Tuning Multiple Motor Travel via Single Motor Velocity

Microtubule-based molecular motors often work in small groups to transport cargos in cells. A key question in understanding transport (and its regulation in vivo) is to identify the sensitivity of multiple-motor-based motion to various single molecule properties. Whereas both single-motor travel distance and microtubule binding rate have been demonstrated to contribute to cargo travel, the role of single-motor velocity is yet to be explored. Here, we recast a previous theoretical study, and make explicit a potential contribution of velocity to cargo travel. We test this possibility experimentally, and demonstrate a strong negative correlation between single-motor velocity and cargo travel for transport driven by two motors. Our study thus discovers a previously unappreciated role of single-motor velocity in regulating multiple-motor transport.     

Studying molecular motor-based cargo transport: what is real and what is noise?

Noise is a major problem in analyzing tracking data of cargos moved by molecular motors. We use Bayesian statistics to incorporate what is known about the noise in parsing the trajectory of a cargo into a series of constant velocity segments. Tracks with just noise and no underlying motion are fit with constant velocity segments to produce a calibration curve of fit quality versus average segment duration. Fits to tracks of moving cargos are compared to the calibration curves with similar noise. The fit with the optimum number of constant velocity states has the least number of segments needed to match the fit quality of the calibration curve. We have tested this approach using tracks with known underlying motion generated by computer simulations and with a specially designed in vitro experiment. We present the results of using this parsing approach to analyze transport of lipid droplets in Drosophila embryos.     

How molecular motors are arranged on a cargo is important for vesicular transport

The spatial organization of the cell depends upon intracellular trafficking of cargos hauled along microtubules and actin filaments by the molecular motor proteins kinesin, dynein, and myosin. Although much is known about how single motors function, there is significant evidence that cargos in vivo are carried by multiple motors. While some aspects of multiple motor function have received attention, how the cargo itself--and motor organization on the cargo--affects transport has not been considered. To address this, we have developed a three-dimensional Monte Carlo simulation of motors transporting a spherical cargo, subject to thermal fluctuations that produce both rotational and translational diffusion. We found that these fluctuations could exert a load on the motor(s), significantly decreasing the mean travel distance and velocity of large cargos, especially at large viscosities. In addition, the presence of the cargo could dramatically help the motor to bind productively to the microtubule: the relatively slow translational and rotational diffusion of moderately sized cargos gave the motors ample opportunity to bind to a microtubule before the motor/cargo ensemble diffuses out of range of that microtubule. For rapidly diffusing cargos, the probability of their binding to a microtubule was high if there were nearby microtubules that they could easily reach by translational diffusion. Our simulations found that one reason why motors may be approximately 100 nm long is to improve their 'on' rates when attached to comparably sized cargos. Finally, our results suggested that to efficiently regulate the number of active motors, motors should be clustered together rather than spread randomly over the surface of the cargo. While our simulation uses the specific parameters for kinesin, these effects result from generic properties of the motors, cargos, and filaments, so they should apply to other motors as well.     

Bidirectional Transport by Molecular Motors: Enhanced Processivity and Response to External Forces

Intracellular transport along cytoskeletal filaments is often mediated by two teams of molecular motors that pull on the same cargo and move in opposite directions along the filaments. We have recently shown theoretically that this bidirectional transport can be understood as a stochastic tug-of-war between the two motor teams. Here, we further develop our theory to investigate the experimentally accessible dynamic behavior of cargos transported by strong motors such as kinesin-1 or cytoplasmic dynein. By studying the run and binding times of such a cargo, we show that the properties of biological motors, such as the large ratio of stall/detachment force and the small ratio of superstall backward/forward velocity, are favorable for bidirectional cargo transport, leading to fast motion and enhanced diffusion. In addition, cargo processivity is shown to be strongly enhanced by transport via several molecular motors even if these motors are engaged in a tug-of-war. Finally, we study the motility of a bidirectional cargo under force. Frictional forces arising, e.g., from the viscous cytoplasm, lead to peaks in the velocity distribution, while external forces as exerted, e.g., by an optical trap, lead to hysteresis effects. Our results, in particular our explicit expressions for the cargo binding time and the distance of the peaks in the velocity relation under friction, are directly accessible to in vitro as well as in vivo experiments.     

Building complexity: an in vitro study of cytoplasmic dynein with in vivo implications

BACKGROUND: Cytoplasmic dynein is the molecular motor responsible for most retrograde microtubule-based vesicular transport. In vitro single-molecule experiments suggest that dynein function is not as robust as that of kinesin-1 or myosin-V because dynein moves only a limited distance (approximately 800 nm) before detaching and can exert a modest (approximately 1 pN) force. However, dynein-driven cargos in vivo move robustly over many microns and exert forces of multiple pN. To determine how to go from limited single-molecule function to robust in vivo transport, we began to build complexity in a controlled manner by using in vitro experiments. RESULTS: We show that a single cytoplasmic dynein motor frequently transitions into an off-pathway unproductive state that impairs net transport. Addition of a second (and/or third) dynein motor, so that cargos are moved by two (or three) motors rather than one, is sufficient to recover several properties of in vivo motion; such properties include long cargo travels, robust motion, and increased forces. Part of this improvement appears to arise from selective suppression of the unproductive state of dynein rather than from a fundamental change in dynein's mechanochemical cycle. CONCLUSIONS: Multiple dyneins working together suppress shortcomings of a single motor and generate robust motion under in vitro conditions. There appears to be no need for additional cofactors (e.g., dynactin) for this improvement. Because cargos are often driven by multiple dyneins in vivo, our results show that changing the number of dynein motors could allow modulation of dynein function from the mediocre single-dynein limit to robust in vivo-like dynein-driven motion.     

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.     

Coordination of opposite-polarity microtubule motors

Many cargoes move bidirectionally, frequently reversing course between plus- and minus-end microtubule travel. For such cargoes, the extent and importance of interactions between the opposite-polarity motors is unknown. In this paper we test whether opposite-polarity motors on lipid droplets in Drosophila embryos are coordinated and avoid interfering with each other's activity, or whether they engage in a tug of war. To this end we impaired the minus-end transport machinery using dynein and dynactin mutations, and then investigated whether plus-end motion was improved or disrupted. We observe a surprisingly severe impairment of plus-end motion due to these alterations of minus-end motor activity. These observations are consistent with a coordination hypothesis, but cannot be easily explained with a tug of war model. Our measurements indicate that dynactin plays a crucial role in the coordination of plus- and minus-end-directed motors. Specifically, we propose that dynactin enables dynein to participate efficiently in bidirectional transport, increasing its ability to stay "on" during minus-end motion and keeping it "off" during plus-end motion.     

Sorting of cargos between axons and dendrites: modelling of differences in cargo transport in these two types of neurites

Explaining how intracellular cargos are sorted between axons and dendrites is important for a mechanistic understanding of what happens in many neurodegenerative disorders. A simple model of cargo sorting relies on differences in microtubule (MT) orientation between axons and dendrites: in mammalian neurons all MTs in axons have their plus ends directed outward while in proximal regions of dendrites the MT polarity is mixed. It can therefore be assumed that cargos that need to be driven into axons associate with kinesin motors while cargos that need to be driven into dendrites associate with dynein motors. This paper develops equations of cargo transport in axons and dendrites based on the above assumptions. Propagation of a pulse of radiolabelled cargos entering an axon and dendrite is simulated. The model equations are solved utilising the Laplace transform method. Differences in cargo transport between axons and dendrites are discussed.     

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.     

A determinant for directionality of organelle transport in Drosophila embryos

BACKGROUND: Motor-driven transport along microtubules is a primary cellular mechanism for moving and positioning organelles. Many cargoes move bidirectionally by using both minus and plus end-directed motors. How such cargoes undergo controlled net transport is unresolved. RESULTS: Using a combination of genetics, molecular biology, and biophysics, we have identified Halo, a novel regulator of lipid droplet transport in early Drosophila embryos. In embryos lacking Halo, net transport of lipid droplets, but not that of other cargoes, is specifically altered; net transport is minus-end directed at developmental stages when it is normally plus-end directed. This reversal is due to an altered balance of motion at the level of individual organelles; without Halo, travel distances and stall forces are reduced for plus-end and increased for minus-end motion. During development, halo mRNA is highly upregulated just as net plus-end transport is initiated (phase II), and its levels drop precipitously shortly before transport becomes minus-end directed (phase III). Exogenously provided Halo prevents the switch to net minus-end transport in phase III in wild-type embryos and induces net plus-end transport during phase II in halo mutant embryos. This mechanism of regulation is likely to be of general importance because the Drosophila genome encodes a family of related proteins with similar sequences, each transiently expressed in distinct domains. CONCLUSIONS: We conclude that Halo acts as a directionality determinant for embryonic droplet transport and is the first member of a new class of transport regulators.     

Cargos Rotate at Microtubule Intersections during Intracellular Trafficking

Intracellular cargos are transported by molecular motors along actin and microtubules, but how their dynamics depends on the complex structure of the cytoskeletal network remains unclear. In this study, we investigated this longstanding question by measuring simultaneously the rotational and translational dynamics of cargos at microtubule intersections in living cells. We engineered two-faced particles that are fluorescent on one hemisphere and opaque on the other and used their optical anisotropy to report the rotation of cargos. We show that cargos undergo brief episodes of unidirectional and rapid rotation while pausing at microtubule intersections. Probability and amplitude of the cargo rotation depend on the geometry of the intersecting filaments. The cargo rotation is not random motion due to detachment from microtubules, as revealed by statistical analyses of the translational and rotational dynamics. Instead, it is an active rotation driven by motor proteins. Although cargos are known to pause at microtubule intersections, this study reveals a different dimension of dynamics at this seemingly static state and, more importantly, provides direct evidence showing the correlation between cargo rotation and the geometry of underlying microtubule intersections.     

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 .     

Cargo transport: two motors are sometimes better than one

Molecular motor proteins are crucial for the proper distribution of organelles and vesicles in cells. Much of our current understanding of how motors function stems from studies of single motors moving cargos in vitro. More recently, however, there has been mounting evidence that the cooperation of multiple motors in moving cargos and the regulation of motor-filament affinity could be key mechanisms that cells utilize to regulate cargo transport. Here, we review these recent advances and present a picture of how the different mechanisms of regulating the number of motors moving a cargo could facilitate cellular functions.     

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.     

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.     

Organelle transport along microtubules in Xenopus melanophores: evidence for cooperation between multiple motors

Xenopus melanophores have pigment organelles or melanosomes which, in response to hormones, disperse in the cytoplasm or aggregate in the perinuclear region. Melanosomes are transported by microtubule motors, kinesin-2 and cytoplasmic dynein, and an actin motor, myosin-V. We explored the regulation of melanosome transport along microtubules in vivo by using a new fast-tracking routine, which determines the melanosome position every 10 ms with 2-nm precision. The velocity distribution of melanosomes transported by cytoplasmic dynein or kinesin-2 under conditions of aggregation and dispersion presented several peaks and could not be fit with a single Gaussian function. We postulated that the melanosome velocity depends linearly on the number of active motors. According to this model, one to three dynein molecules transport each melanosome in the minus-end direction. The transport in the plus-end direction is mainly driven by one to two copies of kinesin-2. The number of dyneins transporting a melanosome increases during aggregation, whereas the number of active kinesin-2 stays the same during aggregation and dispersion. Thus, the number of active dynein molecules regulates the net direction of melanosome transport. The model also shows that multiple motors of the same polarity cooperate during the melanosome transport, whereas motors of opposite polarity do not compete.     

Cargo Transport at Microtubule Crossings: Evidence for Prolonged Tug-of-War between Kinesin Motors

Intracellular transport of cargos along microtubules is often complicated by the topology of the underlying filament network. The fundamental building blocks for this complex arrangement are filament intersections. The navigation of cargos across microtubule intersections remains poorly understood. Here, we demonstrate that kinesin-driven cargos are engaged in a tug-of-war at microtubule intersections. Tug-of-war events result in long pauses that can last from a few seconds to several minutes. We demonstrate that the extent of the tug-of-war and the duration of pauses change with the number of motors on the cargo and can be regulated by ionic strength. We also show that dwell times at intersections depend on the angle between crossing microtubules. Our data suggest that local microtubule geometry can regulate microtubule-based transport.     

Protein localization by actin treadmilling and molecular motors regulates stereocilia shape and treadmilling rate

We present a physical model that describes the active localization of actin-regulating proteins inside stereocilia during steady-state conditions. The mechanism of localization is through the interplay of free diffusion and directed motion, which is driven by coupling to the treadmilling actin filaments and to myosin motors that move along the actin filaments. The resulting localization of both the molecular motors and their cargo is calculated, and is found to have an exponential (or steeper) profile. This localization can be at the base (driven by actin retrograde flow and minus-end myosin motors), or at the stereocilia tip (driven by plus-end myosin motors). The localization of proteins that influence the actin depolymerization and polymerization rates allow us to describe the narrow shape of the stereocilia base, and the observed increase of the actin polymerization rate with the stereocilia height.     

A bidirectional kinesin motor in live Drosophila embryos

Spindle assembly and elongation involve poleward and away-from-the-pole forces produced by microtubule dynamics and spindle-associated motors. Here, we show that a bidirectional Drosophila Kinesin-14 motor that moves either to the microtubule plus or minus end in vitro unexpectedly causes only minor spindle defects in vivo. However, spindles of mutant embryos are longer than wild type, consistent with increased plus-end motor activity. Strikingly, suppressing spindle dynamics by depriving embryos of oxygen causes the bidirectional motor to show increased accumulation at distal or plus ends of astral microtubules relative to wild type, an effect not observed for a mutant motor defective in motility. Increased motor accumulation at microtubule plus ends may be due to increased slow plus-end movement of the bidirectional motor under hypoxia, caused by perturbation of microtubule dynamics or inactivation of the only other known Drosophila minus-end spindle motor, cytoplasmic dynein. Negative-stain electron microscopy images are consistent with highly cooperative motor binding to microtubules, and gliding assays show dependence on motor density for motility. Mutant effects of the bidirectional motor on spindle function may be suppressed under normal conditions by motor: motor interactions and minus-end movement induced by spindle dynamics. These forces may also bias wild-type motor movement toward microtubule minus ends in live cells. Our findings link motor : motor interactions to function in vivo by showing that motor density, together with cellular dynamics, may influence motor function in live cells.