower-level system states. The guided decomposition strategy in our historical cases illustrates the irreducibility of experimentally identified constraints in explaining biological activities of cells. Rather than viewing decomposition and recomposition as separate epistemic activities, we contend that they need to be iteratively integrated to account for the ontological complexity of multi-level systems.

Coordination, cooperation, competition, crowding and congestion of molecular motors: Theoretical models and computer simulations.

Cytoskeletal motor proteins are biological nanomachines that convert chemical energy into mechanical work to carry out various functions such as cell division, cell motility, cargo transport, muscle contraction, beating of cilia and flagella, and ciliogenesis. Most of these processes are driven by the collective operation of several motors in the crowded viscous intracellular environment. Imaging and manipulation of the motors with powerful experimental probes have been complemented by mathematical analysis and computer simulations of the corresponding theoretical models. In this article, we illustrate some of the key theoretical approaches used to understand how coordination, cooperation and competition of multiple motors in the crowded intra-cellular environment drive the processes that are essential for biological function of a cell. In spite of the focus on theory, experimentalists will also find this article as an useful summary of the progress made so far in understanding multiple motor systems.

A Light-Powered Single-Stranded DNA Molecular Motor with Colour-Selective Single-Step Control.

Top-down control of small motion is possible through top-down controlled molecular motors in replacement of larger actuators like MEMS or NEMS (micro- or nano-electromechanical systems) in the current precision technology. Improving top-down control of molecular motors to every single step is desirable for this purpose, and also for synchronization of motor actions for amplified effects. Here we report a designed single-stranded DNA molecular motor powered by alternated ultraviolet and visible light for processive track-walking, with the two light colours each locking the motor in a full directional step to allow saturated driving but no overstepping. This novel nano-optomechanical driving mechanism pushes the top-down control of molecular motors down to every single step, thus providing a key technical capability to advance the molecular motor-based precision technology and also motor synchronization for amplified effects.

Towards the understanding of molecular motors and its relationship with local unfolding.

Molecular motors are machines essential for life since they convert chemical energy into mechanical work. However, the precise mechanism by which nucleotide binding, catalysis, or release of products is coupled to the work performed by the molecular motor is still not entirely clear. This is due, in part, to a lack of understanding of the role of force in the mechanical-structural processes involved in enzyme catalysis. From a mechanical perspective, one promising hypothesis is the Haldane-Pauling hypothesis which considers the idea that part of the enzymatic catalysis is strain-induced. It suggests that enzymes cannot be efficient catalysts if they are fully complementary to the substrates. Instead, they must exert strain on the substrate upon binding, using enzyme-substrate energy interaction (binding energy) to accelerate the reaction rate. A novel idea suggests that during catalysis, significant strain energy is built up, which is then released by a local unfolding/refolding event known as 'cracking'. Recent evidence has also shown that in catalytic reactions involving conformational changes, part of the heat released results in a center-of-mass acceleration of the enzyme, raising the possibility that the heat released by the reaction itself could affect the enzyme's integrity. Thus, it has been suggested that this released heat could promote or be linked to the cracking seen in proteins such as adenylate kinase (AK). We propose that the energy released as a consequence of ligand binding/catalysis is associated with the local unfolding/refolding events (cracking), and that this energy is capable of driving the mechanical work.

The Role of the Host Cytoskeleton in the Formation and Dynamics of Rotavirus Viroplasms.

Rotavirus (RV) replicates within viroplasms, membraneless electron-dense globular cytosolic inclusions with liquid-liquid phase properties. In these structures occur the virus transcription, replication, and packaging of the virus genome in newly assembled double-layered particles. The viroplasms are composed of virus proteins (NSP2, NSP5, NSP4, VP1, VP2, VP3, and VP6), single- and double-stranded virus RNAs, and host components such as microtubules, perilipin-1, and chaperonins. The formation, coalescence, maintenance, and perinuclear localization of viroplasms rely on their association with the cytoskeleton. A stabilized microtubule network involving microtubules and kinesin Eg5 and dynein molecular motors is associated with NSP5, NSP2, and VP2, facilitating dynamic processes such as viroplasm coalescence and perinuclear localization. Key post-translation modifications, particularly phosphorylation events of RV proteins NSP5 and NSP2, play pivotal roles in orchestrating these interactions. Actin filaments also contribute, triggering the formation of the viroplasms through the association of soluble cytosolic VP4 with actin and the molecular motor myosin. This review explores the evolving understanding of RV replication, emphasizing the host requirements essential for viroplasm formation and highlighting their dynamic interplay within the host cell.

Scalable Approach to Molecular Motor-Polymer Conjugates for Light-Driven Artificial Muscles.

The integration of molecular machines and motors into materials represents a promising avenue for creating dynamic and functional molecular systems, with potential applications in soft robotics or reconfigurable biomaterials. However, the development of truly scalable and controllable approaches for incorporating molecular motors into polymeric matrices has remained a challenge. Here, it is shown that light-driven molecular motors with sensitive photo-isomerizable double bonds can be converted into initiators for Cu-mediated controlled/living radical polymerization enabling the synthesis of star-shaped motor-polymer conjugates. This approach enables scalability, precise control over the molecular structure, block copolymer structures, and high-end group fidelity. Moreover, it is demonstrated that these materials can be crosslinked to form gels with quasi-ideal network topology, exhibiting light-triggered contraction. The influence of arm length and polymer structure is investigated, and the first molecular dynamics simulation framework to gain deeper insights into the contraction processes is developed. Leveraging this scalable methodology, the creation of bilayer soft robotic devices and cargo-lifting artificial muscles is showcased, highlighting the versatility and potential applications of this advanced polymer chemistry approach. It is anticipated that the integrated experimental and simulation framework will accelerate scalable approaches for active polymer materials based on molecular machines, opening up new horizons in materials science and bioscience.

Molecular Ratchets and Kinetic Asymmetry: Giving Chemistry Direction.

Over the last two decades ratchet mechanisms have transformed the understanding and design of stochastic molecular systems-biological, chemical and physical-in a move away from the mechanical macroscopic analogies that dominated thinking regarding molecular dynamics in the 1990s and early 2000s (e.g. pistons, springs, etc), to the more scale-relevant concepts that underpin out-of-equilibrium research in the molecular sciences today. Ratcheting has established molecular nanotechnology as a research frontier for energy transduction and metabolism, and has enabled the reverse engineering of biomolecular machinery, delivering insights into how molecules 'walk' and track-based synthesisers operate, how the acceleration of chemical reactions enables energy to be transduced by catalysts (both motor proteins and synthetic catalysts), and how dynamic systems can be driven away from equilibrium through catalysis. The recognition of molecular ratchet mechanisms in biology, and their invention in synthetic systems, is proving significant in areas as diverse as supramolecular chemistry, systems chemistry, dynamic covalent chemistry, DNA nanotechnology, polymer and materials science, molecular biology, heterogeneous catalysis, endergonic synthesis, the origin of life, and many other branches of chemical science. Put simply, ratchet mechanisms give chemistry direction. Kinetic asymmetry, the key feature of ratcheting, is the dynamic counterpart of structural asymmetry (i.e. chirality). Given the ubiquity of ratchet mechanisms in endergonic chemical processes in biology, and their significance for behaviour and function from systems to synthesis, it is surely just as fundamentally important. This Review charts the recognition, invention and development of molecular ratchets, focussing particularly on the role for which they were originally envisaged in chemistry, as design elements for molecular machinery. Different kinetically asymmetric systems are compared, and the consequences of their dynamic behaviour discussed. These archetypal examples demonstrate how chemical systems can be driven inexorably away from equilibrium, rather than relax towards it.

Light-Triggered Disassembly of Molecular Motor-based Supramolecular Polymers Revealed by High-Speed AFM.

Photoresponsive supramolecular polymers have a major potential for applications in responsive materials that are externally triggered by light with spatio-temporal control of their polymerisation state. While changes in macroscopic properties revealed the adaptive nature of these materials, it remains challenging to capture the dynamic depolymerisation process at the molecular level, which requires fast observation techniques combined with in situ irradiation. By implementing in situ UV illumination into a High-Speed Atomic Force Microscope (HS-AFM) setup, we have been able to capture the disassembly of a light-driven molecular motor-based supramolecular polymer. The real-time visualisation of the light-triggered disassembly process not only reveals cooperative depolymerisation, it also shows that this process continues after illumination is halted. Combining the data with cryo-electron microscopy and spectroscopy approaches, we obtain a molecular-level description of the motor-based polymer dynamics reminiscent of actin chain-end depolymerisation. Our detailed understanding of supramolecular depolymerisation will drive the development of future responsive polymer systems.

Highly Ordered Co-Assembly of Bisurea Functionalized Molecular Switches at the Solid-Liquid Interface.

Immobilization of stimulus-responsive systems on solid surfaces is beneficial for controlled signal transmission and adaptive behavior while allowing the characterization of the functional interface with high sensitivity and high spatial resolution. Positioning of the stimuli-responsive units with nanometer-scale precision across the adaptive surface remains one of the bottlenecks in the extraction of cooperative function. Nanoscale organization, cooperativity, and amplification remain key challenges in bridging the molecular and the macroscopic worlds. Here we report on the design, synthesis, and scanning tunneling microscopy (STM) characterization of overcrowded alkene photoswitches merged in self-assembled networks physisorbed at the solid-liquid interface. A detailed anchoring strategy that ensures appropriate orientation of the switches with respect to the solid surface through the use of bis-urea groups is presented. We implement a co-assembly strategy that enables the merging of the photoswitches within physisorbed monolayers of structurally similar 'spacer' molecules. The self-assembly of the individual components and the co-assemblies was examined in detail using (sub)molecular resolution STM which confirms the robust immobilization and controlled orientation of the photoswitches within the spacer monolayers. The experimental STM data is supported by detailed molecular mechanics (MM) simulations. Different designs of the switches and the spacers were investigated which allowed us to formulate guidelines that enable the precise organization of the photoswitches in crystalline physisorbed self-assembled molecular networks.

Structure and Function of Dynein's Non-Catalytic Subunits.

Dynein, an ancient microtubule-based motor protein, performs diverse cellular functions in nearly all eukaryotic cells, with the exception of land plants. It has evolved into three subfamilies-cytoplasmic dynein-1, cytoplasmic dynein-2, and axonemal dyneins-each differentiated by their cellular functions. These megadalton complexes consist of multiple subunits, with the heavy chain being the largest subunit that generates motion and force along microtubules by converting the chemical energy of ATP hydrolysis into mechanical work. Beyond this catalytic core, the functionality of dynein is significantly enhanced by numerous non-catalytic subunits. These subunits are integral to the complex, contributing to its stability, regulating its enzymatic activities, targeting it to specific cellular locations, and mediating its interactions with other cofactors. The diversity of non-catalytic subunits expands dynein's cellular roles, enabling it to perform critical tasks despite the conservation of its heavy chains. In this review, we discuss recent findings and insights regarding these non-catalytic subunits.

Molecular Motor-Driven Light-Controlled Logic-Gated K+ Channel for Cancer Cell Apoptosis.

Developing artificial ion transport systems, which process complicated information and step-wise regulate properties, is essential for deeply comprehending the subtle dynamic behaviors of natural channel proteins (NCPs). Here a photo-controlled logic-gated K+ channel based on single-chain random heteropolymers containing molecular motors, exhibiting multi-core processor-like properties to step-wise control ion transport is reported. Designed with oxygen, deoxygenation, and different wavelengths of light as input signals, complicated logical circuits comprising "YES", "AND", "OR" and "NOT" gate components are established. Implementing these logical circuits with K+ transport efficiencies as output signals, multiple state transitions including "ON", "Partially OFF" and "Totally OFF" in liposomes and cancer cells are realized, further causing step-wise anticancer treatments. Dramatic K+ efflux in the "ON" state (decrease by 50% within 7 min) significantly induces cancer cell apoptosis. This integrated logic-gated strategy will be expanded toward understanding the delicate mechanism underlying NCPs and treating cancer or other diseases is expected.

Microscopic interactions control a structural transition in active mixtures of microtubules and molecular motors.

Microtubules and molecular motors are essential components of the cellular cytoskeleton, driving fundamental processes in vivo, including chromosome segregation and cargo transport. When reconstituted in vitro, these cytoskeletal proteins serve as energy-consuming building blocks to study the self-organization of active matter. Cytoskeletal active gels display rich emergent dynamics, including extensile flows, locally contractile asters, and bulk contraction. However, it is unclear how the protein-protein interaction kinetics set their contractile or extensile nature. Here, we explore the origin of the transition from extensile bundles to contractile asters in a minimal reconstituted system composed of stabilized microtubules, depletant, adenosine 5'-triphosphate (ATP), and clusters of kinesin-1 motors. We show that the microtubule-binding and unbinding kinetics of highly processive motor clusters set their ability to end-accumulate, which can drive polarity sorting of the microtubules and aster formation. We further demonstrate that the microscopic time scale of end-accumulation sets the emergent time scale of aster formation. Finally, we show that biochemical regulation is insufficient to fully explain the transition as generic aligning interactions through depletion, cross-linking, or excluded volume interactions can drive bundle formation despite end-accumulating motors. The extensile-to-contractile transition is well captured by a simple self-assembly model where nematic and polar aligning interactions compete to form either bundles or asters. Starting from a five-dimensional organization phase space, we identify a single control parameter given by the ratio of the different component concentrations that dictates the material-scale organization. Overall, this work shows that the interplay of biochemical and mechanical tuning at the microscopic level controls the robust self-organization of active cytoskeletal materials.

Out-of-Equilibrium Mechanical Disruption of ß-Amyloid-Like Fibers using Light-Driven Molecular Motors.

Artificial molecular motors have the potential to generate mechanical work on their environment by producing autonomous unidirectional motions when supplied with a source of energy. However, the harnessing of this mechanical work to subsequently activate various endoenergetic processes that can be useful in materials science remains elusive. Here, it is shown that by integrating a light-driven rotary motor through hydrogen bonds in a ß-amyloid-like structure forming supramolecular hydrogels, the mechanical work generated during the constant rotation of the molecular machine under UV irradiation is sufficient to disrupt the ß-amyloid fibers and to trigger a gel-to-sol transition at macroscopic scale. This melting of the gel under UV irradiation occurs 25 °C below the temperature needed to melt it by solely using thermal activation. In the dark, a reversible sol-gel transition is observed as the system fully recovers its original microstructure, thus illustrating the possible access to new kinds of motorized materials that can be controlled by advanced out-of-equilibrium thermodynamics.

Light-Activated Synthetic Rotary Motors in Lipid Membranes Induce Shape Changes Through Membrane Expansion.

Membranes are the key structures to separate and spatially organize cellular systems. Their rich dynamics and transformations during the cell cycle are orchestrated by specific membrane-targeted molecular machineries, many of which operate through energy dissipation. Likewise, man-made light-activated molecular rotary motors have previously shown drastic effects on cellular systems, but their physical roles on and within lipid membranes remain largely unexplored. Here, the impact of rotary motors on well-defined biological membranes is systematically investigated. Notably, dramatic mechanical transformations are observed in these systems upon motor irradiation, indicative of motor-induced membrane expansion. The influence of several factors on this phenomenon is systematically explored, such as motor concentration and membrane composition., Membrane fluidity is found to play a crucial role in motor-induced deformations, while only minor contributions from local heating and singlet oxygen generation are observed. Most remarkably, the membrane area expansion under the influence of the motors continues as long as irradiation is maintained, and the system stays out-of-equilibrium. Overall, this research contributes to a comprehensive understanding of molecular motors interacting with biological membranes, elucidating the multifaceted factors that govern membrane responses and shape transitions in the presence of these remarkable molecular machines, thereby supporting their future applications in chemical biology.

A Semi-Markov Approach to Study a Group of Kinesin Motors.

We propose a general mathematical and computational approach to study cellular transport driven by a group of kinesin motors. It is a framework for multi-scale modeling that integrates kinetic models of single kinesin motors, including detachment and reattachment events, to study group behaviors of several motors. By formulating the problem as a semi-Markov process and applying a central limit theorem, asymptotic velocity and diffusivity can be readily calculated, which offers considerable computational advantage over Monte Carlo simulations in tasks such as parameter sensitivity analysis and model selection. We demonstrate the method with some examples. The importance of incorporating the intrinsic microscopic-level dynamics of individual motors is illustrated by showing how changes at the microscopic level propagate to the motor-cargo complex at a mesoscopic level. Particularly, we showcase an example in which changes in the second moment of single-motor characteristics gives rise to different first moment characteristics of the motor group.

Biosensing using antibody-modulated motility of actin filaments on myosin-coated surfaces.

Motor proteins, such as myosin and kinesin, are biological molecular motors involved in force generation and intracellular transport within living cells. The characteristics of molecular motors, i.e., their motility over long distances, their capacity of transporting cargoes, and their very efficient energy consumption, recommend them as potential operational elements of a new class of dynamic nano-devices, with potential applications in biosensing, analyte concentrators, and biocomputation. A possible design of a biosensor based on protein molecular motor comprises a surface with immobilized motors propelling cytoskeletal filaments, which are decorated with antibodies, presented as side-branches. Upon biomolecular recognition of these branches by secondary antibodies, the 'extensions' on the cytoskeletal filaments can achieve considerable lengths (longer than several diameters of the cytoskeletal filament carrier), thus geometrically impairing or halting motility. Because the filaments are several micrometers long, this sensing mechanism converts an event in the nanometer range, i.e., antibody-antigen sizes, into an event in the micrometer range: the visualization of the halting of motility of microns-long cytoskeletal filaments. Here we demonstrate the proof of concept of a sensing system comprising heavy-mero-myosin immobilized on surfaces propelling actin filaments decorated with actin antibodies, whose movement is halted upon the recognition with secondary anti-actin antibodies. Because antibodies to the actin-myosin system are involved in several rare diseases, the first possible application for such a device may be their prognosis and diagnosis. The results also provide insights into guidelines for designing highly sensitive and very fast biosensors powered by motor proteins.

On the use of thermal forces to probe kinesin's response to force.

The stepping dynamics of cytoskeletal motor proteins determines the dynamics of cargo transport. In its native cellular environment, a molecular motor is subject to forces from several sources including thermal forces and forces ensuing from the interaction with other motors bound to the same cargo. Understanding how the individual motors respond to these forces can allow us to predict how they move their cargo when part of a team. Here, using simulation, we show that details of how the kinesin motor responds to small assisting forces-which, at the moment, are not experimentally constrained-can lead to significant changes in cargo dynamics. Using different models of the force-dependent detachment probability of the kinesin motor leads to different predictions on the run-length of the cargo they carry. These differences emerge from the thermal forces acting on the cargo and transmitted to the motor through the motor tail that tethers the motor head to the microtubule. We show that these differences appear for cargo carried by individual motors or motor teams, and use our findings to propose the use of thermal forces as a probe of kinesin's response to force in this otherwise inaccessible force regime.

Thermodynamic and mechanistic analysis of the functional properties of dengue virus NS3 helicase.

The Dengue Virus (DENV) non-structural protein 3 (NS3) is a multi-functional protein critical in the viral life cycle. The DENV NS3 is comprised of a serine protease domain and a helicase domain. The helicase domain itself acts as a molecular motor, either translocating in a unidirectional manner along single-stranded RNA or unwinding double-stranded RNA, processes fueled by the hydrolysis of nucleoside triphosphates. In this brief review, we summarize our contributions and ongoing efforts to uncover the thermodynamic and mechanistic functional properties of the DENV NS3 as an NTPase and helicase.