Multi-motor Transport Research Articles

Horizontal Magnetic Tweezers to Directly Measure the Force-Velocity Relationship for Multiple Kinesin Motors.

Transport of intracellular cargo along cytoskeletal filaments is often achieved by the concerted action of multiple motor molecules. While single-molecule studies have provided profound insight into the mechano-chemical principles and force generation of individual motors, studies on multi-motor systems are less advanced. Here, a horizontal magnetic-tweezers setup is applied, capable of producing up to 150 pN of horizontal force onto 2.8µm superparamagnetic beads, to motor-propelled cytoskeletal filaments. It is found that kinesin-1 driven microtubules decorated with individual beads display frequent transitions in their gliding velocities whichweattribute to dynamic changes in the number of engaged motors. Applying defined temporal force-ramps the force-velocity relationship is directly measured for multi-motor transport. It is found that the stall forces of individual motors are approximately additive and collective backward motion of the transport system under super-stall forces is observed. The magnetic-tweezers apparatus is expected to be readily applicable to a wide range of molecular and cellular motility assays.

High-throughput force measurement of individual kinesin-1 motors during multi-motor transport.

Molecular motors often work in teams to move a cellular cargo. Yet measuring the forces exerted by each motor is challenging. Using a sensor made with denatured ssDNA and multi-color fluorescence, we measured picoNewtons of forces and nanometer distances exerted by individual constrained kinesin-1 motors acting together while driving a common microtubule in vitro. We find that kinesins primarily exerted less than 1 pN force, even while the microtubule is bypassing artificial obstacles of 20-100 nanometer size. Occasionally, individual forces increase upon encountering obstacles, although at other times they do not, with the cargo continuing in a directional manner. Our high-throughput technique, which can measure forces by many motors simultaneously, is expected to be useful for many different types of molecular motors.

An SSDNA Based Force Sensor for Multi Motor Transport as an Alternative to Optical Tweezers

Forces exerted by molecular motors have traditionally been quantified by optical tweezers. The requirement of physical connection to microscopic beads and low-throughput in an optical tweezer experiment limits its applicability in diverse configurations. We present a single-stranded DNA-based force sensor that can measure forces exerted by individual molecular motors in real-time when multiple motors are transporting a common microtubule. An ssDNA molecule, which acts as a force sensing molecule, is sandwiched between motor molecules and the coverslip.

Engineered Icosahedral Protein Cage as a Flexible Model System to Study Properties of Multi-Motor Transport of Viral Cargoes

Engineered Icosahedral Protein Cage as a Flexible Model System to Study Properties of Multi-Motor Transport of Viral Cargoes

In vitro Reconstitution of Kinesin-Driven Vesicle Transport

Intracellular vesicle cargos are transported along microtubules by kinesin and dynein motors, towards the cell periphery and cell center, respectively. Individual motors generally have run lengths of several μm, whereas organelles need to travel tens of μm to reach their destinations. Therefore, vesicle cargos usually have multiple copies of motors such that when one or more motors detach from the microtubule, other motors can continue transport. We previously used a 2D lipid bilayer system to measure the microtubule binding kinetics of membrane-bound kinesin-1 motors, from which we predicted long-range transport of 100 nm diameter vesicles required 35 kinesin-1 motors. Our predicted motor number was higher than what was measured on purified neuronal vesicles, thus we aimed to reconstitute kinesin-driven vesicle transport and characterize vesicle motility behaviors in the present study. GFP-labeled kinesin-1 was linked to POPC liposomes through a DNA linker. As the mixing ratio of kinesin-1 and vesicle increased, the vesicle landing density in AMPPNP increased linearly and then reached a plateau. The average vesicle travel distance in ATP increased continuously with the mixing ratio, indicating that multi-motor transport was achieved. Vesicle transport velocity was insensitive to motor number. This system enables reconstitution of vesicle motility with controlled motor densities and can be extended to study bidirectional transport by kinesins and dyneins.

Motor Dynamics Underlying Cargo Transport by Pairs of Kinesin-1 and Kinesin-3 Motors

Intracellular cargo transport by kinesin family motor proteins is crucial for many cellular processes, particularly vesicle transport in axons and dendrites. In a number of cases, the transport of specific cargo is carried out by two classes of kinesins that move at different speeds and thus compete during transport. Despite advances in single-molecule characterization and modeling approaches, many questions remain regarding the effect of intermotor tension on motor attachment/reattachment rates during cooperative multimotor transport. To understand the motor dynamics underlying multimotor transport, we analyzed the complexes of kinesin-1 and kinesin-3 motors attached through protein scaffolds moving on immobilized microtubules in vitro. To interpret the observed behavior, simulations were carried out using a model that incorporated motor stepping, attachment/detachment rates, and intermotor force generation. In single-molecule experiments, isolated kinesin-3 motors moved twofold faster and had threefold higher landing rates than kinesin-1. When the positively charged loop 12 of kinesin-3 was swapped with that of kinesin-1, the landing rates reversed, indicating that this “K-loop” is a key determinant of the motor reattachment rate. In contrast, swapping loop 12 had negligible effects on motor velocities. Two-motor complexes containing one kinesin-1 and one kinesin-3 moved at different speeds depending on the identity of their loop 12, indicating the importance of the motor reattachment rate on the cotransport speed. Simulations of these loop-swapped motors using experimentally derived motor parameters were able to reproduce the experimental results and identify best fit parameters for the motor reattachment rates for this geometry. Simulation results also supported previous work, suggesting that kinesin-3 microtubule detachment is very sensitive to load. Overall, the simulations demonstrate that the transport behavior of cargo carried by pairs of kinesin-1 and -3 motors are determined by three properties that differ between these two families: the unloaded velocity, the load dependence of detachment, and the motor reattachment rate.

Three-Dimensional Model of Cooperative Transport of Pairs of Kinesin-1 and −2 Motors

Motor proteins are fundamentally important as they are involved in many intercellular activities such as cell division, neurotransmission, and muscle contraction. Due to experimental challenges, the mechanisms of many kinesin family motors remain unclear during multimotor transport. In the past decade, computer simulations have become an important tool in characterizing such cooperative transport by families of kinesin motors, particularly in conjunction with distinct number of motors attached to a DNA scaffold. Here, we present Langevin dynamics simulations and that capture kinesin-1/kinesin-2 motor mechano-chemistry in a three-dimensional scaffold geometry, and show how both motor's distinct force sensitivities to detachment play an important role in multi-motor transport. In particular, our model is able to recapitulate not only the observed velocities of the 2-motor complexes but also the experimentally measured run lengths. Our results also suggest that the variance of the end-to-end reattachment is another key parameter in multi-motor transport besides critical detachment force and reattachment rate.

Slow and fast grouping of cargo velocities in axonal transport due to single versus multi-motor transport

We have recently been exploring the idea that axonal transport velocity is “track and motor limited.” That is, microtubule length as well as microtubule-associated obstructions interact with the number of motors attached to a specific cargo to determine average cargo velocities. We assert that “slow” and “fast” transport as they are commonly referred to in the literature are really single- versus multi-motor transport along interrupted and obstructed track. To this end, we have recently developed a cargo-level motor model that appears to readily reproduce fast and slow transport simply by altering the number of motors. In the work presented here, we explore the ramifications of this model across a wide range of cargo sizes and motor-motor interactions. We find that categorization of cargo transport into “slow” and “fast” might be a natural consequence of track and motor limited transport as cargo load versus average velocity distribution produced by this model are clearly bi-modal with a curved (roughly square-root) relationship between number of motors and cargo load being the best at reproducing experimental data.

Kinesin Motor Domain Dynamics during Single-Motor Stepping and Multi-motor Transport

Kinesin Motor Domain Dynamics during Single-Motor Stepping and Multi-motor Transport

Development of Improved Microscopy and Data Analysis Tools for Understanding Multimotor Transport

Development of Improved Microscopy and Data Analysis Tools for Understanding Multimotor Transport

Motor Reattachment Kinetics Play a Dominant Role in Multimotor-Driven Cargo Transport

Kinesin-based cargo transport in cells frequently involves the coordinated activity of multiple motors, including kinesins from different families that move at different speeds. However, compared to the progress at the single-molecule level, mechanisms by which multiple kinesins coordinate their activity during cargo transport are poorly understood. To understand these multimotor coordination mechanisms, defined pairs of kinesin-1 and kinesin-2 motors were assembled on DNA scaffolds and their motility examined in vitro. Although less processive than kinesin-1 at the single-molecule level, addition of kinesin-2 motors more effectively amplified cargo run lengths. By applying the law of total expectation to cargo binding durations in ADP, the kinesin-2 microtubule reattachment rate was shown to be fourfold faster than that of kinesin-1. This difference in microtubule binding rates was also observed in solution by stopped-flow. High-resolution tracking of a gold-nanoparticle-labeled motor with 1 ms and 2 nm precision revealed that kinesin-2 motors detach and rebind to the microtubule much more frequently than does kinesin-1. Finally, compared to cargo transported by two kinesin-1, cargo transported by two kinesin-2 motors more effectively navigated roadblocks on the microtubule track. These results highlight the importance of motor reattachment kinetics during multimotor transport and suggest a coordinated transport model in which kinesin-1 motors step effectively against loads whereas kinesin-2 motors rapidly unbind and rebind to the microtubule. This dynamic tethering by kinesin-2 maintains the cargo near the microtubule and enables effective navigation along crowded microtubules.

The Role of the Cover-Neck Bundle in Multi-Motor Transport against Load in Cells

Kinesin motor proteins share a highly-conserved catalytic motor domain responsible for coupling the chemical energy of ATP hydrolysis to force generation and microtubule-directed functions. Previous studies of kinesin-1 suggested that ATP binding to the motor domain induces the formation of the cover-neck bundle (CNB), a beta-sheet between β0 at the N-terminus (the coverstrand) and β9 in the first half of the necklinker. The CNB was shown to contribute to force generation by single kinesin-1 motors under purified conditions and has been predicted to function in force generation across the kinesin superfamily. However, differences in coverstrand and necklinker sequences make the role of the CNB across different kinesin families unclear. We adapted artificial cargo systems to delineate key features of the CNB during multi-motor transport against low and high loads in cells. Our results indicate that in teams, kinesin-1 motors can robustly transport cargo against high- and low-load in cells. Molecular modeling was used to predict mutations in the coverstrand and necklinker that disrupt and shorten beta-sheet formation. Experimental testing of these mutations shows that impaired beta-sheet formation compromised the capacity for teams of kinesin-1 motors to transport high-load cargo but not low-load cargo to the periphery of cells. Surprisingly, truncation of the region N-terminal to the coverstrand, predicted to have a minimal role in CNB formation, severely crippled the capacity for teams of kinesin-1 motors to transport both low- and high-load cargoes in cells. We plan to extend these studies to additional kinesin families to identify distinct motility dependencies on formation of the CNB.

Cargo Transport by Two Coupled Myosin Va Motors on Actin Filaments and Bundles

Myosin Va (myoVa) is a processive, actin-based molecular motor essential for intracellular cargo transport. When a cargo is transported by an ensemble of myoVa motors, each motor faces significant physical barriers and directional challenges created by the complex actin cytoskeleton, a network of actin filaments and actin bundles. The principles that govern the interaction of multiple motors attached to the same cargo are still poorly understood. To understand the mechanical interactions between multiple motors, we developed a simple in vitro model in which two individual myoVa motors labeled with different-colored Qdots are linked via a third Qdot that acts as a cargo. The velocity of this two-motor complex was reduced by 27% as compared to a single motor, whereas run length was increased by only 37%, much less than expected from multimotor transport models. Therefore, at low ATP, which allowed us to identify individual motor steps, we investigated the intermotor dynamics within the two-motor complex. The randomness of stepping leads to a buildup of tension in the linkage between motors—which in turn slows down the leading motor—and increases the frequency of backward steps and the detachment rate. We establish a direct relationship between the velocity reduction and the distribution of intermotor distances. The analysis of run lengths and dwell times for the two-motor complex, which has only one motor engaged with the actin track, reveals that half of the runs are terminated by almost simultaneous detachment of both motors. This finding challenges the assumptions of conventional multimotor models based on consecutive motor detachment. Similar, but even more drastic, results were observed with two-motor complexes on actin bundles, which showed a run length that was even shorter than that of a single motor.

Axonal transport cargo motor count versus average transport velocity: is fast versus slow transport really single versus multiple motor transport?

Cargos have been observed exhibiting a "stop-and-go" behavior (i.e. cargo "pause"), and it has generally been assumed that these multi-second pauses can be attributed to equally long pauses of cargo-bound motors during motor procession. We contend that a careful examination of the isolated microtubule experimental record does not support motor pauses. Rather, we believe that the data suggests that motor cargo complexes encounter an obstruction that prevents procession, eventually detach and reattach, with this obstructed-detach-reattach sequence being observed in axon as a "pause." Based on this, along with our quantitative evidence-based contention that slow and fast axonal transport are actually single and multi-motor transport, we have developed a cargo level motor model capable of exhibiting the full range of slow to fast transport solely by changing the number of motors involved. This computational model derived using first-order kinetics is suitable for both kinesin and dynein and includes load-dependence as well as provision for motors encountering obstacles to procession. The model makes the following specific predictions: average distance from binding to obstruction is about 10 μm; average motor maximum velocity is at least 6 μm/s in axon; a minimum of 10 motors is required for the fastest fast transport while only one motor is required for slow transport; individual in-vivo cargo-attached motors may spend as little as 5% of their time processing along a microtubule with the remainder being spent either obstructed or unbound to a microtubule; and at least in the case of neurofilament transport, kinesin and dynein are largely not being in a "tug-of-war" competition.

Multimotor Transport in a System of Active and Inactive Kinesin-1 Motors

Long-range directional transport in cells is facilitated by microtubule-based motor proteins. One example is transport in a nerve cell, where small groups of motor proteins, such as kinesins and cytoplasmic dynein, work together to ensure the supply and clearance of cellular material along the axon. Defects in axonal transport have been linked to Alzheimer’s and other neurodegenerative diseases. However, it is not known in detail how multimotor-based cargo transport is impaired if a fraction of the motors are defective. To mimic impaired multimotor transport in vitro, we performed gliding motility assays with varying fractions of active kinesin-1 motors and inactive kinesin-1 motor mutants. We found that impaired transport manifests in multiple motility regimes: 1), a fast-motility regime characterized by gliding at velocities close to the single-molecule velocity of the active motors; 2), a slow-motility regime characterized by gliding at close-to zero velocity or full stopping; and 3), a regime in which fast and slow motilities coexist. Notably, the transition from the fast to the slow regime occurred sharply at a threshold fraction of active motors. Based on single-motor parameters, we developed a stochastic model and a mean-field theoretical description that explain our experimental findings. Our results demonstrate that impaired multimotor transport mostly occurs in an either/or fashion: depending on the ratio of active to inactive motors, transport is either performed at close to full speed or is out of action.

Bimodality in a System of Active and Passive Kinesin-1 Motors

Long-range directional transport in cells is facilitated by microtubule-based motor proteins. One example is transport in a nerve cell, where small groups of motor proteins, such as kinesin and dynein, work together to perform the supply and clearance of cellular material along the axon. Defects in axonal transport have been linked to Alzheimer and other neurodegenerative diseases. In particular two diseases, Hereditary Spastic Paraplegia (HSP) and Charcot-Marie-Tooth type 2A neuropathy (CMT2A) are connected to mutations of kinesin family members in the motor domain that affect their ATPase activity. However, it is not known how in detail multi-motor based cargo transport is impacted if the motor function of a fraction of motors is inhibited. In order to mimic hindered multi-motor transport in-vitro, we performed gliding motility assays with varying fractions of active kinesin-1 and passivated kinesin-1 (rigor mutants). We found that hindered gliding manifests in three motility regimes: gliding at the velocity of single motors, simultaneous gliding and stopping (bistable movement), and stopping. Notably, an abrupt transition from gliding to stopping occurred at a certain threshold fraction. Furthermore, we developed a theoretical description based on single motor parameters. Our model explains the bimodal microtubule movement as well as the sharp transition from gliding to stopping. Our results demonstrate that hindered transport is acting in a bimodal either-or-fashion: depending on the fraction of passive motors, transport by a multi-motor system is either performed close to full speed or not at all.

Intracellular Transport: New Tools Provide Insights into Multi-motor Transport

Teams of kinesin and dynein motors drive bidirectional transport of intracellular cargoes along the microtubule cytoskeleton. How do opposite-polarity motors interact to achieve targeted trafficking? A new study uses tools from synthetic biology to probe collective motor function.

Long‐Range Transport of Giant Vesicles along Microtubule Networks

We report on a minimal system to mimic intracellular transport of membrane-bounded, vesicular cargo. In a cell-free assay, purified kinesin-1 motor proteins were directly anchored to the membrane of giant unilamellar vesicles, and their movement studied along two-dimensional microtubule networks. Motion-tracking of vesicles with diameters of 1-3 μm revealed traveling distances up to the millimeter range. The transport velocities were identical to velocities of cargo-free motors. Using total internal reflection fluorescence (TIRF) microscopy, we were able to estimate the number of GFP-labeled motors involved in the transport of a single vesicle. We found that the vesicles were transported by the cooperative activity of typically 5-10 motor molecules. The presented assay is expected to open up further applications in the field of synthetic biology, aiming at the in vitro reconstitution of sub-cellular multi-motor transport systems. It may also find applications in bionanotechnology, where the controlled long-range transport of artificial cargo is a promising means to advance current lab-on-a-chip systems.

Force–velocity relationship for multiple kinesin motors pulling a magnetic bead

Although the velocity of single kinesin motors against an opposing force F of 0-10 pN is well known, the behavior of multiple kinesin motors working to overcome a larger load is still poorly understood. We have carried out gliding assays in which 3-7 Drosophila kinesin-1 motors moved a microtubule at 200-700 μm/s against a 0-31 pN load at saturating [ATP]. The load F was generated by applying a spatially uniform magnetic field gradient to a superparamagnetic bead attached to the (+) end of the microtubule. When F was scaled by the average number of motors [Symbol: see text]n[Symbol: see text], the force-velocity relationship for multiple motors was similar to the force-velocity relationship for a single motor, supporting a minimal load-sharing model. The velocity distribution at low load has a single mode consistent with rapid fluctuations of n. However, against a load of 2.5-4.7 pN/motor, additional modes appeared at lower velocity. These observations support the Klumpp-Lipowsky model of multimotor transport [Proc Natl Acad Sci USA 102. 17284-17289 (2005)].

Monte—Carlo Simulation of Multiple-Molecular-Motor Transport

Multimotor transport is studied by Monte-Carlo simulation with consideration of motor detachment from the filament. Our work shows, in the case of low load, the velocity of multi-motor system can decrease or increase with increasing motor numbers depending on the single motor force-velocity curve. The stall force and run-length reduced greatly compared to other models. Especially in the case of low ATP concentrations, the stall force of multi motor transport even smaller than the single motor's stall force.