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.     

Measuring Molecular Motor Forces In Vivo: Implications for Tug-of-War Models of Bidirectional Transport

Molecular motor proteins use the energy released from ATP hydrolysis to generate force and haul cargoes along cytoskeletal filaments. Thus, measuring the force motors generate amounts to directly probing their function. We report on optical trapping methodology capable of making precise in vivo stall-force measurements of individual cargoes hauled by molecular motors in their native environment. Despite routine measurement of motor forces in vitro, performing and calibrating such measurements in vivo has been challenging. We describe the methodology recently developed to overcome these difficulties, and used to measure stall forces of both kinesin-1 and cytoplasmic dynein-driven lipid droplets in Drosophila embryos. Critically, by measuring the cargo dynamics in the optical trap, we find that there is memory: it is more likely for a cargo to resume motion in the same direction—rather than reverse direction—after the motors transporting it detach from the microtubule under the force of the optical trap. This suggests that only motors of one polarity are active on the cargo at any instant in time and is not consistent with the tug-of-war models of bidirectional transport where both polarity motors can bind the microtubules at all times. We further use the optical trap to measure in vivo the detachment rates from microtubules of kinesin-1 and dynein-driven lipid droplets. Unlike what is commonly assumed, we find that dynein’s but not kinesin’s detachment time in vivo increases with opposing load. This suggests that dynein’s interaction with microtubules behaves like a catch bond.     

Assessing the Impact of Electrostatic Drag on Processive Molecular Motor Transport

The bidirectional movement of intracellular cargo is usually described as a tug-of-war among opposite-directed families of molecular motors. While tug-of-war models have enjoyed some success, recent evidence suggests underlying motor interactions are more complex than previously understood. For example, these tug-of-war models fail to predict the counterintuitive phenomenon that inhibiting one family of motors can decrease the functionality of opposite-directed transport. In this paper, we use a stochastic differential equations modeling framework to explore one proposed physical mechanism, called microtubule tethering, that could play a role in this “co-dependence” among antagonistic motors. This hypothesis includes the possibility of a trade-off: weakly bound trailing molecular motors can serve as tethers for cargoes and processing motors, thereby enhancing motor–cargo run lengths along microtubules; however, this introduces a cost of processing at a lower mean velocity. By computing the small- and large-time mean-squared displacement of our theoretical model and comparing our results to experimental observations of dynein and its “helper protein” dynactin, we find some supporting evidence for microtubule tethering interactions. We extrapolate these findings to predict how dynein–dynactin might interact with the opposite-directed kinesin motors and introduce a criterion for when the trade-off is beneficial in simple systems.     

Finite-particle tracking reveals submicroscopic-size changes of mitochondria during transport in mitral cell dendrites

The mechanisms of molecular motor regulation during bidirectional organelle transport are still uncertain. There is, for instance, the unsettled question of whether opposing motor proteins can be engaged in a tug-of-war. Clearly, any non-synchronous activation of the molecular motors of one cargo can principally lead to changes in the cargo's shape and size; the cargo's size and shape parameters would certainly be observables of such changes. We therefore set out to measure position, shape and size parameters of fluorescent mitochondria (during their transport) in dendrites of cultured neurons using a finite-particle tracking algorithm. Our data clearly show transport-related submicroscopic-size changes of mitochondria. The observed displacements of the mitochondrial front and rear ends are consistent with a model in which microtubule plus- and minus-end-directed motor proteins or motors of the same type but moving along anti-parallel microtubules are often out-of-phase and occasionally engaged in a tug-of-war. Mostly the leading and trailing ends of mitochondria undergo similar characteristic movements but with a substantial time delay between the displacements of both ends, a feature reminiscent of an inchworm-like motility mechanism. More generally, we demonstrate that observing the position, shape and size of actively transported finite objects such as mitochondria can yield information on organelle transport that is generally not accessible by tracking the organelles' centroid alone.     

Load‐dependent detachment kinetics plays a key role in bidirectional cargo transport by kinesin and dynein

Bidirectional cargo transport along microtubules is carried out by opposing teams of kinesin and dynein motors. Despite considerable study, the factors that determine whether these competing teams achieve net anterograde or retrograde transport in cells remain unclear. The goal of this work is to use stochastic simulations of bidirectional transport to determine the motor properties that most strongly determine overall cargo velocity and directionality. Simulations were carried out based on published optical tweezer characterization of kinesin‐1 and kinesin‐2, and for available data for cytoplasmic dynein and the dynein‐dynactin‐BicD2 (DDB) complex. By varying dynein parameters and analyzing cargo trajectories, we find that net cargo transport is predicted to depend minimally on the dynein stall force, but strongly on dynein load‐dependent detachment kinetics. In simulations, dynein is dominated by kinesin‐1, but DDB and kinesin‐1 are evenly matched, recapitulating recent experimental work. Kinesin‐2 competes less well against dynein and DDB, and overall, load‐dependent motor detachment is the property that most determines a motor's ability to compete in bidirectional transport. It follows that the most effective intracellular regulators of bidirectional transport are predicted to be those that alter motor detachment kinetics rather than motor velocity or stall force.      

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.     

Modeling myosin Va liposome transport through actin filament networks reveals a percolation threshold that modulates transport properties

Myosin Va (myoVa) motors transport membrane-bound cargo through three-dimensional, intracellular actin filament networks. We developed a coarse-grained, in silico model to predict how actin filament density (3-800 filaments) within a randomly oriented actin network affects fluid-like liposome (350 nm vs. 1750 nm) transport by myoVa motors. Five thousand simulated liposomes transported within each network adopted one of three states: transport, tug-of-war, or diffusion. Diffusion due to liposome detachment from actin rarely occurred given at least 10 motors on the liposome surface. However, with increased actin density, liposomes transitioned from primarily directed transport on single actin filaments to an apparent random walk, resulting from a mixture of transport and tug-of-wars as the probability of encountering additional actin filaments increased. This phase transition arises from a percolation phase transition at a critical number of accessible actin filaments, N_c. N_c is a geometric property of the actin network that depends only on the position and polarity of the actin filaments, transport distance, and the liposome diameter, as evidenced by a fivefold increase in liposome diameter resulting in a fivefold decrease in N_c. Thus in cells, actin network density and cargo size may be regulated to match cargo delivery to the cell’s physiological demands.     

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.     

Filament-Filament Switching Can Be Regulated by Separation Between Filaments Together with Cargo Motor Number

How intracellular transport controls the probability that cargos switch at intersections between filaments is not well understood. In one hypothesis some motors on the cargo attach to one filament while others attach to the intersecting filament, and the ensuing tug-of-war determines which filament is chosen. We investigate this hypothesis using 3D computer simulations, and discover that switching at intersections increases with the number of motors on the cargo, but is not strongly dependent on motor number when the filaments touch. Thus, simply controlling the number of active motors on the cargo cannot account for in vivo observations that found reduced switching with increasing motor number, suggesting additional mechanisms of regulation. We use simulations to show that one possible way to regulate switching is by simultaneously adjusting the separation between planes containing the crossing filaments and the total number of active motors on the cargo. Heretofore, the effect of filament-filament separation on switching has been unexplored. We find that the switching probability decreases with increasing filament separation. This effect is particularly strong for cargos with only a modest number of motors. As the filament separation increases past the maximum head-to-head distance of the motor, individual motors walking along a filament will be unable to reach the intersecting filament. Thus, any switching requires that other motors on the cargo attach to the intersecting filament and haul the cargo along it, while motor(s) engaged on the original filament detach. Further, if the filament separation is large enough, the cargo can have difficulty proceeding along the initial filament because the engaged motors can walk underneath the intersecting filament, but the cargo itself cannot fit between the filaments. Thus, the cargo either detaches entirely from the original filament, or must dip to the side of the initial filament and then pass below the crossing filament.     

Lipid Droplets Purified from Drosophila Embryos as an Endogenous Handle for Precise Motor Transport Measurements

Molecular motor proteins are responsible for long-range transport of vesicles and organelles. Recent works have elucidated the richness of the transport complex, with multiple teams of similar and dissimilar motors and their cofactors attached to individual cargoes. The interaction among these different proteins, and with the microtubules along which they translocate, results in the intricate patterns of cargo transport observed in cells. High-precision and high-bandwidth measurements are required to capture the dynamics of these interactions, yet the crowdedness in the cell necessitates performing such measurements in vitro. Here, we show that endogenous cargoes, lipid droplets purified from Drosophila embryos, can be used to perform high-precision and high-bandwidth optical trapping experiments to study motor regulation in vitro. Purified droplets have constituents of the endogenous transport complex attached to them and exhibit long-range motility. A novel method to determine the quality of the droplets for high-resolution measurements in an optical trap showed that they compare well with plastic beads in terms of roundness, homogeneity, position sensitivity, and trapping stiffness. Using high-resolution and high-bandwidth position measurements, we demonstrate that we can follow the series of binding and unbinding events that lead to the onset of active transport.     

Transport Properties of Melanosomes along Microtubules Interpreted by a Tug-of-War Model with Loose Mechanical Coupling

In this work, we explored theoretically the transport of organelles driven along microtubules by molecular motors of opposed polarities using a stochastic model that considers a Langevin dynamics for the cargo, independent cargo-motor linkers and stepping motion for the motors. It has been recently proposed that the stiffness of the motor plays an important role when multiple motors collectively transport a cargo. Therefore, we considered in our model the recently reported values for the stiffness of the cargo-motor linker determined in living cells (～0.01 pN/nm, [1]) which is significantly lower than the motor stiffness obtained in in vitro assays and used in previous studies. Our model could reproduce the multimodal velocity distributions and typical trajectory characteristics including the properties of the reversions in the overall direction of motion observed during melanosome transport along microtubules in Xenopus laevis melanophores. Moreover, we explored the contribution of the different motility states of the cargo-motor system to the different modes of the velocity distributions and could identify the microscopic mechanisms of transport leading to trajectories compatible with those observed in living cells. Finally, by changing the attachment and detachment rates, the model could reproduce the different velocity distributions observed during melanosome transport along microtubules in Xenopus laevis melanophores stimulated for aggregation and dispersion. Our analysis suggests that active tug-of-war processes with loose mechanical coupling can account for several aspects of cargo transport along microtubules in living cells.     

Lipid Droplets Purified from Drosophila Embryos as an Endogenous Handle for Precise Motor Transport Measurements

Molecular motor proteins are responsible for long-range transport of vesicles and organelles. Recent works have elucidated the richness of the transport complex, with multiple teams of similar and dissimilar motors and their cofactors attached to individual cargoes. The interaction among these different proteins, and with the microtubules along which they translocate, results in the intricate patterns of cargo transport observed in cells. High-precision and high-bandwidth measurements are required to capture the dynamics of these interactions, yet the crowdedness in the cell necessitates performing such measurements in vitro. Here, we show that endogenous cargoes, lipid droplets purified from Drosophila embryos, can be used to perform high-precision and high-bandwidth optical trapping experiments to study motor regulation in vitro. Purified droplets have constituents of the endogenous transport complex attached to them and exhibit long-range motility. A novel method to determine the quality of the droplets for high-resolution measurements in an optical trap showed that they compare well with plastic beads in terms of roundness, homogeneity, position sensitivity, and trapping stiffness. Using high-resolution and high-bandwidth position measurements, we demonstrate that we can follow the series of binding and unbinding events that lead to the onset of active transport.     

Mechanical coupling of microtubule-dependent motor teams during peroxisome transport in Drosophila S2 cells

BackgroundIntracellular transport requires molecular motors that step along cytoskeletal filaments actively dragging cargoes through the crowded cytoplasm. Here, we explore the interplay of the opposed polarity motors kinesin-1 and cytoplasmic dynein during peroxisome transport along microtubules in Drosophila S2 cells.MethodsWe used single particle tracking with nanometer accuracy and millisecond time resolution to extract quantitative information on the bidirectional motion of organelles. The transport performance was studied in cells expressing a slow chimeric plus-end directed motor or the kinesin heavy chain. We also analyzed the influence of peroxisomes membrane fluidity in methyl-β-ciclodextrin treated cells. The experimental data was also confronted with numerical simulations of two well-established tug of war scenarios.Results and conclusionsThe velocity distributions of retrograde and anterograde peroxisomes showed a multimodal pattern suggesting that multiple motor teams drive transport in either direction. The chimeric motors interfered with the performance of anterograde transport and also reduced the speed of the slowest retrograde team. In addition, increasing the fluidity of peroxisomes membrane decreased the speed of the slowest anterograde and retrograde teams.General significanceOur results support the existence of a crosstalk between opposed-polarity motor teams. Moreover, the slowest teams seem to mechanically communicate with each other through the membrane to trigger transport.     

The influence of direct motor-motor interaction in models for cargo transport by a single team of motors

We analyze theoretically the effects of excluded-volume interactions between motors on the dynamics of a cargo driven by multiple motors. The model considered shares much in common with others recently proposed in the literature, with the addition of direct interaction between motors and motor back steps. The cargo is assumed to follow a continuum Langevin dynamics, while individual motors evolve following a Monte Carlo algorithm based on experimentally accessible probabilities for discrete forward and backward jumps, and attachment and detachment rates. The links between cargo and motors are considered as nonlinear springs. By means of numerical simulations we compute the relevant quantities characterizing the dynamical properties of the system, and we compare the results to those for noninteracting motors. We find that interactions lead to quite relevant changes in the force-velocity relation for cargo, with a considerable reduction of the stall force, and also cause a notable decrease of the run length. These effects are mainly due to traffic-like phenomena in the microtubule. The consideration of several parallel tracks for motors reduces such effects. However, we find that for realistic values of the number of motors and the number of tracks, the influence of interactions on the global parameters of transport of cargo are far from being negligible. Our studies also provide an analysis of the relevance of motor back steps on the modeling, and of the influence of different assumptions for the detachment rates. In particular, we discuss these two aspects in connection with the possibility of observing processive back motion of cargo at large load forces.     

Models of motor-assisted transport of intracellular particles

One-dimensional models are presented for the macroscopic intracellular transport of vesicles and organelles by molecular motors on a network of aligned intracellular filaments. A motor-coated vesicle or organelle is described as a diffusing particle binding intermittently to filaments, when it is transported at the motor velocity. Two models are treated in detail: 1) a unidirectional model, where only one kind of motor is operative and all filaments have the same polarity; and 2) a bidirectional model, in which filaments of both polarities exist (for example, a randomly polarized actin network for myosin motors) and/or particles have plus-end and minus-end motors operating on unipolar filaments (kinesin and dynein on microtubules). The unidirectional model provides net particle transport in the absence of a concentration gradient. A symmetric bidirectional model, with equal mixtures of filament polarities or plus-end and minus-end motors of the same characteristics, provides rapid transport down a concentration gradient and enhanced dispersion of particles from a point source by motor-assisted diffusion. Both models are studied in detail as a function of the diffusion constant and motor velocity of bound particles, and their rates of binding to and detachment from filaments. These models can form the basis of more realistic models for particle transport in axons, melanophores, and the dendritic arms of melanocytes, in which networks of actin filaments and microtubules coexist and motors for both types of filament are implicated.     

Endogenous GSK‐3/Shaggy Regulates Bidirectional Axonal Transport of the Amyloid Precursor Protein

Neurons rely on microtubule (MT) motor proteins such as kinesin‐1 and dynein to transport essential cargos between the cell body and axon terminus. Defective axonal transport causes abnormal axonal cargo accumulations and is connected to neurodegenerative diseases, including Alzheimer's disease (AD). Glycogen synthase kinase 3 (GSK‐3) has been proposed to be a central player in AD and to regulate axonal transport by the MT motor protein kinesin‐1. Using genetic, biochemical and biophysical approaches in Drosophila melanogaster, we find that endogenous GSK‐3 is a required negative regulator of both kinesin‐1‐mediated and dynein‐mediated axonal transport of the amyloid precursor protein (APP), a key contributor to AD pathology. GSK‐3 also regulates transport of an unrelated cargo, embryonic lipid droplets. By measuring the forces motors generate in vivo, we find that GSK‐3 regulates transport by altering the activity of kinesin‐1 motors but not their binding to the cargo. These findings reveal a new relationship between GSK‐3 and APP, and demonstrate that endogenous GSK‐3 is an essential in vivo regulator of bidirectional APP transport in axons and lipid droplets in embryos. Furthermore, they point to a new regulatory mechanism in which GSK‐3 controls the number of active motors that are moving a cargo .     

Opposite-polarity motors activate one another to trigger cargo transport in live cells

Intracellular transport is typically bidirectional, consisting of a series of back and forth movements. Kinesin-1 and cytoplasmic dynein require each other for bidirectional transport of intracellular cargo along microtubules; i.e., inhibition or depletion of kinesin-1 abolishes dynein-driven cargo transport and vice versa. Using Drosophila melanogaster S2 cells, we demonstrate that replacement of endogenous kinesin-1 or dynein with an unrelated, peroxisome-targeted motor of the same directionality activates peroxisome transport in the opposite direction. However, motility-deficient versions of motors, which retain the ability to bind microtubules and hydrolyze adenosine triphosphate, do not activate peroxisome motility. Thus, any pair of opposite-polarity motors, provided they move along microtubules, can activate one another. These results demonstrate that mechanical interactions between opposite-polarity motors are necessary and sufficient for bidirectional organelle transport in live cells.     

Actin bundle architecture and mechanics regulate myosin II force generation

The actin cytoskeleton is a soft, structural material that underlies biological processes such as cell division, motility, and cargo transport. The cross-linked actin filaments self-organize into a myriad of architectures, from disordered meshworks to ordered bundles, which are hypothesized to control the actomyosin force generation that regulates cell migration, shape, and adhesion. Here, we use fluorescence microscopy and simulations to investigate how actin bundle architectures with varying polarity, spacing, and rigidity impact myosin II dynamics and force generation. Microscopy reveals that mixed-polarity bundles formed by rigid cross-linkers support slow, bidirectional myosin II filament motion, punctuated by periods of stalled motion. Simulations reveal that these locations of stalled myosin motion correspond to sustained, high forces in regions of balanced actin filament polarity. By contrast, mixed-polarity bundles formed by compliant, large cross-linkers support fast, bidirectional motion with no traps. Simulations indicate that trap duration is directly related to force magnitude and that the observed increased velocity corresponds to lower forces resulting from both the increased bundle compliance and filament spacing. Our results indicate that the microstructures of actin assemblies regulate the dynamics and magnitude of myosin II forces, highlighting the importance of architecture and mechanics in regulating forces in biological materials.     

Cargo properties play a critical role in myosin Va-driven cargo transport along actin filaments

High-resolution experiments revealed that a single myosin-Va motor can transport micron-sized cargo on actin filaments in a stepwise manner. However, intracellular cargo transport is mediated through the dense actin meshwork by a team of myosin Va motors. The mechanism of how motors interact mechanically to bring about efficient cargo transport is still poorly understood. This study describes a stochastic model where a quantitative understanding of the collective behaviors of myosin Va motors is developed based on cargo stiffness. To understand how cargo properties affect the overall cargo transport, we have designed a model in which two myosin Va motors were coupled by wormlike chain tethers with persistence length ranging from 10 to 80 nm and contour length from 100 to 200 nm, and predicted distributions of velocity, run length, and tether force. Our analysis showed that these parameters are sensitive to both the contour and persistence length of cargo. While the velocity of two couple motors is decreased compared to a single motor (from 531 ± 251 nm/s to as low as 318 ± 287 nm/s), the run length (716 ± 563 nm for a single motor) decreased for short, rigid tethers (to as low as 377 ± 187 μm) and increased for long, flexible tethers (to as high as 1.74 ± 1.50 μm). The sensitivity of processive properties to tether rigidity (persistence length) was greatest for short tethers, which caused the motors to exhibit close, yet anti-cooperative coordination. Motors coupled by longer tethers stepped more independently regardless of tether rigidity. Therefore, the properties of the cargo or linkage must play an essential role in motor-motor communication and cargo transport.     

Two kinesins transport cargo primarily via the action of one motor: implications for intracellular transport

The number of microtubule motors attached to vesicles, organelles, and other subcellular commodities is widely believed to influence their motile properties. There is also evidence that cells regulate intracellular transport by tuning the number and/or ratio of motor types on cargos. Yet, the number of motors responsible for cargo motion is not easily characterized, and the extent to which motor copy number affects intracellular transport remains controversial. Here, we examined the load-dependent properties of structurally defined motor assemblies composed of two kinesin-1 molecules. We found that a group of kinesins can produce forces and move with velocities beyond the abilities of single kinesin molecules. However, such capabilities are not typically harnessed by the system. Instead, two-kinesin assemblies adopt a range of microtubule-bound configurations while transporting cargos against an applied load. The binding arrangement of motors on their filament dictates how loads are distributed within the two-motor system, which in turn influences motor-microtubule affinities. Most configurations promote microtubule detachment and prevent both kinesins from contributing to force production. These results imply that cargos will tend to be carried by only a fraction of the total number of kinesins that are available for transport at any given time, and provide an alternative explanation for observations that intracellular transport depends weakly on kinesin number in vivo.