Microtubule Tracks Research Articles

Mechanism and regulation of kinesin motors.

Kinesins are a diverse superfamily of microtubule-based motors that perform fundamental roles in intracellular transport, cytoskeletal dynamics and cell division. These motors share a characteristic motor domain that powers unidirectional motility and force generation along microtubules, and they possess unique tail domains that recruit accessory proteins and facilitate oligomerization, regulation and cargo recognition. The location, direction and timing of kinesin-driven processes are tightly regulated by various cofactors, adaptors, microtubule tracks and microtubule-associated proteins. This Review focuses on recent structural and functional studies that reveal how members of the kinesin superfamily use the energy of ATP hydrolysis to transport cargoes, depolymerize microtubules and regulate microtubule dynamics. I also survey how accessory proteins and post-translational modifications regulate the autoinhibition, cargo binding and motility of some of the best-studied kinesins. Despite much progress, the mechanism and regulation of kinesins are still emerging, and unresolved questions can now be tackled using newly developed approaches in biophysics and structural biology.

Modelling intracellular transport in crowded environments: effects of motor association to cargos.

In intracellular transports, motor proteins transport macromolecules as cargos to desired locations by moving on biopolymers such as microtubules. Recent experiments suggest that, while moving in crowded environments, cargos that can associate motor proteins during their translocation have larger run-length and association time compared to free motors. Here, we model the dynamics of a cargo that can associate at the most m free motors present on the microtubule track as obstacles to its motion. The proposed models display competing effects of association and crowding, leading to a peak in the run-length with the free-motor density. For and 3, we show that this feature is governed by the largest eigenvalue of the transition matrix describing the cargo dynamics. In all the above cases, free motors are assumed to be present on the microtubule as stalled obstacles. We finally compare simulation results for the run-length for general scenarios where the free motors undergo processive motion in addition to binding and unbinding to or from the microtubule.

Competition between physical search and a weak-to-strong transition rate-limits kinesin binding times.

The self-organization of cells relies on the profound complexity of protein-protein interactions. Challenges in directly observing these events have hindered progress toward understanding their diverse behaviors. One notable example is the interaction between molecular motors and cytoskeletal systems that combine to perform a variety of cellular functions. In this work, we leverage theory and experiments to identify and quantify the rate-limiting mechanism of the initial association between a cargo-bound kinesin motor and a microtubule track. Recent advances in optical tweezers provide binding times for several lengths of kinesin motors trapped at varying distances from a microtubule, empowering the investigation of competing models. We first explore a diffusion-limited model of binding. Through Brownian dynamics simulations and simulation-based inference, we find this simple diffusion model fails to explain the experimental binding times, but an extended model that accounts for the ADP state of the molecular motor agrees closely with the data, even under the scrutiny of penalizing for additional model complexity. We provide quantification of both kinetic rates and biophysical parameters underlying the proposed binding process. Our model suggests that a typical binding event is limited by ADP state rather than physical search. Lastly, we predict how these association rates can be modulated in distinct ways through variation of environmental concentrations and physical properties.

A model for cooperativity of kinesin-4 motors by communicating through the microtubule track

Kinesin-4 motor proteins at nanomolar concentrations can accumulate at microtubule plus ends, regulating the microtubule length. Recent experimental studies showed that at picomolar motor concentrations, under which nearly no motor crowding can occur, the run length of the kinesin-4 motor increases whereas the velocity decreases with the motor concentration, indicating the presence of a long-range communication between the motors through the microtubule track. However, the mechanism of the long-range communication is illusive. Here, based on the proposal that large conformational changes of the tubulin induced by the strong interaction with a kinesin head can propagate along the microtubule such that influence the tubulin-binding energy of other motors distant away, the dynamics of the kinesin-4 motor at the picomolar motor concentrations is studied theoretically, explaining quantitatively the experimental data. Moreover, the lifetime of the motor when multiple motors are jammed near the plus ends at high motor concentrations is studied theoretically.

Myosin F controls actin organization and dynamics in Toxoplasma gondii.

Intracellular cargo transport is a ubiquitous cellular process in all eukaryotes. In many cell types, membrane bound cargo is associated with molecular motors which transport cargo along microtubule and actin tracks. In Toxoplasma gondii (T. gondii), an obligate intracellular parasite in the phylum Apicomplexa, organization of the endomembrane pathway depends on actin and an unconventional myosin motor, myosin F (MyoF). Loss of MyoF and actin disrupts vesicle transport, organelle positioning, and division of the apicoplast, a nonphotosynthetic plastid organelle. How this actomyosin system contributes to these cellular functions is still unclear. Using live-cell imaging, we observed that MyoF-EmeraldFP (MyoF-EmFP) displayed a dynamic and filamentous-like organization in the parasite cytosol, reminiscent of cytosolic actin filament dynamics. MyoF was not associated with the Golgi, apicoplast or dense granule surfaces, suggesting that it does not function using the canonical cargo transport mechanism. Instead, we found that loss of MyoF resulted in a dramatic rearrangement of the actin cytoskeleton in interphase parasites accompanied by significantly reduced actin dynamics. However, actin organization during parasite replication and motility was unaffected by the loss of MyoF. These findings revealed that MyoF is an actin organizing protein in Toxoplasma and facilitates cargo movement using an unconventional transport mechanism.

Persistent PKA activation redistributes NaV1.5 to the cell surface of adult rat ventricular myocytes.

During chronic stress, persistent activation of cAMP-dependent protein kinase (PKA) occurs, which can contribute to protective or maladaptive changes in the heart. We sought to understand the effect of persistent PKA activation on NaV1.5 channel distribution and function in cardiomyocytes using adult rat ventricular myocytes as the main model. PKA activation with 8CPT-cAMP and okadaic acid (phosphatase inhibitor) caused an increase in Na+ current amplitude without altering the total NaV1.5 protein level, suggesting a redistribution of NaV1.5 to the myocytes' surface. Biotinylation experiments in HEK293 cells showed that inhibiting protein trafficking from intracellular compartments to the plasma membrane prevented the PKA-induced increase in cell surface NaV1.5. Additionally, PKA activation induced a time-dependent increase in microtubule plus-end binding protein 1 (EB1) and clustering of EB1 at myocytes' peripheral surface and intercalated discs (ICDs). This was accompanied by a decrease in stable interfibrillar microtubules but an increase in dynamic microtubules along the myocyte surface. Imaging and coimmunoprecipitation experiments revealed that NaV1.5 interacted with EB1 and β-tubulin, and both interactions were enhanced by PKA activation. We propose that persistent PKA activation promotes NaV1.5 trafficking to the peripheral surface of myocytes and ICDs by providing dynamic microtubule tracks and enhanced guidance by EB1. Our proposal is consistent with an increase in the correlative distribution of NaV1.5, EB1, and β-tubulin at these subcellular domains in PKA-activated myocytes. Our study suggests that persistent PKA activation, at least during the initial phase, can protect impulse propagation in a chronically stressed heart by increasing NaV1.5 at ICDs.

Timing of Phagosome Maturation Depends on Their Transport Switching from Actin to Microtubule Tracks.

Phagosomes, specialized membrane compartments responsible for digesting internalized pathogens, undergo sequential dynamic and biochemical changes as they mature from nascent phagosomes to degradative phagolysosomes. Maturation of phagosomes depends on their transport along actin filaments and microtubules. However, the specific quantitative relationship between the biochemical transformation and transport dynamics remains poorly characterized. The autonomous nature of phagosomes, moving and maturing at different rates, makes understanding this relationship challenging. Addressing this challenge, in this study we engineered particle sensors to image and quantify single phagosomes' maturation. We found that as phagosomes move from the actin cortex to microtubule tracks, the timing of their actin-to-microtubule transition governs the duration of the early phagosome stage before acquiring degradative capacities. Prolonged entrapment of phagosomes in the actin cortex extends the early phagosome stage by delaying the dissociation of early endosome markers and phagosome acidification. Conversely, a shortened transition from actin- to microtubule-based movements causes the opposite effect on phagosome maturation. These results suggest that the actin- and microtubule-based transport of phagosomes functions like a "clock" to coordinate the timing of biochemical events during phagosome maturation, which is crucial for effective pathogen degradation.

SpindlesTracker: An Automatic and Low-cost Labeled Workflow for Spindle Analysis.

Quantitative analysis of spindle dynamics in mitosis through fluorescence microscopy requires tracking spindle elongation in noisy image sequences. Deterministic methods, which use typical microtubule detection and tracking methods, perform poorly in the sophisticated background of spindles. In addition, the expensive data labeling cost also limits the application of machine learning in this field. Here we present a fully automatic and low-cost labeled workflow that efficiently analyzes the dynamic spindle mechanism of time-lapse images, called SpindlesTracker. In this workflow, we design a network named YOLOX-SP which can accurately detect the location and endpoint of each spindle under box-level data supervision. We then optimize the algorithm SORT and MCP for spindle's tracking and skeletonization. As there was no publicly available dataset, we annotated a S.pombe dataset that was entirely acquired from the real world for both training and evaluation. Extensive experiments demonstrate that SpindlesTracker achieves excellent performance in all aspects, while reducing label costs by 60%. Specifically, it achieves 84.1% mAP in spindle detection and over 90% accuracy in endpoint detection. Furthermore, the improved algorithm enhances tracking accuracy by 1.3% and tracking precision by 6.5%. Statistical results also indicate that the mean error of spindle length is within 1 μm. In summary, SpindlesTracker holds significant implications for the study of mitotic dynamic mechanisms and can be readily extended to the analysis of other filamentous objects. The code and the dataset are both released on https://github.com/lizhogn/SpindlesTrackerGitHub.

Simulations suggest robust microtubule attachment of kinesin and dynein in antagonistic pairs

Intracellular transport is propelled by kinesin and cytoplasmic dynein motors that carry membrane-bound vesicles and organelles bidirectionally along microtubule tracks. Much is known about these motors at the molecular scale, but many questions remain regarding how kinesin and dynein cooperate and compete during bidirectional cargo transport at the cellular level. The goal of the present study was to use a stochastic stepping model constructed by using published load-dependent properties of kinesin-1 and dynein-dynactin-BicD2 (DDB) to identify specific motor properties that determine the speed, directionality, and transport dynamics of a cargo carried by one kinesin and one dynein motor. Model performance was evaluated by comparing simulations to recently published experiments of kinesin-DDB pairs connected by complementary oligonucleotide linkers. Plotting the instantaneous velocity distributions from kinesin-DDB experiments revealed a single peak centered around zero velocity. In contrast, velocity distributions from simulations displayed a central peak around 100 nm/s, along with two side peaks corresponding to the unloaded kinesin and DDB velocities. We hypothesized that frequent motor detachment events and relatively slow motor reattachment rates resulted in periods in which only one motor is attached. To investigate this hypothesis, we varied specific model parameters and compared the resulting instantaneous velocity distributions, and we confirmed this systematic investigation using a machine-learning approach that minimized the residual sum of squares between the experimental and simulation velocity distributions. The experimental data were best recapitulated by a model in which the kinesin and dynein stall forces are matched, the motor detachment rates are independent of load, and the kinesin-1 reattachment rate is 50 s−1. These results provide new insights into motor dynamics during bidirectional transport and put forth hypotheses that can be tested by future experiments.

Anterograde transport of synaptic vesicles is modulated by vesicular reversals and stationary cargo clusters.

Stationary clusters of vesicles are a prominent feature of axonal transport, but little is known about their physiological and functional relevance to axonal transport. We investigate the role of vesicle motility characteristics in modulating the formation and lifetimes of such stationary clusters, and their effect on cargo flow. We develop a simulation model describing key features of axonal cargo transport, benchmarking the model against experiments in the Posterior Lateral Mechanosensory (PLM) neurons of C. elegans. Our simulations include multiple microtubule tracks, varied cargo motion states and accounts for dynamic cargo-cargo interactions. Our model also incorporates static obstacles to vesicle transport in the form of microtubule ends, stalled vesicles, and stationary mitochondria. We demonstrate, both in simulations and in an experimental system, that a reduction in reversal rates is associated with a higher proportion of long-lived stationary vesicle clusters and reduced net anterograde transport. Our simulations support the view that stationary clusters function as dynamic reservoirs of cargo vesicles, and reversals aid cargo in navigating obstacles and regulate cargo transport by modulating the proportion of stationary vesicle clusters along the neuronal process.

Quantifying Yeast Microtubules and Spindles Using the Toolkit for Automated Microtubule Tracking (TAMiT).

Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such as microtubules, a line of fluorescence and even non-linear structures results. While much progress has been made in techniques for imaging and microscopy, image analysis is less well-developed. Current analysis of fluorescent microtubules uses either manual tools, such as kymographs, or automated software. As a result, our ability to quantify microtubule dynamics and organization from light microscopy remains limited. Despite the development of automated microtubule analysis tools for in vitro studies, analysis of images from cells often depends heavily on manual analysis. One of the main reasons for this disparity is the low signal-to-noise ratio in cells, where background fluorescence is typically higher than in reconstituted systems. Here, we present the Toolkit for Automated Microtubule Tracking (TAMiT), which automatically detects, optimizes, and tracks fluorescent microtubules in living yeast cells with sub-pixel accuracy. Using basic information about microtubule organization, TAMiT detects linear and curved polymers using a geometrical scanning technique. Images are fit via an optimization problem for the microtubule image parameters that are solved using non-linear least squares in Matlab. We benchmark our software using simulated images and show that it reliably detects microtubules, even at low signal-to-noise ratios. Then, we use TAMiT to measure monopolar spindle microtubule bundle number, length, and lifetime in a large dataset that includes several S. pombe mutants that affect microtubule dynamics and bundling. The results from the automated analysis are consistent with previous work and suggest a direct role for CLASP/Cls1 in bundling spindle microtubules. We also illustrate automated tracking of single curved astral microtubules in S. cerevisiae, with measurement of dynamic instability parameters. The results obtained with our fully-automated software are similar to results using hand-tracked measurements. Therefore, TAMiT can facilitate automated analysis of spindle and microtubule dynamics in yeast cells.

The Mobility of Neurofilaments in Mature Myelinated Axons of Adult Mice.

Studies in cultured neurons have shown that neurofilaments are cargoes of axonal transport that move rapidly but intermittently along microtubule tracks. However, the extent to which axonal neurofilaments move in vivo has been controversial. Some researchers have proposed that most axonally transported neurofilaments are deposited into a persistently stationary network and that only a small proportion of axonal neurofilaments are transported in mature axons. Here we use the fluorescence photoactivation pulse-escape technique to test this hypothesis in intact peripheral nerves of adult male hThy1-paGFP-NFM mice, which express low levels of mouse neurofilament protein M tagged with photoactivatable GFP. Neurofilaments were photoactivated in short segments of large, myelinated axons, and the mobility of these fluorescently tagged polymers was determined by analyzing the kinetics of their departure. Our results show that >80% of the fluorescence departed the window within 3 h after activation, indicating a highly mobile neurofilament population. The movement was blocked by glycolytic inhibitors, confirming that it was an active transport process. Thus, we find no evidence for a substantial stationary neurofilament population. By extrapolation of the decay kinetics, we predict that 99% of the neurofilaments would have exited the activation window after 10 h. These data support a dynamic view of the neuronal cytoskeleton in which neurofilaments cycle repeatedly between moving and pausing states throughout their journey along the axon, even in mature myelinated axons. The filaments spend a large proportion of their time pausing, but on a timescale of hours, most of them move.

Causes, costs and consequences of kinesin motors communicating through the microtubule lattice.

Microtubules are critical for a variety of important functions in eukaryotic cells. During intracellular trafficking, molecular motor proteins of the kinesin superfamily drive the transport of cellular cargoes by stepping processively along the microtubule surface. Traditionally, the microtubule has been viewed as simply a track for kinesin motility. New work is challenging this classic view by showing that kinesin-1 and kinesin-4 proteins can induce conformational changes in tubulin subunits while they are stepping. These conformational changes appear to propagate along the microtubule such that the kinesins can work allosterically through the lattice to influence other proteins on the same track. Thus, the microtubule is a plastic medium through which motors and other microtubule-associated proteins (MAPs) can communicate. Furthermore, stepping kinesin-1 can damage the microtubule lattice. Damage can be repaired by the incorporation of new tubulin subunits, but too much damage leads to microtubule breakage and disassembly. Thus, the addition and loss of tubulin subunits are not restricted to the ends of the microtubule filament but rather, the lattice itself undergoes continuous repair and remodeling. This work leads to a new understanding of how kinesin motors and their microtubule tracks engage in allosteric interactions that are critical for normal cell physiology.

The role of neurofilament transport in the radial growth of myelinated axons.

The cross-sectional area of myelinated axons increases greatly during postnatal development in mammals and is an important influence on axonal conduction velocity. This radial growth is driven primarily by an accumulation of neurofilaments, which are cytoskeletal polymers that serve a space-filling function in axons. Neurofilaments are assembled in the neuronal cell body and transported into axons along microtubule tracks. The maturation of myelinated axons is accompanied by an increase in neurofilament gene expression and a decrease in neurofilament transport velocity, but the relative contributions of these processes to the radial growth are not known. Here, we address this question by computational modeling of the radial growth of myelinated motor axons during postnatal development in rats. We show that a single model can explain the radial growth of these axons in a manner consistent with published data on axon caliber, neurofilament and microtubule densities, and neurofilament transport kinetics in vivo. We find that the increase in the cross-sectional area of these axons is driven primarily by an increase in the influx of neurofilaments at early times and by a slowing of neurofilament transport at later times. We show that the slowing can be explained by a decline in the microtubule density.

Kinesin-1, -2 and -3 use family-specific mechanochemical strategies to effectively compete with dynein during bidirectional transport.

Bidirectional cargo transport in neurons requires competing activity of motors from the kinesin-1, -2 and -3 superfamilies against cytoplasmic dynein-1. Previous studies demonstrated that when kinesin-1 attached to dynein-dynactin-BicD2 (DDB) complex, the tethered motors move slowly with a slight plus-end bias, suggesting kinesin-1 overpowers DDB but DDB generates a substantial hindering load. Compared to kinesin-1, motors from the kinesin-2 and -3 families display a higher sensitivity to load in single-molecule assays and are thus predicted to be overpowered by dynein complexes in cargo transport. To test this prediction, we used a DNA scaffold to pair DDB with members of the kinesin-1, -2 and -3 families to recreate bidirectional transport in vitro, and tracked the motor pairs using two-channel TIRF microscopy. Unexpectedly, we find that when both kinesin and dynein are engaged and stepping on the microtubule, kinesin-1, -2, and -3 motors are able to effectively withstand hindering loads generated by DDB. Stochastic stepping simulations reveal that kinesin-2 and -3 motors compensate for their faster detachment rates under load with faster reattachment kinetics. The similar performance between the three kinesin transport families highlights how motor kinetics play critical roles in balancing forces between kinesin and dynein, and emphasizes the importance of motor regulation by cargo adaptors, regulatory proteins, and the microtubule track for tuning the speed and directionality of cargo transport in cells.

Employing Live-Cell Imaging to Study Motor-Mediated Transport.

Microtubule-based transport is a highly regulated process, requiring kinesin and/or dynein motors, a multitude of motor-associated regulatory proteins including activating adaptors and scaffolding proteins, and microtubule tracks that also provide regulatory cues. While in vitro studies are invaluable, fully replicating the physiological conditions under which motility occurs in cells is not yet possible. Here, we describe two methods that can be employed to study motor-based transport and motor regulation in a cellular context. Live-cell imaging of organelle transport in neurons leverages the uniform polarity of microtubules in axons to better understand the factors regulating microtubule-based motility. Peroxisome recruitment assays allow users to examine the net effect of motors and motor-regulatory proteins on organelle distribution. Together, these methods open the door to motility experiments that more fully interrogate the complex cellular environment.

A synthetic pregnenolone analog promotes microtubule dynamics and neural development

BackgroundPregnenolone (P5) is a neurosteroid that promotes microtubule polymerization. It also reduces stress and negative symptoms of schizophrenia, promotes memory, as well as recovery from spinal cord injury. P5 is the first substance in the steroid-synthetic pathway; it can be further metabolized into other steroids. Therefore, it is difficult to differentiate the roles of P5 versus its metabolites in the brain. To alleviate this problem, we synthesized and screened a series of non-metabolizable P5 derivatives for their ability to polymerize microtubules similar to P5.ResultsWe identified compound #43 (3-beta-pregnenolone acetate), which increased microtubule polymerization. We showed that compound #43 modified microtubule dynamics in live cells, increased neurite outgrowth and changed growth cone morphology in mouse cerebellar granule neuronal culture. Furthermore, compound #43 promoted the formation of stable microtubule tracks in zebrafish developing cerebellar axons.ConclusionsWe have developed compound #43, a nonmetabolized P5 analog, that recapitulates P5 functions in vivo and can be a new therapeutic candidate for the treatment of neurodevelopmental diseases.

Using Fluorescence Recovery After Photobleaching data to uncover filament dynamics.

Fluorescence Recovery After Photobleaching (FRAP) has been extensively used to understand molecular dynamics in cells. This technique when applied to soluble, globular molecules driven by diffusion is easily interpreted and well understood. However, the classical methods of analysis cannot be applied to anisotropic structures subjected to directed transport, such as cytoskeletal filaments or elongated organelles transported along microtubule tracks. A new mathematical approach is needed to analyze FRAP data in this context and determine what information can be obtain from such experiments. To address these questions, we analyze fluorescence intensity profile curves after photobleaching of fluorescently labelled intermediate filaments anterogradely transported along microtubules. We apply the analysis to intermediate filament data to determine information about the filament motion. Our analysis consists of deriving equations for fluorescence intensity profiles and developing a mathematical model for the motion of filaments and simulating the model. Two closed forms for profile curves were derived, one for filaments of constant length and one for filaments with constant velocity, and three types of simulation were carried out. In the first type of simulation, the filaments have random velocities which are constant for the duration of the simulation. In the second type, filaments have random velocities which instantaneously change at random times. In the third type, filaments have random velocities and exhibit pausing between velocity changes. Our analysis shows: the most important distribution governing the shape of the intensity profile curves obtained from filaments is the distribution of the filament velocity. Furthermore, filament length which is constant during the experiment, had little impact on intensity profile curves. Finally, gamma distributions for the filament velocity with pauses give the best fit to asymmetric fluorescence intensity profiles of intermediate filaments observed in FRAP experiments performed in polarized migrating astrocytes. Our analysis also shows that the majority of filaments are stationary. Overall, our data give new insight into the regulation of intermediate filament dynamics during cell migration.

Unraveling axonal mechanisms of traumatic brain injury

Axonal swellings (AS) are one of the neuropathological hallmark of axonal injury in several disorders from trauma to neurodegeneration. Current evidence proposes a role of perturbed Ca2+ homeostasis in AS formation, involving impaired axonal transport and focal distension of the axons. Mechanisms of AS formation, in particular moments following injury, however, remain unknown. Here we show that AS form independently from intra-axonal Ca2+ changes, which are required primarily for the persistence of AS in time. We further show that the majority of axonal proteins undergoing de/phosphorylation immediately following injury belong to the cytoskeleton. This correlates with an increase in the distance of the actin/spectrin periodic rings and with microtubule tracks remodeling within AS. Observed cytoskeletal rearrangements support axonal transport without major interruptions. Our results demonstrate that the earliest axonal response to injury consists in physiological adaptations of axonal structure to preserve function rather than in immediate pathological events signaling axonal destruction.

Kinesin-1, -2, and -3 motors use family-specific mechanochemical strategies to effectively compete with dynein during bidirectional transport.

Bidirectional cargo transport in neurons requires competing activity of motors from the kinesin-1, -2, and -3 superfamilies against cytoplasmic dynein-1. Previous studies demonstrated that when kinesin-1 attached to dynein-dynactin-BicD2 (DDB) complex, the tethered motors move slowly with a slight plus-end bias, suggesting kinesin-1 overpowers DDB but DDB generates a substantial hindering load. Compared to kinesin-1, motors from the kinesin-2 and -3 families display a higher sensitivity to load in single-molecule assays and are thus predicted to be overpowered by dynein complexes in cargo transport. To test this prediction, we used a DNA scaffold to pair DDB with members of the kinesin-1, -2, and -3 families to recreate bidirectional transport in vitro, and tracked the motor pairs using two-channel TIRF microscopy. Unexpectedly, we find that when both kinesin and dynein are engaged and stepping on the microtubule, kinesin-1, -2, and -3 motors are able to effectively withstand hindering loads generated by DDB. Stochastic stepping simulations reveal that kinesin-2 and -3 motors compensate for their faster detachment rates under load with faster reattachment kinetics. The similar performance between the three kinesin transport families highlights how motor kinetics play critical roles in balancing forces between kinesin and dynein, and emphasizes the importance of motor regulation by cargo adaptors, regulatory proteins, and the microtubule track for tuning the speed and directionality of cargo transport in cells.