Properties Of Single Motors Research Articles

Cargo surface fluidity can reduce inter-motor mechanical interference, promote load-sharing and enhance processivity in teams of molecular motors.

In cells, multiple molecular motors work together as teams to carry cargoes such as vesicles and organelles over long distances to their destinations by stepping along a network of cytoskeletal filaments. How motors that typically mechanically interfere with each other, work together as teams is unclear. Here we explored the possibility that purely physical mechanisms, such as cargo surface fluidity, may potentially enhance teamwork, both at the single motor and cargo level. To explore these mechanisms, we developed a three dimensional simulation of cargo transport along microtubules by teams of kinesin-1 motors. We accounted for cargo membrane fluidity by explicitly simulating the Brownian dynamics of motors on the cargo surface and considered both the load and ATP dependence of single motor functioning. Our simulations show that surface fluidity could lead to the reduction of negative mechanical interference between kinesins and enhanced load sharing thereby increasing the average duration of single motors on the filament. This, along with a cooperative increase in on-rates as more motors bind leads to enhanced collective processivity. At the cargo level, surface fluidity makes more motors available for binding, which can act synergistically with the above effects to further increase transport distances though this effect is significant only at low ATP or high motor density. Additionally, the fluid surface allows for the clustering of motors at a well defined location on the surface relative to the microtubule and the fluid-coupled motors can exert more collective force per motor against loads. Our work on understanding how teamwork arises in cargo-coupled motors allows us to connect single motor properties to overall transport, sheds new light on cellular processes, reconciles existing observations, encourages new experimental validation efforts and can also suggest new ways of improving the transport of artificial cargo powered by motor teams.

Force sharing and force generation by two teams of elastically coupled molecular motors

Many active cellular processes such as long-distance cargo transport, spindle organization, as well as flagellar and ciliary beating are driven by molecular motors. These motor proteins act collectively and typically work in small teams. One particularly interesting example is two teams of antagonistic motors that pull a common cargo into opposite directions, thereby generating mutual interaction forces. Important issues regarding such multiple motor systems are whether or not motors from the same team share their load equally, and how the collectively generated forces depend on the single motor properties. Here we address these questions by introducing a stochastic model for cargo transport by an arbitrary number of elastically coupled molecular motors. We determine the state space of this motor system and show that this space has a rather complex and nested structure, consisting of multiple activity states and a large number of elastic substates, even for the relatively small system of two identical motors working against one antagonistic motor. We focus on this latter case because it represents the simplest tug-of-war that involves force sharing between motors from the same team. We show that the most likely motor configuration is characterized by equal force sharing between identical motors and that the most likely separation of these motors corresponds to a single motor step. These likelihoods apply to different types of motors and to different elastic force potentials acting between the motors. Furthermore, these features are observed both in the steady state and during the initial build-up of elastic strains. The latter build-up is non-monotonic and exhibits a maximum at intermediate times, a striking consequence of mutual unbinding of the elastically coupled motors. Mutual strain-induced unbinding also reduces the magnitude of the collectively generated forces. Our computational approach is quite general and can be extended to other motor systems such as motor teams working against an optical trap or mixed teams of motors with different single motor properties.

Collective Dynamics of Cooperating Homodimeric and Heterotrimeric Kinesin-2 Motor Proteins Unraveled

Intracellular transport takes place in the crowded environment of the cell, densely packed with proteins, cytoskeletal filaments, vesicles and organelles. In most transport systems, multiple motors team up to transport a single cargo, resulting in collective motor behavior that cannot be explained only from single-motor properties. Intraflagellar transport (IFT) in C. elegans cilia is an example of functional coordination and cooperation of two motor proteins with distinct motility properties operating together in groups to transport a single cargo: homodimeric OSM-3 (fast and processive) and heterotrimeric Kinesin-II (slow and less processive). To study the mechanism of the collective dynamics of OSM-3 and kinesin-II in cilia, we use fluorescence microscopy and high-density in-vitro motility assays. Using an advanced analysis technique based on correlation of the fluorescence intensities, we extracted the quantitative motor parameters (such as motor density, velocity and average run length) from the image sequences in an automated way with a high level of precision. Our experiments and analyses show that OSM-3 and Kinesin-II motors are affected differently by the presence of the other motor. For example, OSM-3 velocity and run length are substantially reduced in the presence of the Kinesin-II. Numerical calculations based on TASEP-LK model (Totally Asymmetric Simple Exclusion Process-Langmuir Kinetics) underline the importance of intermolecular interactions, such as the change of the OSM-3 affinity to the microtubule dependent on the Kinesin-II density. Based on this approach, a general picture of collective dynamics of OSM-3 and Kinesin-II motor proteins in IFT is formulated.

Collective Effects of Motors and Microtubules Geometry in Gliding Assays

Sub-cellular transport over long distances appears is mainly achieved by molecular motor and cytoskeletal polymerization. However in processes such as the assembly of the spindle, growth and regeneration of cilia and neurites and cell polarization, the transport of cytoskeletal filaments itself becomes of vital importance. Here, we have examined the transport of radial arrays of microtubules in experiments with centrosomes in Xenopus meiotic egg extracts. We find the transport of such centrosomal asters towards chromatin to be directed. In a Langevin-dynamics simulation where we model explicit motor mechanics corresponding to dynein and MT dynamic instability, this transport still requires the symmetry of the system to be broken. Some of the biologically feasible mechanisms to achieve this involve modulating MT lengths, or localizing motors in a spatially inhomogeneous manner. Based on previous experimental evidence for gradients of regulatory proteins, we explore models of length asymmetry. On comparing the model predictions to mouse meiotic spindles however, we find such a model to be insufficient. As a result we invoke a motor asymmetry model.This work appears to suggest general principles in the transport of MT arrays, when their geometry deviates from linear filaments. We discuss some of the causes for a predominance of one mechanism over the other, based on single-motor properties and attempt to test the prediction results by examining MT aster transport in the presence of spatially symmetric motor distributions.

Teamwork in microtubule motors

Diverse cellular processes are driven by the collective force from multiple motor proteins. Disease-causing mutations cause aberrant function of motors, but the impact is observed at a cellular level and beyond, therefore necessitating an understanding of cell mechanics at the level of motor molecules. One way to do this is by measuring the force generated by ensembles of motors in vivo at single-motor resolution. This has been possible for microtubule motor teams that transport intracellular organelles, revealing unexpected differences between collective and single-molecule function. Here we review how the biophysical properties of single motors, and differences therein, may translate into collective motor function during organelle transport and perhaps in other processes outside transport.

Characterization of bidirectional molecular motor-assisted transport models

Intracellular cargos that are transported by groups of molecular motors often display bidirectional movement. This can be seen experimentally by tracking the trajectories of individual cargos in vivo. Typically, the cargo trajectories display many turning events that result from the stochastic nature of the involved motor processes. In this paper, we simulate cargo trajectories for different binding mechanisms. We introduce a series of statistical tools to analyze and quantitatively characterize these trajectories. As we demonstrate for specified single-motor properties, the novel statistical methods allow us to quantitatively distinguish between different models for bidirectional transport. In this way, the tools provide a quantitative connection between the statistical properties of the cargo trajectories and the molecular properties of the motor proteins. Such methods are also applicable to experimentally measured cargo trajectories and should be helpful in elucidating the mechanisms that lead to bidirectional transport.

Critical Motor Number for Fractional Steps of Cytoskeletal Filaments in Gliding Assays

In gliding assays, filaments are pulled by molecular motors that are immobilized on a solid surface. By varying the motor density on the surface, one can control the number of motors that pull simultaneously on a single filament. Here, such gliding assays are studied theoretically using Brownian (or Langevin) dynamics simulations and taking the local force balance between motors and filaments as well as the force-dependent velocity of the motors into account. We focus on the filament stepping dynamics and investigate how single motor properties such as stalk elasticity and step size determine the presence or absence of fractional steps of the filaments. We show that each gliding assay can be characterized by a critical motor number, . Because of thermal fluctuations, fractional filament steps are only detectable as long as . The corresponding fractional filament step size is where is the step size of a single motor. We first apply our computational approach to microtubules pulled by kinesin-1 motors. For elastic motor stalks that behave as linear springs with a zero rest length, the critical motor number is found to be , and the corresponding distributions of the filament step sizes are in good agreement with the available experimental data. In general, the critical motor number depends on the elastic stalk properties and is reduced to for linear springs with a nonzero rest length. Furthermore, is shown to depend quadratically on the motor step size . Therefore, gliding assays consisting of actin filaments and myosin-V are predicted to exhibit fractional filament steps up to motor number . Finally, we show that fractional filament steps are also detectable for a fixed average motor number as determined by the surface density (or coverage) of the motors on the substrate surface.

Casein kinase 2 reverses tail-independent inactivation of kinesin-1

Kinesin-1 is a plus-end microtubule-based motor, and defects in kinesin-based transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a head-tail interaction, but is believed to be active otherwise. Here we report a tail-independent inactivation of kinesin, reversible by the disease-relevant signaling protein, casein kinase 2 (CK2). The majority of initially active kinesin (native or tail-less) loses its ability to interact with microtubules in vitro, and CK2 reverses this inactivation (~ 4-fold) without altering kinesin’s single motor properties. This activation pathway does not require motor phosphorylation, and is independent of head-tail auto-inhibition. In cultured mammalian cells, reducing CK2 expression, but not its kinase activity, decreases the force required to stall lipid droplet transport, consistent with a decreased number of active kinesin motors. Our results provide the first direct evidence of a protein kinase up-regulating kinesin-based transport, and suggest a novel pathway for regulating the activity of cargo-bound kinesin.

2011 ASCB Annual Meeting abstracts

Kinesin-1 is a plus-end microtubule-based motor, and defects in kinesin-based transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a direct head-tail interaction, but is believed to be active otherwise. Here we report a tail-independent inactivation of kinesin, reversible by the disease-relevant signaling protein, casein kinase 2 (CK2). The majority of initially active kinesin (native or tail-less) loses its ability to bind/interact with microtubules in vitro, and CK2 reverses this inactivation (~ 4-fold) without altering kinesin’s single motor properties. This activation pathway does not require motor phosphorylation, and is independent of head-tail autoinhibition. In cultured mammalian cells, reducing CK2 expression, but not kinase activity, decreases the force required to stall lipid droplet transport, consistent with a reduction in the number of active motors. These results provide the first direct evidence of a protein kinase up-regulating kinesin-based transport, and suggest a novel pathway for regulating the activity of cargo-bound kinesin.

A Micromechanical Model of Cargo Transport by Multiple Microtubule Motors

Although the motions of many sub-cellular cargos are known to be driven by teams of microtubule motor proteins, understanding the role collective motor function plays in intracellular transport has been difficult to characterize. One confounding issue is that a team of molecular motors can adopt a spectrum of microtubule-associated configurations, depending on the number of bound motors and their organization on the microtubule. Since each microstate configuration can confer different mechanical and dynamic properties to a cargo, understanding how molecular motors function collectively ultimately requires knowledge of their relative contribution to cargo motility. To address this issue, we have developed methods to examine the load-dependent transport properties of structurally-defined multiple motor systems composed of two kinesin-1 molecules. Herein, we describe a discrete microstate transition-rate model of two-kinesin mechanics that can explain several unexpected behaviors observed in our assays. While this model accounts for a comprehensive spectrum of geometric arrangements of motors between a cargo and the microtubule, transition rates between microstate configurations are almost exclusively parameterized using data from single-kinesin optical trapping measurements. Overall, our model shows strong agreement with our data and recapitulates the central experimental finding that configurations where two kinesins both assume a portion of the applied load are rare and short-lived, causing two kinesins to exhibit negative cooperativity and properties that resemble the action of a single motor molecule. The bottom-up construction of our model also allows us to explore the effects of changes in single motor properties such as compliance, force-velocity relationship, and forward/backward stepping behavior on the dynamics of the system as a whole. This now provides a foundation for analyses of transport behaviors produced by more complex systems composed of different numbers, types and geometric arrangements of motors.

Casein Kinase 2 Reverses Tail-Independent Inhibition of Kinesin-1

Kinesin-1 is a plus-end microtubule-based molecular motor, and defects in kinesin transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a direct head-tail interaction, but is believed to be active otherwise. In contrast, this study uncovers a fast but reversible inhibition distinct from the canonical auto-inhibition pathway. The majority of the initially active kinesin (full-length or tail-less) loses its ability to bind/interact with microtubule, and Casein Kinase 2 (CK2) reverses this inactivation (up to 4-fold) without altering kinesin's single motor properties. Motor phosphorylation is not required for this CK2 -mediated kinesin activation. In cultured mammalian cells, knockdown of CK2 level, but not kinase activity, was sufficient to decrease the force required to stall lipid droplet transport, consistent with a reduction in the number of active motors. We propose that CK2 forms a positive regulating complex with the motor. This study provides the first direct evidence of a protein kinase positively regulating kinesin-transport, and uncovers a pathway whereby inactive cargo-bound kinesin can be activated.

Direct Measurement of the Force-Velocity Relationship for Multiple Kinesin-1 Motors by Magnetic Tweezers

Transport of intracellular cargo is known to be achieved by the concerted operation of multiple motor proteins. However, force generation by multiple motors remains a matter of debate even though the load-bearing properties of single motors have intensively been characterized by various in vitro assays. Here, we report a novel assay to study cooperative transport in the presence of external load. In particular, we designed a magnetic-tweezers setup that is capable of exerting horizontal forces of up to 100 pN on superparamagnetic beads that are attached to microtubules gliding on a surface coated with kinesin-1 motors.

Stochastic kinetics of ribosomes: Single motor properties and collective behavior

Syntheses of protein molecules in a cell are carried out by ribosomes. A ribosome can be regarded as a molecular motor which utilizes the input chemical energy to move on a messenger RNA (mRNA) track that also serves as a template for the polymerization of the corresponding protein. The forward movement, however, is characterized by an alternating sequence of translocation and pause. Using a quantitative model, which captures the mechanochemical cycle of an individual ribosome, we derive an exact analytical expression for the distribution of its dwell times at the successive positions on the mRNA track. Inverse of the average dwell time satisfies a "Michaelis-Menten-type" equation and is consistent with the general formula for the average velocity of a molecular motor with an unbranched mechanochemical cycle. Extending this formula appropriately, we also derive the exact force-velocity relation for a ribosome. Often many ribosomes simultaneously move on the same mRNA track, while each synthesizes a copy of the same protein. We extend the model of a single ribosome by incorporating steric exclusion of different individuals on the same track. We draw the phase diagram of this model of ribosome traffic in three-dimensional spaces spanned by experimentally controllable parameters. We suggest new experimental tests of our theoretical predictions.