Kinesin II mediates Vg1 mRNA transport in Xenopus oocytes

The subcellular localization of specific mRNAs is a widespread mechanism for regulating gene expression. In Xenopus oocytes microtubules are required for localization of Vg1 mRNA to the vegetal cortex during the late RNA localization pathway. The factors that mediate microtubule-based RNA transport during the late pathway have been elusive. Here we show that heterotrimeric kinesin II becomes enriched at the vegetal cortex of stage III/IV Xenopus oocytes concomitant with the localization of endogenous Vg1 mRNA. In addition, expression of a dominant negative mutant peptide fragment or injection of a function-blocking antibody, both of which impair the function of heterotrimeric kinesin II, block localization of Vg1 mRNA. We also show that exogenous Vg1 RNA or Xcat-2, another RNA that can use the late pathway, recruits endogenous kinesin II to the vegetal pole and colocalizes with it at the cortex. These data support a model in which kinesin II mediates the transport of specific RNA complexes destined for the vegetal cortex.     

Directional Transport Is Mediated by a Dynein-Dependent Step in an RNA Localization Pathway

Cytoplasmic RNA localization is a key biological strategy for establishing polarity in a variety of organisms and cell types. However, the mechanisms that control directionality during asymmetric RNA transport are not yet clear. To gain insight into this crucial process, we have analyzed the molecular machinery directing polarized transport of RNA to the vegetal cortex in Xenopus oocytes. Using a novel approach to measure directionality of mRNA transport in live oocytes, we observe discrete domains of unidirectional and bidirectional transport that are required for vegetal RNA transport. While kinesin-1 appears to promote bidirectional transport along a microtubule array with mixed polarity, dynein acts first to direct unidirectional transport of RNA towards the vegetal cortex. Thus, vegetal RNA transport occurs through a multistep pathway with a dynein-dependent directional cue. This provides a new framework for understanding the mechanistic basis of cell and developmental polarity.     

Coupling viruses to dynein and kinesin-1

It is now clear that transport on microtubules by dynein and kinesin family motors has an important if not critical role in the replication and spread of many different viruses. Understanding how viruses hijack dynein and kinesin motors using a limited repertoire of proteins offers a great opportunity to determine the molecular basis of motor recruitment. In this review, we discuss the interactions of dynein and kinesin-1 with adenovirus, the α herpes viruses: herpes simplex virus (HSV1) and pseudorabies virus (PrV), human immunodeficiency virus type 1 (HIV-1) and vaccinia virus. We highlight where the molecular links to these opposite polarity motors have been defined and discuss the difficulties associated with identifying viral binding partners where the basis of motor recruitment remains to be established. Ultimately, studying microtubule-based motility of viruses promises to answer fundamental questions as to how the activity and recruitment of the dynein and kinesin-1 motors are coordinated and regulated during bi-directional transport.     

The family-specific K-loop influences the microtubule on-rate but not the superprocessivity of kinesin-3 motors

The kinesin-3 family (KIF) is one of the largest among the kinesin superfamily and an important driver of a variety of cellular transport events. Whereas all kinesins contain the highly conserved kinesin motor domain, different families have evolved unique motor features that enable different mechanical and functional outputs. A defining feature of kinesin-3 motors is the presence of a positively charged insert, the K-loop, in loop 12 of their motor domains. However, the mechanical and functional output of the K-loop with respect to processive motility of dimeric kinesin-3 motors is unknown. We find that, surprisingly, the K-loop plays no role in generating the superprocessive motion of dimeric kinesin-3 motors (KIF1, KIF13, and KIF16). Instead, we find that the K-loop provides kinesin-3 motors with a high microtubule affinity in the motor''s ADP-bound state, a state that for other kinesins binds only weakly to the microtubule surface. A high microtubule affinity results in a high landing rate of processive kinesin-3 motors on the microtubule surface. We propose that the family-specific K-loop contributes to efficient kinesin-3 cargo transport by enhancing the initial interaction of dimeric motors with the microtubule track.     

Autoinhibition of the kinesin-2 motor KIF17 via dual intramolecular mechanisms

Long-distance transport in cells is driven by kinesin and dynein motors that move along microtubule tracks. These motors must be tightly regulated to ensure the spatial and temporal fidelity of their transport events. Transport motors of the kinesin-1 and kinesin-3 families are regulated by autoinhibition, but little is known about the mechanisms that regulate kinesin-2 motors. We show that the homodimeric kinesin-2 motor KIF17 is kept in an inactive state in the absence of cargo. Autoinhibition is caused by a folded conformation that enables nonmotor regions to directly contact and inhibit the enzymatic activity of the motor domain. We define two molecular mechanisms that contribute to autoinhibition of KIF17. First, the C-terminal tail interferes with microtubule binding; and second, a coiled-coil segment blocks processive motility. The latter is a new mechanism for regulation of kinesin motors. This work supports the model that autoinhibition is a general mechanism for regulation of kinesin motors involved in intracellular trafficking events.     

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

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

Kinesin-mediated transport of Smad2 is required for signaling in response to TGF-beta ligands

During vertebrate development, Activin/Nodal-related ligands signal through Smad2, leading to its activation and accumulation in the nucleus. Here, we demonstrate that Smad2 constantly shuttles between the cytoplasm and nucleus both in early Xenopus embryo explants and in living zebrafish embryos, providing a mechanism whereby the intracellular components of the pathway constantly monitor receptor activity. We have gone on to demonstrate that an intact microtubule network and kinesin ATPase activity are required for Smad2 phosphorylation and nuclear accumulation in response to Activin/Nodal in early vertebrate embryos and TGF-beta in mammalian cells. The kinesin involved is kinesin-1, and Smad2 interacts with the kinesin-1 light chain subunit. Interfering with kinesin activity in Xenopus and zebrafish embryos phenocopies loss of Nodal signaling. Our results reveal that kinesin-mediated transport of Smad2 along microtubules to the receptors is an essential step in ligand-induced Smad2 activation.     

Kinesin motors and primary cilia

Cilia and flagella play important roles in human health by contributing to cellular motility as well as sensing and responding to environmental cues. Defects in ciliary assembly and/or function can lead to a range of human diseases, collectively known as the ciliopathies, including polycystic kidney, liver and pancreatic diseases, sterility, obesity, situs inversus, hydrocephalus and retinal degeneration. A basic understanding of how cilia form and function is essential for deciphering ciliopathies and generating therapeutic treatments. The cilium is a unique compartment that contains a distinct complement of protein and lipid. However, the molecular mechanisms by which soluble and membrane protein components are targeted to and trafficked into the cilium are not well understood. Cilia are generated and maintained by IFT (intraflagellar transport) in which IFT cargoes are transported along axonemal microtubules by kinesin and dynein motors. A variety of genetic, biochemical and cell biological approaches has established the heterotrimeric kinesin-2 motor as the 'core' IFT motor, whereas other members of the kinesin-2, kinesin-3 and kinesin-4 families function as 'accessory' motors for the transport of specific cargoes in diverse cell types. Motors of the kinesin-9 and kinesin-13 families play a non-IFT role in regulating ciliary beating or axonemal length, respectively. Entry of kinesin motors and their cargoes into the ciliary compartment requires components of the nuclear import machinery, specifically importin-β2 (transportin-1) and Ran-GTP (Ran bound to GTP), suggesting that similar mechanisms may regulate entry into the nuclear and ciliary compartments.     

Transport by Populations of Fast and Slow Kinesins Uncovers Novel Family-Dependent Motor Characteristics Important for In?Vivo Function

Intracellular cargo transport frequently involves multiple motor types, either having opposite directionality or having the same directionality but different speeds. Although significant progress has been made in characterizing kinesin motors at the single-molecule level, predicting their ensemble behavior is challenging and requires tight coupling between experiments and modeling to uncover the underlying motor behavior. To understand how diverse kinesins attached to the same cargo coordinate their movement, we carried out microtubule gliding assays using pairwise mixtures of motors from the kinesin-1, -2, -3, -5, and -7 families engineered to have identical run lengths and surface attachments. Uniform motor densities were used and microtubule gliding speeds were measured for varying proportions of fast and slow motors. A coarse-grained computational model of gliding assays was developed and found to recapitulate the experiments. Simulations incorporated published force-dependent velocities and run lengths, along with mechanical interactions between motors bound to the same microtubule. The simulations show that the force-dependence of detachment is the key parameter that determines gliding speed in multimotor assays, while motor compliance, surface density, and stall force all play minimal roles. Simulations also provide estimates for force-dependent dissociation rates, suggesting that kinesin-1 and the mitotic motors kinesin-5 and -7 maintain microtubule association against loads, whereas kinesin-2 and -3 readily detach. This work uncovers unexpected motor behavior in multimotor ensembles and clarifies functional differences between kinesins that carry out distinct mechanical tasks in cells.     

The Effect of Monastrol on the Processive Motility of a Dimeric Kinesin-5 Head/Kinesin-1 Stalk Chimera

Controlled activity of several kinesin motors is required for the proper assembly of the mitotic spindle. Eg5, a homotetrameric bipolar kinesin-5 from Xenopus laevis, can cross-link and slide anti-parallel microtubules apart by a motility mechanism comprising diffusional and directional modes. How this mechanism is regulated, possibly by the tail domains of the opposing motors, is poorly understood. In order to explore the basic unregulated kinesin-5 motor activity, we generated a stably dimeric kinesin-5 construct, Eg5Kin, consisting of the motor domain and neck linker of Eg5 and the neck coiled coil of Drosophila melanogaster kinesin-1 (DmKHC). In single-molecule motility assays, we found this chimera to be highly processive. In addition, we studied the effect of the kinesin-5-specific inhibitor monastrol using single-molecule fluorescence assays. We found that monastrol reduced the length of processive runs, but strikingly did not affect velocity. Quantitative analysis of monastrol dose dependence suggests that two bound monastrol molecules are required to be bound to an Eg5Kin dimer to terminate a run.     

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.     

Transport by Populations of Fast and Slow Kinesins Uncovers Novel Family-Dependent Motor Characteristics Important for In Vivo Function

Intracellular cargo transport frequently involves multiple motor types, either having opposite directionality or having the same directionality but different speeds. Although significant progress has been made in characterizing kinesin motors at the single-molecule level, predicting their ensemble behavior is challenging and requires tight coupling between experiments and modeling to uncover the underlying motor behavior. To understand how diverse kinesins attached to the same cargo coordinate their movement, we carried out microtubule gliding assays using pairwise mixtures of motors from the kinesin-1, -2, -3, -5, and -7 families engineered to have identical run lengths and surface attachments. Uniform motor densities were used and microtubule gliding speeds were measured for varying proportions of fast and slow motors. A coarse-grained computational model of gliding assays was developed and found to recapitulate the experiments. Simulations incorporated published force-dependent velocities and run lengths, along with mechanical interactions between motors bound to the same microtubule. The simulations show that the force-dependence of detachment is the key parameter that determines gliding speed in multimotor assays, while motor compliance, surface density, and stall force all play minimal roles. Simulations also provide estimates for force-dependent dissociation rates, suggesting that kinesin-1 and the mitotic motors kinesin-5 and -7 maintain microtubule association against loads, whereas kinesin-2 and -3 readily detach. This work uncovers unexpected motor behavior in multimotor ensembles and clarifies functional differences between kinesins that carry out distinct mechanical tasks in cells.     

Evidence for overlapping, but not identical, protein machineries operating in vegetal RNA localization along early and late pathways in Xenopus oocytes

RNAs that localize to the vegetal cortex of Xenopus oocytes are involved in early embryonic patterning and cell fate specification. Two mechanistically distinct pathways lead to RNA enrichment at the vegetal cortex: the early and the late. While several candidate proteins that seem to operate in the late localization pathway have been identified, proteins involved in the early pathway remain to be identified. In this study, we report on the isolation of a novel vegetally localized RNA in Xenopus oocytes that makes use of the early pathway and encodes a protein with a conserved but functionally uncharacterized NIF-motif. The localization signal of XNIF was mapped to a 300-nucleotide region in the 5'-UTR, which is able to mediate both accumulation to the mitochondrial cloud in stage I oocytes, as well as vegetal transport in later stage oocytes. The XNIF-LE contains 16 copies of the previously defined CAC-containing signal motifs for RNA localization. A critical number of such repeats seems to be required for accumulation in the mitochondrial cloud along the early pathway, but additional repeats seem to be required for localization along the late pathway. Cross-linking experiments identify two novel proteins of 62 and 64 kDa that interact with the XNIF-LE but not with the Vg1-LE that operates in the late pathway. Conversely, at least two of the previously identified VgRBPs, Vg1RBP1 and Prrp, also bind to the XNIF-LE. Thus, overlapping, but not identical, protein machineries mediate vegetal RNA localization along early and late pathways in Xenopus oocytes.     

Obstacles on the Microtubule Reduce the Processivity of Kinesin-1 in a Minimal In Vitro System and in Cell Extract

Inside cells, a multitude of molecular motors and other microtubule-associated proteins are expected to compete for binding to a limited number of binding sites available on microtubules. Little is known about how competition for binding sites affects the processivity of molecular motors and, therefore, cargo transport, organelle positioning, and microtubule organization, processes that all depend on the activity of more or less processive motors. Very few studies have been performed in the past to address this question directly. Most studies reported only minor effects of crowding on the velocity of motors. However, a controversy appears to exist regarding the effect of crowding on motor processivity. Here, we use single-molecule imaging of mGFP-labeled minimal dimeric kinesin-1 constructs in vitro to study the effects of competition on kinesin's processivity. For competitors, we use kinesin rigor mutants as static roadblocks, minimal wild-type kinesins as motile obstacles, and a cell extract as a complex mixture of microtubule-associated proteins. We find that mGFP-labeled kinesin-1 detaches prematurely from microtubules when it encounters obstacles, leading to a strong reduction of its processivity, a behavior that is largely independent of the type of obstacle used here. Kinesin has a low probability to wait briefly when encountering roadblocks. Our data suggest, furthermore, that kinesin can occasionally pass obstacles on the protofilament track.     

Tight Functional Coupling of Kinesin-1A and Dynein Motors in the Bidirectional Transport of Neurofilaments

We have tested the hypothesis that kinesin-1A (formerly KIF5A) is an anterograde motor for axonal neurofilaments. In cultured sympathetic neurons from kinesin-1A knockout mice, we observed a 75% reduction in the frequency of both anterograde and retrograde neurofilament movement. This transport defect could be rescued by kinesin-1A, and with successively decreasing efficacy by kinesin-1B and kinesin-1C. In wild-type neurons, headless mutants of kinesin-1A and kinesin-1C inhibited both anterograde and retrograde movement in a dominant-negative manner. Because dynein is thought to be the retrograde motor for axonal neurofilaments, we investigated the effect of dynein inhibition on anterograde and retrograde neurofilament transport. Disruption of dynein function by using RNA interference, dominant-negative approaches, or a function-blocking antibody also inhibited both anterograde and retrograde neurofilament movement. These data suggest that kinesin-1A is the principal but not exclusive anterograde motor for neurofilaments in these neurons, that there may be some functional redundancy among the kinesin-1 isoforms with respect to neurofilament transport, and that the activities of the anterograde and retrograde neurofilament motors are tightly coordinated.     

Functional analysis of human microtubule-based motor proteins, the kinesins and dyneins, in mitosis/cytokinesis using RNA interference

Microtubule (MT)-based motor proteins, kinesins and dyneins, play important roles in multiple cellular processes including cell division. In this study, we describe the generation and use of an Escherichia coli RNase III-prepared human kinesin/dynein esiRNA library to systematically analyze the functions of all human kinesin/dynein MT motor proteins. Our results indicate that at least 12 kinesins are involved in mitosis and cytokinesis. Eg5 (a member of the kinesin-5 family), Kif2A (a member of the kinesin-13 family), and KifC1 (a member of the kinesin-14 family) are crucial for spindle formation; KifC1, MCAK (a member of the kinesin-13 family), CENP-E (a member of the kinesin-7 family), Kif14 (a member of the kinesin-3 family), Kif18 (a member of the kinesin-8 family), and Kid (a member of the kinesin-10 family) are required for chromosome congression and alignment; Kif4A and Kif4B (members of the kinesin-4 family) have roles in anaphase spindle dynamics; and Kif4A, Kif4B, MKLP1, and MKLP2 (members of the kinesin-6 family) are essential for cytokinesis. Using immunofluorescence analysis, time-lapse microscopy, and rescue experiments, we investigate the roles of these 12 kinesins in detail.     

Aspergillus myosin-V supports polarized growth in the absence of microtubule-based transport

In the filamentous fungus Aspergillus nidulans, both microtubules and actin filaments are important for polarized growth at the hyphal tip. Less clear is how different microtubule-based and actin-based motors work together to support this growth. Here we examined the role of myosin-V (MYOV) in hyphal growth. MYOV-depleted cells form elongated hyphae, but the rate of hyphal elongation is significantly reduced. In addition, although wild type cells without microtubules still undergo polarized growth, microtubule disassembly abolishes polarized growth in MYOV-depleted cells. Thus, MYOV is essential for polarized growth in the absence of microtubules. Moreover, while a triple kinesin null mutant lacking kinesin-1 (KINA) and two kinesin-3s (UNCA and UNCB) undergoes hyphal elongation and forms a colony, depleting MYOV in this triple mutant results in lethality due to a severe defect in polarized growth. These results argue that MYOV, through its ability to transport secretory cargo, can support a significant amount of polarized hyphal tip growth in the absence of any microtubule-based transport. Finally, our genetic analyses also indicate that KINA (kinesin-1) rather than UNCA (kinesin-3) is the major kinesin motor that supports polarized growth in the absence of MYOV.     

Vg1 RNA binding protein mediates the association of Vg1 RNA with microtubules in Xenopus oocytes.

Localized RNAs are found in a variety of somatic and developing cell types. In many cases, microtubules have been implicated as playing a role in facilitating transport of these RNAs. Here we report that Vg1 RNA, which is localized to the vegetal cortex of Xenopus laevis oocytes, is associated with microtubules in vivo. Because of the ubiquitous nature of tubulin, the association of specific RNAs with microtubules is likely to involve factors that recognize both RNA and microtubules. Vg1 RNA binding protein (Vg1 RBP), previously shown to bind with high affinity to the vegetal localization site in Vg1 RNA, appears to function in this capacity. Vg1 RBP is associated with microtubules: it is enriched in microtubule extracts of oocytes and is also co-precipitated by heterologous, polymerized tubulin. Furthermore, Vg1 RBP binding activity is required for the specific association of Vg1 RNA to microtubules in vitro. These data suggest a general model for how specific RNAs can be localized to particular sites via common cytoskeletal elements.     

Kinesin-dependent transport results in polarized migration of the nucleus in oocytes and inward movement of yolk granules in meiotic embryos

During female meiosis, meiotic spindles are positioned at the oocyte cortex to allow expulsion of chromosomes into polar bodies. In C. elegans, kinesin-dependent translocation of the entire spindle to the cortex precedes dynein-dependent rotation of one spindle pole toward the cortex. To elucidate the role of kinesin-1 in spindle translocation, we examined the localization of kinesin subunits in meiotic embryos. Surprisingly, kinesin-1 was not associated with the spindle and instead was restricted to the cytoplasm in the middle of the embryo. Yolk granules moved on linear tracks, in a kinesin-dependent manner, away from the cortex, resulting in their concentration in the middle of the embryo where the kinesin was concentrated. These results suggest that cytoplasmic microtubules might be arranged with plus ends extending inward, away from the cortex. This microtubule arrangement would not be consistent with direct transport of the meiotic spindle toward the cortex by kinesin-1. In maturing oocytes, the nucleus underwent kinesin-dependent migration to the future site of spindle attachment at the anterior cortex. Thus the spindle translocation defect observed in kinesin-1 mutants may be a result of failed nuclear migration, which places the spindle too far from the cortex for the spindle translocation mechanism to function.     

Dynactin is required for bidirectional organelle transport

Kinesin II is a heterotrimeric plus end-directed microtubule motor responsible for the anterograde movement of organelles in various cell types. Despite substantial literature concerning the types of organelles that kinesin II transports, the question of how this motor associates with cargo organelles remains unanswered. To address this question, we have used Xenopus laevis melanophores as a model system. Through analysis of kinesin II-mediated melanosome motility, we have determined that the dynactin complex, known as an anchor for cytoplasmic dynein, also links kinesin II to organelles. Biochemical data demonstrates that the putative cargo-binding subunit of Xenopus kinesin II, Xenopus kinesin II-associated protein (XKAP), binds directly to the p150Glued subunit of dynactin. This interaction occurs through aa 530-793 of XKAP and aa 600-811 of p150Glued. These results reveal that dynactin is required for transport activity of microtubule motors of opposite polarity, cytoplasmic dynein and kinesin II, and may provide a new mechanism to coordinate their activities.