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.     

Kinesin and dynein-dynactin at intersecting microtubules: motor density affects dynein function

Kinesin and cytoplasmic dynein are microtubule-based motor proteins that actively transport material throughout the cell. Microtubules can intersect at a variety of angles both near the nucleus and at the cell periphery, and the behavior of molecular motors at these intersections has implications for long-range transport efficiency and accuracy. To test motor function at microtubule intersections, crossovers were arranged in vitro using flow to orient successive layers of filaments. Single kinesin and cytoplasmic dynein-dynactin molecules fused with green-fluorescent protein, and artificial bead cargos decorated with multiple motors, were observed while they encountered intersections. Single kinesins tend to cross intersecting microtubules, whereas single dynein-dynactins have a more varied response. For bead cargos, kinesin motion is independent of motor number. Dynein beads with high motor numbers pause, but their actions become more varied as the motor number decreases. These results suggest that regulating the number of active dynein molecules could change a motile cargo into one that is anchored at an intersection, consistent with dynein's proposed transport and tethering functions in the cell.     

Assembled capsules transportation driven by motor proteins

An active biomimetic system by integrating kinesin motor, microtubule, and man-made biomimetic microcapsule has been constructed. Biomimetic microcapsules were fabricated by using the layer-by-layer technique and could serve as cargos in this active biomimetic system. Both of the hollow and filled capsules as cargos can be transported by kinesin motors along microtubules. It may help to create kinesin-powered complex hybrid micro- and nanodevices.     

Microtubule‐binding protein doublecortin‐like kinase 1 (DCLK1) guides kinesin‐3‐mediated cargo transport to dendrites

In neurons, the polarized distribution of vesicles and other cellular materials is established through molecular motors that steer selective transport between axons and dendrites. It is currently unclear whether interactions between kinesin motors and microtubule‐binding proteins can steer polarized transport. By screening all 45 kinesin family members, we systematically addressed which kinesin motors can translocate cargo in living cells and drive polarized transport in hippocampal neurons. While the majority of kinesin motors transport cargo selectively into axons, we identified five members of the kinesin‐3 (KIF1) and kinesin‐4 (KIF21) subfamily that can also target dendrites. We found that microtubule‐binding protein doublecortin‐like kinase 1 (DCLK1) labels a subset of dendritic microtubules and is required for KIF1‐dependent dense‐core vesicles (DCVs) trafficking into dendrites and dendrite development. Our study demonstrates that microtubule‐binding proteins can provide local signals for specific kinesin motors to drive polarized cargo transport.     

MAPping out distribution routes for kinesin couriers

In the crowded environment of eukaryotic cells, diffusion is an inefficient distribution mechanism for cellular components. Long‐distance active transport is required and is performed by molecular motors including kinesins. Furthermore, in highly polarised, compartmentalised and plastic cells such as neurons, regulatory mechanisms are required to ensure appropriate spatio‐temporal delivery of neuronal components. The kinesin machinery has diversified into a large number of kinesin motor proteins as well as adaptor proteins that are associated with subsets of cargo. However, many mechanisms contribute to the correct delivery of these cargos to their target domains. One mechanism is through motor recognition of sub‐domain‐specific microtubule (MT) tracks, sign‐posted by different tubulin isoforms, tubulin post‐translational modifications, tubulin GTPase activity and MT‐associated proteins (MAPs). With neurons as a model system, a critical review of these regulatory mechanisms is presented here, with a particular focus on the emerging contribution of compartmentalised MAPs. Overall, we conclude that – especially for axonal cargo – alterations to the MT track can influence transport, although in vivo, it is likely that multiple track‐based effects act synergistically to ensure accurate cargo distribution.     

One axon,many kinesins: What's the logic?

A large number of membrane-bounded organelles, protein complexes, and mRNAs are transported along microtubules to different locations within the neuronal axon. Axonal transport in the anterograde direction is carried out by members of a superfamily of specialized motor proteins, the kinesins. All kinesins contain a conserved motor domain that hydrolyses ATP to generate movement along microtubules. Regions outside the motor domain are responsible for cargo binding and regulation of motor activity. Present in a soluble, inactive form in the cytoplasm, kinesins are activated upon cargo binding. Selective targeting of different types of kinesin motors to specific cargoes is directed by amino acid sequences situated in their variable tails. Cargo proteins with specific function at their destination, bind directly to specific kinesins for transport. Whereas most kinesins move to microtubule plus-ends, a small number of them move to microtubule minus-ends, and may participate in retrograde axonal transport. Axonal transport by kinesins has a logic: Fully assembled, multisubunit, functional complexes (e.g., ion channel complexes, signaling complexes, RNA-protein complexes) are transported to their destination by kinesin motors that interact transiently (i.e., during transport only) with one of the complexes' subunits.     

Modulation of Kinesin’s Load-Bearing Capacity by Force Geometry and the Microtubule Track

Kinesin motors and their associated microtubule tracks are essential for long-distance transport of cellular cargos. Intracellular activity and proper recruitment of kinesins is regulated by biochemical signaling, cargo adaptors, microtubule-associated proteins, and mechanical forces. In this study, we found that the effect of opposing forces on the kinesin-microtubule attachment duration depends strongly on experimental assay geometry. Using optical tweezers and the conventional single-bead assay, we show that detachment of kinesin from the microtubule is likely accelerated by forces vertical to the long axis of the microtubule due to contact of the single bead with the underlying microtubule. We used the three-bead assay to minimize the vertical force component and found that when the opposing forces are mainly parallel to the microtubule, the median value of attachment durations between kinesin and microtubules can be up to 10-fold longer than observed using the single-bead assay. Using the three-bead assay, we also found that not all microtubule protofilaments are equivalent interacting substrates for kinesin and that the median value of attachment durations of kinesin varies by more than 10-fold, depending on the relative angular position of the forces along the circumference of the microtubule. Thus, depending on the geometry of forces across the microtubule, kinesin can switch from a fast detaching motor (median attachment duration <0.2 s) to a persistent motor that sustains attachment (median attachment duration >3 s) at high forces (5 pN). Our data show that the load-bearing capacity of the kinesin motor is highly variable and can be dramatically affected by off-axis forces and forces across the microtubule lattice, which has implications for a range of cellular activities, including cell division and organelle transport.     

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.     

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

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

Motor proteins: kinesin can replace Myosin

Directional transport of specific cargos is tuned to specific molecular motors and specific cytoskeletal tracks. Myosin V transports its cargo on actin cables, whereas kinesin or dynein transport their cargo on microtubules. A recent study shows that an engineered kinesin can substitute for myosin V and its cargo-specific transport and subsequent cellular functions.     

Kinesin motors as molecular machines

Molecular motor proteins, fueled by energy from ATP hydrolysis, move along actin filaments or microtubules, performing work in the cell. The kinesin microtubule motors transport vesicles or organelles, assemble bipolar spindles or depolymerize microtubules, functioning in basic cellular processes. The mechanism by which motor proteins convert energy from ATP hydrolysis into work is likely to differ in basic ways from man-made machines. Several mechanical elements of the kinesin motors have now been tentatively identified, permitting researchers to begin to decipher the mechanism of motor function. The force-producing conformational changes of the motor and the means by which they are amplified are probably different for the plus- and minus-end kinesin motors.     

Dynein anchors its mRNA cargo after apical transport in the Drosophila blastoderm embryo

Molecular motors actively transport many types of cargo along the cytoskeleton in a wide range of organisms. One class of cargo is localized mRNAs, which are transported by myosin on actin filaments or by kinesin and dynein on microtubules. How the cargo is kept at its final intracellular destination and whether the motors are recycled after completion of transport are poorly understood. Here, we use a new RNA anchoring assay in living Drosophila blastoderm embryos to show that apical anchoring of mRNA after completion of dynein transport does not depend on actin or on continuous active transport by the motor. Instead, apical anchoring of RNA requires microtubules and involves dynein as a static anchor that remains with the cargo at its final destination. We propose a general principle that could also apply to other dynein cargo and to some other molecular motors, whereby cargo transport and anchoring reside in the same molecule.     

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.     

Hither and yon: a review of bi-directional microtubule-based transport

Active transport is critical for cellular organization and function, and impaired transport has been linked to diseases such as neuronal degeneration. Much long distance transport in cells uses opposite polarity molecular motors of the kinesin and dynein families to move cargos along microtubules. It is increasingly clear that many cargos are moved by both sets of motors, and frequently reverse course. This review compares this bi-directional transport to the more well studied uni-directional transport. It discusses some bi-directionally moving cargos, and critically evaluates three different physical models for how such transport might occur. It then considers the evidence for the number of active motors per cargo, and how the net or average direction of transport might be controlled. The likelihood of a complex linking the activities of kinesin and dynein is also discussed. The paper concludes by reviewing elements of apparent universality between different bi-directionally moving cargos and by briefly considering possible reasons for the existence of bi-directional transport.     

Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport.These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles ¹. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available.Building on the classic inverted motility assay ², the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo ³. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface.The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA⁴, quantum dots ⁵ or a wide variety of antigens via biotinylated antibodies ^4-6.Download video file.^{(57M, mov)}     

Processive movement of single kinesins on crowded microtubules visualized using quantum dots

Kinesin-1 is a processive molecular motor transporting cargo along microtubules. Inside cells, several motors and microtubule-associated proteins compete for binding to microtubules. Therefore, the question arises how processive movement of kinesin-1 is affected by crowding on the microtubule. Here we use total internal reflection fluorescence microscopy to image in vitro the runs of single quantum dot-labelled kinesins on crowded microtubules under steady-state conditions and to measure the degree of crowding on a microtubule at steady-state. We find that the runs of kinesins are little affected by high kinesin densities on a microtubule. However, the presence of high densities of a mutant kinesin that is not able to step efficiently reduces the average speed of wild-type kinesin, while hardly changing its processivity. This indicates that kinesin waits in a strongly bound state on the microtubule when encountering an obstacle until the obstacle unbinds and frees the binding site for kinesin's next step. A simple kinetic model can explain quantitatively the behaviour of kinesin under both crowding conditions.     

Direction and speed of microtubule movements driven by kinesin motors arranged on catchin thick filaments

Conventional kinesin (Kinesin-1) is a microtubule-based molecular motor that supports intracellular vesicle/organelle transport in various eukaryotic cells. To arrange kinesin motors similarly to myosin motors on thick filaments in muscles, the motor domain of rat conventional kinesin (amino acid residues 1-430) fused to the C-terminal 829 amino acid residues of catchin (KHC430Cat) was bacterially expressed and attached to catchin filaments that can attach to and arrange myosin molecules in a bipolar manner on their surface. Unlike the case of myosin where actin filaments move toward the center much faster than in the opposite direction along the catchin filaments, microtubules moved at the same speed in both directions. In addition, many microtubules moved across the filaments at the same speed with various angles between the axes of the microtubule and catchin filament. Kinesin/catchin chimera proteins with a shorter kinesin neck domain were also prepared. Those without the whole hinge 1 domain and the C-terminal part of the neck helix moved microtubules toward the center of the catchin filaments significantly, but only slightly, faster than in the opposite direction, although the movements in both directions were slower than those of the KHC430Cat construct. The results suggest that kinesin has substantial mechanical flexibility within the motor domain, possibly within the neck linker, enabling its interaction with microtubules having any orientation.     

Conventional kinesin mediates microtubule-microtubule interactions in vivo

Conventional kinesin is a ubiquitous organelle transporter that moves cargo toward the plus-ends of microtubules. In addition, several in vitro studies indicated a role of conventional kinesin in cross-bridging and sliding microtubules, but in vivo evidence for such a role is missing. In this study, we show that conventional kinesin mediates microtubule-microtubule interactions in the model fungus Ustilago maydis. Live cell imaging and ultrastructural analysis of various mutants in Kin1 revealed that this kinesin-1 motor is required for efficient microtubule bundling and participates in microtubule bending in vivo. High levels of Kin1 led to increased microtubule bending, whereas a rigor-mutation in the motor head suppressed all microtubule motility and promoted strong microtubule bundling, indicating that kinesin can form cross-bridges between microtubules in living cells. This effect required a conserved region in the C terminus of Kin1, which was shown to bind microtubules in vitro. In addition, a fusion protein of yellow fluorescent protein and the Kin1tail localized to microtubule bundles, further supporting the idea that a conserved microtubule binding activity in the tail of conventional kinesins mediates microtubule-microtubule interactions in vivo.     

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.     

Microtubule motor proteins and the organization of the pollen tube cytoplasm

Molecular motors are molecules that drive a wide range of activities (for example, organelle movement, chromosome segregation, and flagellar movement) in cells. Thus, they play essential roles in diverse cellular functions. Understanding their structures, mechanisms of action and different roles is therefore of great practical importance. The role of microtubules during pollen tube growth is presently not identified, nor are basic properties. We do not know, for example, where microtubules are organized, the extent of microtubule dynamics, and the polarity of microtubules in the pollen tube. Roles of microtubules and related motors in organelle trafficking are not clear. Regardless of scarce information, microtubule-based motors of both the kinesin and dynein families have been identified in the pollen tube. Most of these microtubule motors have also been found in association with membrane-bounded organelles, which suggest that these proteins could translocate organelles or vesicles along microtubules. The biochemical features of these proteins are typical of the motor protein class. Immunofluorescence microscopy of pollen tubes probed with antibodies that cross-react with microtubule motors indicate that these proteins are localized in different regions of the pollen tube; therefore, they could have different roles. Although a number of microtubule motors have been identified in the pollen tube, the role of these proteins during pollen tube germination and growth or organelle movement is not yet recognized, as tube elongation and organelle movement in the pollen tube depend mostly on actin filaments. In the effort to understand the specific role that microtubules and related motors have in the pollen tube, it is therefore necessary to identify the molecular machinery that interacts with microtubules. Furthermore, it is crucial to clearly establish the types of interaction between organelles and microtubules. This review summarizes the current state of the art on microtubule motors in the pollen tube, mainly surrounding the putative roles of microtubule motors in organelle movement and cytoplasmic organization. Some hypotheses and speculations are also presented.