Single Molecule Imaging Reveals Differences in Microtubule Track Selection Between Kinesin Motors

Cells generate diverse microtubule populations by polymerization of a common α/β-tubulin building block. How microtubule associated proteins translate microtubule heterogeneity into specific cellular functions is not clear. We evaluated the ability of kinesin motors involved in vesicle transport to read microtubule heterogeneity by using single molecule imaging in live cells. We show that individual Kinesin-1 motors move preferentially on a subset of microtubules in COS cells, identified as the stable microtubules marked by post-translational modifications. In contrast, individual Kinesin-2 (KIF17) and Kinesin-3 (KIF1A) motors do not select subsets of microtubules. Surprisingly, KIF17 and KIF1A motors that overtake the plus ends of growing microtubules do not fall off but rather track with the growing tip. Selection of microtubule tracks restricts Kinesin-1 transport of VSVG vesicles to stable microtubules in COS cells whereas KIF17 transport of Kv1.5 vesicles is not restricted to specific microtubules in HL-1 myocytes. These results indicate that kinesin families can be distinguished by their ability to recognize microtubule heterogeneity. Furthermore, this property enables kinesin motors to segregate membrane trafficking events between stable and dynamic microtubule populations.     

Processive cytoskeletal motors studied with single-molecule fluorescence techniques

Processive cytoskeletal motors from the myosin, kinesin, and dynein families walk on actin filaments and microtubules to drive cellular transport and organization in eukaryotic cells. These remarkable molecular machines are able to take hundreds of successive steps at speeds of up to several microns per second, allowing them to effectively move vesicles and organelles throughout the cytoplasm. Here, we focus on single-molecule fluorescence techniques and discuss their wide-ranging applications to the field of cytoskeletal motor research. We cover both traditional fluorescence and sub-diffraction imaging of motors, providing examples of how fluorescence data can be used to measure biophysical parameters of motors such as coordination, stepping mechanism, gating, and processivity. We also outline some remaining challenges in the field and suggest future directions.     

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.     

Dynein, dynactin, and kinesin II's interaction with microtubules is regulated during bidirectional organelle transport

The microtubule motors, cytoplasmic dynein and kinesin II, drive pigmented organelles in opposite directions in Xenopus melanophores, but the mechanism by which these or other motors are regulated to control the direction of organelle transport has not been previously elucidated. We find that cytoplasmic dynein, dynactin, and kinesin II remain on pigment granules during aggregation and dispersion in melanophores, indicating that control of direction is not mediated by a cyclic association of motors with these organelles. However, the ability of dynein, dynactin, and kinesin II to bind to microtubules varies as a function of the state of aggregation or dispersion of the pigment in the cells from which these molecules are isolated. Dynein and dynactin bind to microtubules when obtained from cells with aggregated pigment, whereas kinesin II binds to microtubules when obtained from cells with dispersed pigment. Moreover, the microtubule binding activity of these motors/dynactin can be reversed in vitro by the kinases and phosphatase that regulate the direction of pigment granule transport in vivo. These findings suggest that phosphorylation controls the direction of pigment granule transport by altering the ability of dynein, dynactin, and kinesin II to interact with microtubules.     

Molecular motors in axonal transport

Neurons require a large amount of intracellular transport. Cytoplasmic polypeptides and membrane-bounded organelles move from the perikaryon, down the length of the axon, and to the synaptic terminals. This movement occurs at distinct rates and is termed axonal transport. Axonal transport is divided into the slow transport of cytoplasmic proteins including glycolytic enzymes and cytoskeletal structures and the fast transport of membrane-bounded organelles along linear arrays of microtubules. The polypeptide compositions of the rate classes of axonal transport have been well characterized, but the underlying molecular mechanisms of this movement are less clear. Progress has been particularly slow toward understanding force-generation in slow transport, but recent developments have provided insight into the molecular motors involved in fast axonal transport. Recent advances in the cellular and molecular biology of one fast axonal transport motor, kinesin, have provided a clearer understanding of organelle movement along microtubules. The availability of cellular and molecular probes for kinesin and other putative axonal transport motors have led to a reevaluation of our understanding of intracellular motility.     

Dynactin: coordinating motors with opposite inclinations

In eukaryotic cells, many organelles are transported bidirectionally along microtubules by kinesin and dynein. These opposite-polarity motors appear to be coordinated to avoid interfering with each other's function. New work has provided the first molecular insight into how such coordination might occur.     

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.     

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.     

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.     

Modeling bidirectional transport of quantum dot nanoparticles in membrane nanotubes

This paper develops a model of transport of quantum dot (QD) nanoparticles in membrane nanotubes (MNTs). It is assumed that QDs are transported inside intracellular organelles (called here nanoparticle-loaded vesicles, NLVs) that are propelled by either kinesin or dynein molecular motors while moving on microtubules (MTs). A vesicle may have both types of motors attached to it, but the motors are assumed to work in a cooperative fashion, meaning that at a given time the vesicle is moved by either kinesin or dynein motors. The motors are assumed not to work against each other, when one type of motors is pulling the vesicle, the other type is inactive. From time to time the motors may switch their roles: passive motors can become active motors and vice versa, resulting in the change of the vesicle’s direction of motion. It is further assumed that QDs can escape NLVs and become free QDs, which are then transported by diffusion. Free QDs can be internalized by NLVs. The effects of two possible types of MT orientation in MNTs are investigated: when all MTs have a uniform polarity orientation, with their plus-ends directed toward one of the cells connected by an MNT, and when MTs have a mixed polarity orientation, with half of MTs having their plus-ends directed toward one of the cells and the other half having their plus-ends directed toward the other cell. Computational results are presented for three cases. The first case is when organelles are as likely to be transported by kinesin motors as by dynein motors. The second case is when organelles are more likely to be transported by kinesin motors than by dynein motors, and the third case is when NLVs do not associate with dynein motors at all.     

Myosin-dependent transport in neurons

Axonal transport in neurons has been shown to be microtubule dependent, driven by the molecular motor proteins kinesin and dynein. However, organelles undergoing fast transport can often pause or rapidly change directions without apparent dissociation from their transport tracks. Cytoskeletal polymers such as neurofilaments and microtubules have also been shown to make infrequent but rapid movements in axons indicating that their transport is likely to involve molecular motors. In addition, neurons have multiple compartments that are devoid of microtubules where transport of organelles is still seen to occur. These areas are rich in other cytoskeletal polymers such as actin filaments. Transported organelles have been shown to associate with multiple motor proteins including myosins. This suggests that nonmicrotubule-based transport may be myosin driven. In this review we will focus our attention on myosin motors known to be present in neurons and evaluate the evidence that they contribute to transport or other functions in the different compartments of the neuron.     

Kinesin‐2 motors adapt their stepping behavior for processive transport on axonemes and microtubules

Two structurally distinct filamentous tracks, namely singlet microtubules in the cytoplasm and axonemes in the cilium, serve as railroads for long‐range transport processes in vivo. In all organisms studied so far, the kinesin‐2 family is essential for long‐range transport on axonemes. Intriguingly, in higher eukaryotes, kinesin‐2 has been adapted to work on microtubules in the cytoplasm as well. Here, we show that heterodimeric kinesin‐2 motors distinguish between axonemes and microtubules. Unlike canonical kinesin‐1, kinesin‐2 takes directional, off‐axis steps on microtubules, but it resumes a straight path when walking on the axonemes. The inherent ability of kinesin‐2 to side‐track on the microtubule lattice restricts the motor to one side of the doublet microtubule in axonemes. The mechanistic features revealed here provide a molecular explanation for the previously observed partitioning of oppositely moving intraflagellar transport trains to the A‐ and B‐tubules of the same doublet microtubule. Our results offer first mechanistic insights into why nature may have co‐evolved the heterodimeric kinesin‐2 with the ciliary machinery to work on the specialized axonemal surface for two‐way traffic.     

Fluorescence correlation spectroscopy reveals the dynamics of kinesins interacting with organelles during microtubule-dependent transport in cells

Microtubule-dependent motors usually work together to transport organelles through the crowded intracellular milieu. Thus, transport performance depends on how motors organize on the cargo. Unfortunately, the lack of methodologies capable of measuring this organization in cells determines that many aspects of the collective action of motors remain elusive. Here, we combined fluorescence fluctuations and single particle tracking techniques to address how kinesins organize on rod-like mitochondria moving along microtubules in cells. This methodology simultaneously provides mitochondria trajectories and EGFP-tagged kinesin-1 intensity at different mitochondrial positions with millisecond resolution. We show that kinesin exchange at the mitochondrion surface is within ~100 ms and depends on the organelle speed. During anterograde transport, the mitochondrial leading tip presents slower motor exchange in comparison to the rear tip. In contrast, retrograde mitochondria show similar exchange rates of kinesins at both tips. Numerical simulations provide theoretical support to these results and evidence that motors do not share the load equally during intracellular transport.     

Computational Modeling Reveals Optimal Strategy for Kinase Transport by Microtubules to Nerve Terminals

Intracellular transport of proteins by motors along cytoskeletal filaments is crucial to the proper functioning of many eukaryotic cells. Since most proteins are synthesized at the cell body, mechanisms are required to deliver them to the growing periphery. In this article, we use computational modeling to study the strategies of protein transport in the context of JNK (c-JUN NH2-terminal kinase) transport along microtubules to the terminals of neuronal cells. One such strategy for protein transport is for the proteins of the JNK signaling cascade to bind to scaffolds, and to have the whole protein-scaffold cargo transported by kinesin motors along microtubules. We show how this strategy outperforms protein transport by diffusion alone, using metrics such as signaling rate and signal amplification. We find that there exists a range of scaffold concentrations for which JNK transport is optimal. Increase in scaffold concentration increases signaling rate and signal amplification but an excess of scaffolds results in the dilution of reactants. Similarly, there exists a range of kinesin motor speeds for which JNK transport is optimal. Signaling rate and signal amplification increases with kinesin motor speed until the speed of motor translocation becomes faster than kinase/scaffold-motor binding. Finally, we suggest experiments that can be performed to validate whether, in physiological conditions, neuronal cells do indeed adopt such an optimal strategy. Understanding cytoskeletal-assisted protein transport is crucial since axonal and cell body accumulation of organelles and proteins is a histological feature in many human neurodegenerative diseases. In this paper, we have shown that axonal transport performance changes with altered transport component concentrations and transport speeds wherein these aspects can be modulated to improve axonal efficiency and prevent or slowdown axonal deterioration.     

Molecular motors: strategies to get along

The majority of active transport in the cell is driven by three classes of molecular motors: the kinesin and dynein families that move toward the plus-end and minus-end of microtubules, respectively, and the unconventional myosin motors that move along actin filaments. Each class of motor has different properties, but in the cell they often function together. In this review we summarize what is known about their single-molecule properties and the possibilities for regulation of such properties. In view of new results on cytoplasmic dynein, we attempt to rationalize how these different classes of motors might work together as part of the intracellular transport machinery. We propose that kinesin and myosin are robust and highly efficient transporters, but with somewhat limited room for regulation of function. Because cytoplasmic dynein is less efficient and robust, to achieve function comparable to the other motors it requires a number of accessory proteins as well as multiple dyneins functioning together. This necessity for additional factors, as well as dynein's inherent complexity, in principle allows for greatly increased control of function by taking the factors away either singly or in combination. Thus, dynein's contribution relative to the other motors can be dynamically tuned, allowing the motors to function together differently in a variety of situations.     

Effects of Obstacles on the Dynamics of Kinesins,Including Velocity and Run Length,Predicted by a Model of Two Dimensional Motion

Kinesins are molecular motors which walk along microtubules by moving their heads to different binding sites. The motion of kinesin is realized by a conformational change in the structure of the kinesin molecule and by a diffusion of one of its two heads. In this study, a novel model is developed to account for the 2D diffusion of kinesin heads to several neighboring binding sites (near the surface of microtubules). To determine the direction of the next step of a kinesin molecule, this model considers the extension in the neck linkers of kinesin and the dynamic behavior of the coiled-coil structure of the kinesin neck. Also, the mechanical interference between kinesins and obstacles anchored on the microtubules is characterized. The model predicts that both the kinesin velocity and run length (i.e., the walking distance before detaching from the microtubule) are reduced by static obstacles. The run length is decreased more significantly by static obstacles than the velocity. Moreover, our model is able to predict the motion of kinesin when other (several) motors also move along the same microtubule. Furthermore, it suggests that the effect of mechanical interaction/interference between motors is much weaker than the effect of static obstacles. Our newly developed model can be used to address unanswered questions regarding degraded transport caused by the presence of excessive tau proteins on microtubules.     

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.     

Single-molecule investigation of the interference between kinesin,tau and MAP2c

Motor proteins and microtubule-associated proteins (MAPs) play important roles in cellular transport, regulation of shape and polarity of cells. While motor proteins generate motility, MAPs are thought to stabilize the microtubule tracks. However, the proteins also interfere with each other, such that MAPs are able to inhibit transport of vesicles and organelles in cells. In order to investigate the mechanism of MAP-motor interference in molecular detail, we have studied single kinesin molecules by total internal reflection fluorescence microscopy in the presence of different neuronal MAPs (tau, MAP2c). The parameters observed included run-length (a measure of processivity), velocity and frequency of attachment. The main effect of MAPs was to reduce the attachment frequency of motors. This effect was dependent on the concentration, the affinity to microtubules and the domain composition of MAPs. In contrast, once attached, the motors did not show a change in speed, nor in their run-length. The results suggest that MAPs can regulate motor activity on the level of initial attachment, but not during motion.     

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.     

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.