Single-molecule studies of nucleic acid motors

Nucleic acid motors comprise a variety of structurally, mechanistically and functionally very different enzymes. These motor proteins have in common the ability to directionally move DNA or RNA, or to move along DNA or RNA using a chemical energy source such as ATP. Recently, it became possible to study the action of a single motor on single DNA or RNA molecules in real time; this has provided unprecedented insight into the behavior and mechanism of these motors. As a result, the past few years have witnessed an enormous increase in such single-molecule studies of a variety of different motor systems. Particular highlights have included the investigation of the sequence-dependent behavior and helical tracking of motors, and the attainment of the ultimate (i.e. single base pair) resolution, which enables the detection of individual single base motor steps.     

Nonprocessive Motor Dynamics at the Microtubule Membrane Tube Interface

Key cellular processes such as cell division, membrane compartmentalization, and intracellular transport rely on motor proteins. Motors have been studied in detail on the single motor level such that information on their step size, stall force, average run length, and processivity are well known. However, in vivo, motors often work together, so that the question of their collective coordination has raised great interest. Here, we specifically attach motors to giant vesicles and examine collective motor dynamics during membrane tube formation. Image correlation spectroscopy reveals directed motion as processive motors walk at typical speeds (≤500 nm/s) along an underlying microtubule and accumulate at the tip of the growing membrane tube. In contrast, nonprocessive motors exhibit purely diffusive behavior, decorating the entire length of a microtubule lattice with diffusion constants at least 1000 times smaller than a freely-diffusing lipid-motor complex in a lipid bilayer (1 μm²/s); fluorescence recovery after photobleaching experiments confirm the presence of the slower-moving motor population at the microtubule-membrane tube interface. We suggest that nonprocessive motors dynamically bind and unbind to maintain a continuous interaction with the microtubule. This dynamic and continuous interaction is likely necessary for nonprocessive motors to mediate bidirectional membrane tube dynamics reported previously.     

Stepping and Crowding of Molecular Motors: Statistical Kinetics from an Exclusion Process Perspective

Motor enzymes are remarkable molecular machines that use the energy derived from the hydrolysis of a nucleoside triphosphate to generate mechanical movement, achieved through different steps that constitute their kinetic cycle. These macromolecules, nowadays investigated with advanced experimental techniques to unveil their molecular mechanisms and the properties of their kinetic cycles, are implicated in many biological processes, ranging from biopolymerization (e.g., RNA polymerases and ribosomes) to intracellular transport (motor proteins such as kinesins or dyneins). Although the kinetics of individual motors is well studied on both theoretical and experimental grounds, the repercussions of their stepping cycle on the collective dynamics still remains unclear. Advances in this direction will improve our comprehension of transport process in the natural intracellular medium, where processive motor enzymes might operate in crowded conditions. In this work, we therefore extend contemporary statistical kinetic analysis to study collective transport phenomena of motors in terms of lattice gas models belonging to the exclusion process class. Via numerical simulations, we show how to interpret and use the randomness calculated from single particle trajectories in crowded conditions. Importantly, we also show that time fluctuations and non-Poissonian behavior are intrinsically related to spatial correlations and the emergence of large, but finite, clusters of comoving motors. The properties unveiled by our analysis have important biological implications on the collective transport characteristics of processive motor enzymes in crowded conditions.     

Stepping and Crowding of Molecular Motors: Statistical Kinetics from an Exclusion Process Perspective

Motor enzymes are remarkable molecular machines that use the energy derived from the hydrolysis of a nucleoside triphosphate to generate mechanical movement, achieved through different steps that constitute their kinetic cycle. These macromolecules, nowadays investigated with advanced experimental techniques to unveil their molecular mechanisms and the properties of their kinetic cycles, are implicated in many biological processes, ranging from biopolymerization (e.g., RNA polymerases and ribosomes) to intracellular transport (motor proteins such as kinesins or dyneins). Although the kinetics of individual motors is well studied on both theoretical and experimental grounds, the repercussions of their stepping cycle on the collective dynamics still remains unclear. Advances in this direction will improve our comprehension of transport process in the natural intracellular medium, where processive motor enzymes might operate in crowded conditions. In this work, we therefore extend contemporary statistical kinetic analysis to study collective transport phenomena of motors in terms of lattice gas models belonging to the exclusion process class. Via numerical simulations, we show how to interpret and use the randomness calculated from single particle trajectories in crowded conditions. Importantly, we also show that time fluctuations and non-Poissonian behavior are intrinsically related to spatial correlations and the emergence of large, but finite, clusters of comoving motors. The properties unveiled by our analysis have important biological implications on the collective transport characteristics of processive motor enzymes in crowded conditions.     

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

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

Bidirectional transport along microtubules

Active transport by microtubule motors has a plethora of crucial roles in eukaryotic cells. Organelles often move bidirectionally, employing both plus-end and minus-end directed motors. Bidirectional motion is widespread and may allow dynamic regulation, error correction and the establishment of polarized organelle distributions. Emerging evidence suggests that motors for both directions are simultaneously present on cellular 'cargo', but that their activity is coordinated so that when plus-end motors are active, minus-end motors are not, and vice versa. Both the dynein cofactor dynactin and the Klarsicht (Klar) protein appear to be important for such coordination. The direction of net transport depends on the balance between plus-end directed and minus-end directed motion. In several model systems, factors crucial for setting this balance have now been identified, setting the stage for a molecular dissection of the underlying regulatory mechanisms. These analyses will likely provide insight into motor cooperation in general.     

Computational identification of protein-protein interactions in rice based on the predicted rice interactome network

Plant protein-protein interaction networks have not been identified by large-scale experiments. In order to better understand the protein interactions in rice, the Predicted Rice Interactome Network (PRIN; http://bis.zju.edu.cn/prin/) presented 76,585 predicted interactions involving 5,049 rice proteins. After mapping genomic features of rice (GO annotation, subcellular localization prediction, and gene expression), we found that a well-annotated and biologically significant network is rich enough to capture many significant functional linkages within higher-order biological systems, such as pathways and biological processes. Furthermore, we took MADS-box domain-containing proteins and circadian rhythm signaling pathways as examples to demonstrate that functional protein complexes and biological pathways could be effectively expanded in our predicted network. The expanded molecular network in PRIN has considerably improved the capability of these analyses to integrate existing knowledge and provide novel insights into the function and coordination of genes and gene networks.     

A method for multiprotein assembly in cells reveals independent action of kinesins in complex

Teams of processive molecular motors are critical for intracellular transport and organization, yet coordination between motors remains poorly understood. Here, we develop a system using protein components to generate assemblies of defined spacing and composition inside cells. This system is applicable to studying macromolecular complexes in the context of cell signaling, motility, and intracellular trafficking. We use the system to study the emergent behavior of kinesin motors in teams. We find that two kinesin motors in complex act independently (do not help or hinder each other) and can alternate their activities. For complexes containing a slow kinesin-1 and fast kinesin-3 motor, the slow motor dominates motility in vitro but the fast motor can dominate on certain subpopulations of microtubules in cells. Both motors showed dynamic interactions with the complex, suggesting that motor–cargo linkages are sensitive to forces applied by the motors. We conclude that kinesin motors in complex act independently in a manner regulated by the microtubule track.     

Bidirectional transport of organelles: unity and struggle of opposing motors

Bidirectional transport along microtubules is ensured by opposing motor proteins: cytoplasmic dynein that drives cargo to the minus-ends and various kinesins that generally move to the plus-ends of microtubules. Regulation of motor proteins that are simultaneously bound to the same organelle is required to maintain directional transport and prevent pausing of cargo pulled away by motors of opposite polarity. Debates of the recent decade have been focused on two possible mechanisms of such regulation: (i) coordination, which implies that only one type of motors is active at a given time, and (ii) tug-of-war, which assumes that both motors are active at the same time and that direction of transport depends on the outcome of motor's confrontation. The initial idea of coordination has been challenged by observations of simultaneous activity of plus- and minus-end-directed motors applied to the same cargo. Analysis of the available data indicates that coordination and tug-of-war theories rather complement than contradict each other: cargo interacts with two teams of active motors, the resulting direction and the winner team are determined by coordination complexes, but the activity of the loser team is never completely inhibited and remains at some background level. Such persisting activity might enhance the overall efficiency of transport by increasing processivity or helping to overcome the obstacles on microtubule track.     

Viscoelasticity of living materials: mechanics and chemistry of muscle as an active macromolecular system

At the molecular and cellular level, mechanics and chemistry are two aspects of the same macromolecular system. We present a bottom-up approach to such systems based on Kramers' diffusion theory of chemical reactions, the theory of polymer dynamics, and the recently developed models for molecular motors. Using muscle as an example, we develop a viscoelastic theory of muscle in terms of an simple equation for single motor protein movement. Both A.V. Hill's contractile component and A.F. Huxley's equation of sliding-filament motion are shown to be special cases of the general viscoelastic theory of the active material. Some disparity between the mechanical and the chemical views of cross-bridges and motor proteins are noted, and a duality between force and energy in discrete states and transitions of macromolecular systems is discussed.     

From protein networks to biological systems

A system-level understanding of any biological process requires a map of the relationships among the various molecules involved. Technologies to detect and predict protein interactions have begun to produce very large maps of protein interactions, some including most of an organism's proteins. These maps can be used to study how proteins work together to form molecular machines and regulatory pathways. They also provide a framework for constructing predictive models of how information and energy flow through biological networks. In many respects, protein interaction maps are an entrée into systems biology.     

Pushing, pulling and trapping--modes of motor protein supported protein translocation

Protein translocation across the cellular membranes is an ubiquitous and crucial activity of cells. This process is mediated by translocases that consist of a protein conducting channel and an associated motor protein. Motor proteins interact with protein substrates and utilize the free energy of ATP binding and hydrolysis for protein unfolding, translocation and unbinding. Since motor proteins are found either at the cis- or trans-side of the membrane, different mechanisms for translocation have been proposed. In the Power stroke model, cis-acting motors are thought to push, while trans-motors pull on the substrate protein during translocation. In the Brownian ratchet model, translocation occurs by diffusion of the unfolded polypeptide through the translocation pore while directionality is achieved by trapping and refolding. Recent insights in the structure and function of the molecular motors suggest that different mechanisms can be employed simultaneously.     

Membrane-associated nanomotors for macromolecular transport

Nature has endowed cells with powerful nanomotors to accomplish intricate mechanical tasks, such as the macromolecular transport across membranes occurring in cell division, bacterial conjugation, and in a wide variety of secretion systems. These biological motors couple the chemical energy provided by ATP hydrolysis to the mechanical work needed to transport DNA and/or protein effectors. Here, we review what is known about the molecular mechanisms of these membrane-associated machines. Sequence and structural comparison between these ATPases reveal that they share a similar motor domain, suggesting a common evolutionary ancestor. Learning how these machines operate will lead the design of nanotechnology devices with unique applications in medicine and engineering.     

Thermodynamics and kinetics of a molecular motor ensemble

If, contrary to conventional models of muscle, it is assumed that molecular forces equilibrate among rather than within molecular motors, an equation of state and an expression for energy output can be obtained for a near-equilibrium, coworking ensemble of molecular motors. These equations predict clear, testable relationships between motor structure, motor biochemistry, and ensemble motor function, and we discuss these relationships in the context of various experimental studies. In this model, net work by molecular motors is performed with the relaxation of a near-equilibrium intermediate step in a motor-catalyzed reaction. The free energy available for work is localized to this step, and the rate at which this free energy is transferred to work is accelerated by the free energy of a motor-catalyzed reaction. This thermodynamic model implicitly deals with a motile cell system as a dynamic network (not a rigid lattice) of molecular motors within which the mechanochemistry of one motor influences and is influenced by the mechanochemistry of other motors in the ensemble.     

Mechanical Operation and Intersubunit Coordination of Ring-Shaped Molecular Motors: Insights from Single-Molecule Studies

Ring NTPases represent a large and diverse group of proteins that couple their nucleotide hydrolysis activity to a mechanical task involving force generation and some type of transport process in the cell. Because of their shape, these enzymes often operate as gates that separate distinct cellular compartments to control and regulate the passage of chemical species across them. In this manner, ions and small molecules are moved across membranes, biopolymer substrates are segregated between cells or moved into confined spaces, double-stranded nucleic acids are separated into single strands to provide access to the genetic information, and polypeptides are unfolded and processed for recycling. Here we review the recent advances in the characterization of these motors using single-molecule manipulation and detection approaches. We describe the various mechanisms by which ring motors convert chemical energy to mechanical force or torque and coordinate the activities of individual subunits that constitute the ring. We also examine how single-molecule studies have contributed to a better understanding of the structural elements involved in motor-substrate interaction, mechanochemical coupling, and intersubunit coordination. Finally, we discuss how these molecular motors tailor their operation—often through regulation by other cofactors—to suit their unique biological functions.     

Motor Force Homeostasis in Skeletal Muscle Contraction

In active biological contractile processes such as skeletal muscle contraction, cellular mitosis, and neuronal growth, an interesting common observation is that multiple motors can perform coordinated and synchronous actions, whereas individual myosin motors appear to randomly attach to and detach from actin filaments. Recent experiment has demonstrated that, during skeletal muscle shortening at a wide range of velocities, individual myosin motors maintain a force of ∼6 pN during a working stroke. To understand how such force-homeostasis can be so precisely regulated in an apparently chaotic system, here we develop a molecular model within a coupled stochastic-elastic theoretical framework. The model reveals that the unique force-stretch relation of myosin motor and the stochastic behavior of actin-myosin binding cause the average number of working motors to increase in linear proportion to the filament load, so that the force on each working motor is regulated at ∼6 pN, in excellent agreement with experiment. This study suggests that it might be a general principle to use catch bonds together with a force-stretch relation similar to that of myosin motors to regulate force homeostasis in many biological processes.     

Mechanical Operation and Intersubunit Coordination of Ring-Shaped Molecular Motors: Insights from Single-Molecule Studies

Ring NTPases represent a large and diverse group of proteins that couple their nucleotide hydrolysis activity to a mechanical task involving force generation and some type of transport process in the cell. Because of their shape, these enzymes often operate as gates that separate distinct cellular compartments to control and regulate the passage of chemical species across them. In this manner, ions and small molecules are moved across membranes, biopolymer substrates are segregated between cells or moved into confined spaces, double-stranded nucleic acids are separated into single strands to provide access to the genetic information, and polypeptides are unfolded and processed for recycling. Here we review the recent advances in the characterization of these motors using single-molecule manipulation and detection approaches. We describe the various mechanisms by which ring motors convert chemical energy to mechanical force or torque and coordinate the activities of individual subunits that constitute the ring. We also examine how single-molecule studies have contributed to a better understanding of the structural elements involved in motor-substrate interaction, mechanochemical coupling, and intersubunit coordination. Finally, we discuss how these molecular motors tailor their operation—often through regulation by other cofactors—to suit their unique biological functions.     

Self-organized density patterns of molecular motors in arrays of cytoskeletal filaments

The stationary states of systems with many molecular motors are studied theoretically for uniaxial and centered (asterlike) arrangements of cytoskeletal filaments using Monte Carlo simulations and a two-state model. Mutual exclusion of motors from binding sites of the filaments is taken into account. For small overall motor concentration, the density profiles are exponential and algebraic in uniaxial and centered filament systems, respectively. For uniaxial systems, exclusion leads to the coexistence of regions of high and low densities of bound motors corresponding to motor traffic jams, which grow upon increasing the overall motor concentration. These jams are insensitive to the motor behavior at the end of the filament. In centered systems, traffic jams remain small and an increase in the motor concentration leads to a flattening of the profile if the motors move inwards, and to the buildup of a concentration maximum in the center of the aster if motors move outwards. In addition to motor density patterns, we also determine the corresponding patterns of the motor current.     

Rotary protein motors

Three protein motors have been unambiguously identified as rotary engines: the bacterial flagellar motor and the two motors that constitute ATP synthase (F(0)F(1) ATPase). Of these, the bacterial flagellar motor and F(0) motors derive their energy from a transmembrane ion-motive force, whereas the F(1) motor is driven by ATP hydrolysis. Here, we review the current understanding of how these protein motors convert their energy supply into a rotary torque.     

The mechanochemistry of integrated motor protein complexes

The assembly of molecular motor proteins into multi-unit protein complexes plays an important role in determining the intracellular transport and trafficking properties of many subcellular commodities. Yet, it is not known how proteins within these complexes interact and function collectively. Considering the established ties between motor transport and diseases, it has become increasingly important to investigate the functional properties of these essential transport ‘motifs’. Doing so requires that the composite motile and force-generating properties of multi-unit motor assemblies are characterized. However, such analyses are typically confounded by a lack of understanding of the links between the structural and mechanical properties of many motor complexes. New experimental challenges also emerge when one examines motor cooperation. Distributions in the mechanical microstates available to motor ensembles must be examined in order to fully understand the transport behavior of multi-motor complexes. Furthermore, mechanisms by which motors communicate must be explored to determine whether motor groups can move cargo together in a truly cooperative fashion. Resolving these issues requires the development of experimental methods that allow the dynamics of complex systems of transport proteins to be monitored with the same precision available to single-molecule biophysical assays. Herein, we discuss key fundamental principles governing the function of motor complexes and their relation to mechanisms that regulate intracellular cargo transport. We also outline new experimental strategies to resolve these essential features of intracellular transport.