Microtubular Motor Research Articles

Crystal structure of the stalk region of axonemal inner-arm dynein-d reveals unique features in the coiled-coil and microtubule-binding domain.

Axonemal dynein is an ATP-dependent microtubular motor protein responsible for cilia and flagella beating, and its dysfunction can cause diseases such as primary ciliary dyskinesia and sperm dysmotility. Despite its biological importance, structure-based mechanisms underlying axonemal dynein motors remain unclear. Here, we determined the X-ray crystal structure of the human inner-arm dynein-d (DNAH1) stalk region, which contains a long antiparallel coiled-coil and a microtubule-binding domain (MTBD), at 2.7 Å resolution. Notably, differences in the relative orientation of the coiled-coil and MTBD in comparison with other dyneins, as well as the diverse orientations of the MTBD flap region among various isoforms, lead us to propose a 'spike shoe model' with an altered stepping angle for the interaction between IAD-d and microtubules. Based on these findings, we discuss isoform-specific functions of the axonemal dynein stalk MTBDs.

Bacteria-propelled microtubular motors for efficient penetration and targeting delivery of thrombolytic agents

Effective thrombolysis is critical to rapidly rebuild blood flow for thrombosis patients. Drug delivery systems have been developed to address inadequate pharmacokinetics of thrombolytic agents, but challenges still remain in the timely removal of blood clots regarding the dense fibrin networks. Herein, rod-shaped tubular micromotors were developed to achieve efficient penetration and thorough destruction of thrombi. By using electrospun fiber fragments as the template, urokinase (uPA)-loaded polydopamine (PDA) microtubes with surface decorated fucoidan (FuPDAuPA) were prepared at the aspect ratio of around 2. One E. coli Nissle 1917 (EcN) was assembled into one microtube to construct a FuPDAuPA@EcN hybrid micromotor through PDA adhesion and L-aspartate induction. The pharmacokinetic analysis indicates that the encapsulation of uPA into micromotors extends the half-life from 0.4 to 5.6 h and increases the bioavailability over 10 times. EcN-propelled motion elevates adsorption capacities of FuPDAuPA@EcN for more than four times compared with that of FuPDAuPA. The fucoidan-mediated targeting causes 2-fold higher thrombolysis capacity in vitro and over 10-fold higher uPA accumulation in thrombi in vivo. In the treatment of venous thrombi at mouse hindlimbs, intravenous administration of FuPDAuPA@EcN completely removed blood clots with almost full recovery of blood flows and apparently alleviated tail bleeding. It should be noted that FuPDAuPA@EcN treatment at a reduced uPA dose caused no significant difference in the blood flow rate compared with those of FuPDAuPA. The synergistic action of fucoidan-induced targeting and EcN-driven motion provides a prerequisite for promoting thrombolytic efficacy and reducing uPA dose and bleeding side effect. Statement of significanceThe standard treatment to thrombosis patient is intravenous infusion of thrombolytic agents, but the associated bleeding complications and impairment of normal haemostasis greatly offset the therapeutic benefits. Drug delivery systems have been developed to address the limitations of inadequate pharmacokinetics of thrombolytic agents, but challenges still exist in less efficient penetration into dense networks for thorough destruction of thrombi. Up to now only few attempts have been made to construct nano-/micromotors for combating thrombosis and there is no single case that antithrombosis is assisted by bacteria or cells-propelled motors. Herein, bacteria-propelled microtubes were developed to carry urokinase for efficient penetration into blood clots and effective thrombolysis. The synergistic action of bacteria-driven motion and specific ligand-induced targeting holds a promising treatment strategy for life-threatening cardiovascular diseases such as thrombosis and atherosclerosis.

Mechanical Counterbalance of Kinesin and Dynein Motors in a Microtubular Network Regulates Cell Mechanics, 3D Architecture, and Mechanosensing.

Microtubules (MTs) and MT motor proteins form active 3D networks made of unstretchable cables with rod-like bending mechanics that provide cells with a dynamically changing structural scaffold. In this study, we report an antagonistic mechanical balance within the dynein–kinesin microtubular motor system. Dynein activity drives the microtubular network inward compaction, while isolated activity of kinesins bundles and expands MTs into giant circular bands that deform the cell cortex into discoids. Furthermore, we show that dyneins recruit MTs to sites of cell adhesion, increasing the topographic contact guidance of cells, while kinesins antagonize it via retraction of MTs from sites of cell adhesion. Actin-to-microtubule translocation of septin-9 enhances kinesin–MT interactions, outbalances the activity of kinesins over that of dyneins, and induces the discoid architecture of cells. These orthogonal mechanisms of MT network reorganization highlight the existence of an intricate mechanical balance between motor activities of kinesins and dyneins that controls cell 3D architecture, mechanics, and cell–microenvironment interactions.

Cargo Transportation and Methylene Blue Degradation by Using Fuel-Powered Micromotors.

In the past two decades, micromotors have experienced rapid development, especially in environmental remediation, the biomedical field, and in cargo delivery. In this study micromotors have been synthesized from a variety of materials. Different functional layers and catalytic layers are formed through template electrodeposition (the bottom‐up method). At the same time, the article analyzes the influence of hydrogen peroxide concentration, surfactant type and concentration on the speed of the micromotors. Cargo transportation through tubular micromotors has always been a problem that people are eager to solve. In this article, we electrodeposit a layer of Ni in the microtubes, which effectively guides the microtubular motors to complete the cargo transportation. The potential applications of micromotors are also being explored. We added the prepared micromotors to the methylene blue solution to effectively enhance the degradation.

Design and Fabrication of Tubular Micro/Nanomotors via 3D Laser Lithography.

Catalytic tubular micro/nanomachines convert chemical energy from a surrounding aqueous fuel solution into mechanical energy to generate autonomous movements, propelled by the oxygen bubbles decomposed by hydrogen peroxide and expelled from the microtubular cavity. With the development of nanotechnology, micro/nanomotors have attracted more and more interest due to their numerous potential for in vivo and in vitro applications. Here, highly efficient chemical catalytic microtubular motors were fabricated via 3D laser lithography and their motion behavior under the action of driving force in fluids was demonstrated. The frequency of catalytically-generated bubbles ejection was influenced by the geometrical shape of the micro/nanomotor and surrounding chemical fuel environment, resulting in the variation in motion speed. The micro/nanomotors generated with a rocket-like shape displayed a more active motion compared with that of a single tubular micro/nanomotor, providing a wider range of practical micro-/nanoscale applications in the future.

Structure–Function Dependence on Template-Based Micromotors

Catalytically bubble propelled micromotors are one of the most frequently used self-propelled micromachines in the literature. Typically, the structure–function relationship is not investigated. We present detailed study of how the structural geometry influences the propulsion of these micromachines. We prepared different micromotors with membrane template-assisted electrodeposition by applying varied electroplating time. We obtained a variety of differently sized micromotors with a range of propulsion dynamics. We used varied electroplating time to tailor the length of the micromotor, but also the wall thickness, microtube opening diameter, and even the structural integrity of the micromotor. The study of propulsion dynamics of micromotors in different H2O2 concentrations indicated that both geometric parameters and the chemical environment affect the velocity of micromotors. Microtubular motors prepared by short electrodeposition time (e.g., 300 s) require higher fuel concentration to activate and move....

The Motor KIF5C Links the Requirements of Stable Microtubules and IGF-1 Receptor Membrane Insertion for Neuronal Polarization

Three early signals of asymmetry have been described to occur in a single neurite of neurons at stage 2 of differentiation (before polarization) and shown to be essential for neuronal polarization: (i) accumulation of stable microtubules, (ii) enrichment of the plasma membrane with activatable IGF-1r, and (iii) polarized transport of the microtubular motor KIF5C. Here, we studied the possible relationship between these three phenomena. Our results show that the activatable (membrane-inserted) IGF-1r and stable microtubules accumulate in the same neurite of cells at stage 2. The polarized insertion of IGF-1r depends on microtubule dynamics as shown using drugs which modify microtubule stability. Silencing of KIF5C expression prevents the polarized insertion of IGF-1r into the neuronal plasmalemma and neuronal polarization. Syntaxin 6 and VAMP4, necessary for the polarized insertion of the IGF-1r, are associated to vesicles carried by the microtubular motor KIF5C and is transported preferentially to the neurite where KIF5C accumulates. We conclude that the enrichment of stable microtubules in the future axon enhances KIF5C-mediated vesicular transport of syntaxin 6 and VAMP4, which in turn mediates the polarized insertion of IGF-1r in the plasmalemma, a key step for neuronal polarization. We herewith establish a mechanistic link between three early polarity events necessary for the establishment of neuronal polarity.

Locomotive Studies on Electrodeposited Bimetallic Microtubular Motors for pH Sensing

In order to harness the potential of self-propelled miniaturized machines, there has been a sustained effort to increase locomotion in chemically-powered micromotors. Catalytic microdevices, propelled by hydrogen peroxide decomposition to oxygen bubbles, has been the most ubiquitous of them all. The microtubular motors were achieved through a templated-electrodeposition approach with a silver-sputtered polycarbonate membrane, serving as a working electrode in a three-electrode setup. Bimetallic components were accomplished with the sequential electrodeposition of copper, followed by platinum, which serves as the inner catalytic lining. The use of pH to alter the mobility of micromotors is demonstrated in this work through the manipulation of hydrogen peroxide chemistry in different acidity/alkalinity. The addition of sodium hydroxide to increase the pH of the solution led to a consequent increase in activity of micromotors. Meanwhile, introduction of hydrochloric acid led to corrosion of the microstructures, culminating in locomotive changes. Such dramatic changes in activity and velocities of the micromotors allow the usage of this behavior for pH detection. This concept was illustrated in the electrosynthesized tubular bimetallic Cu/Pt micromotors. Alteration of pH shall serve as a useful general strategy for manipulating hydrogen peroxide decomposition in oxygen-bubble propelled catalytic micromotors.

Motor Coordination in Long-Distance Transport in Axons

Retrograde transport of nerve growth factor signaling endosomes by microtubular motors, from the axon terminals to cell bodies, is vital for the survival of neurons. The robustness of this fast long-distance axonal transport and biased directionality could be attributed to the cooperative mechanics of multiple motors and/or intracellular regulation mechanisms. Here, we present a comprehensive motion analysis of retrograde nerve growth factor (NGF)-endosome trajectories in axons to show that cooperative motor mechanics and intracellular motor regulation are both important factors determining the endosome directionality. We used quantum dot (QD) to fluorescently label NGF and acquired trajectories of retrograde QD-NGF-endosomes with < 20 nm accuracy at 32 Hz, using pseudo-total internal reflection fluorescence imaging. Using a combination of transient motion analysis and Bayesian parsing, we segregated the trajectories into sustained periods of retrograde (dynein-driven) motion, constrained pauses and brief anterograde reversals. Mean square displacement analysis and the temperature dependence of transient reversals confirm that motors of opposite polarities (dyneins and kinesins) are both active on the endosomes during retrograde transport. Stochastic multi-motor model simulations show that the biased directionality as well as several statistical metrics of NGF-endosome transport can only be simulated reasonably by assuming that the microtubule-binding affinity of kinesin is down-regulated. Specifically, the simulations suggest that the NGF-endosomes are driven on average by 4-7 active dyneins and 1-3 down-regulated kinesins. These observations are corroborated by the dynamics of endosomes detaching under load in axons; showcasing the cooperativity of multiple dyneins and the subdued activity of kinesins. We discuss the ramifications of our results for intracellular transport regulation, in conjunction with recent studies on cellular cargo in a wide range of motility (bidirectional to unidirectional) regimes.

Kif3a Guides Microtubular Dynamics, Migration and Lumen Formation of MDCK Cells

The microtubular motor Kinesin-2 and its subunit Kif3a are essential for the formation of primary cilia, an organelle implicated in a wide spectrum of developmental abnormalities. Outside cilia, Kinesin-2 mediated transport has been implicated in vesicle and N-cadherin transport, but it is unknown if and how extraciliary Kif3a affects basic cellular functions such as migration or the formation of multicellular structures. Here we show that tetracycline inducible depletion of Kif3a in MDCK cells slows epithelial cell migration. Microtubules at the leading edge of Kif3a depleted cells failed to grow perpendicularly into the leading edge and microtubular dynamics were dampened in Kif3a depleted cells. Loss of Kif3a retarded lateral membrane specification and completely prevented the formation of three-dimensional spheres in collagen. These data uncover that Kif3a regulates the microtubular cytoskeleton in the cell periphery and imply that extra-ciliary Kif3a has an unexpected function in morphogenesis.

Magnetic and meniscus-effect control of catalytic rolled-up micromotors

Rolled-up catalytic micromotors with tubular structures are fabricated by rolling up strained Pt/Co/Ti metallic nanomembranes through selectively etching of the sacrificial lift off resist (LOR). The rolled-up micromotors present distinct motion behaviors in an organic/aqueous mixture fuel compared to those in a pure aqueous fuel. These self-propelled micromotors can move in pure aqueous fuel with a speed up to several millimeters per second. However, when placed into an organic/aqueous mixture fuel, the micromotors walk on the liquid surface. An intermediate Co layer facilitates a magnetically guided motion of these microtubular motors. The motion at the air–liquid interface can be regulated and suspended by the meniscus-climbing effect. Such meniscus-navigated motion represents a novel approach for regulation of micromotors at the air–liquid interface and opens the door to new and exciting operations of these micro-scale machines.

Small Peptide Inhibitors Disrupt a High-Affinity Interaction between Cytoplasmic Dynein and a Viral Cargo Protein

Several viruses target the microtubular motor system in early stages of the viral life cycle. African swine fever virus (ASFV) protein p54 hijacks the microtubule-dependent transport by interaction with a dynein light chain (DYNLL1/DLC8). This was shown to be a high-affinity interaction, and the residues gradually disappearing were mapped on DLC8 to define a putative p54 binding surface by nuclear magnetic resonance (NMR) spectroscopy. The potential of short peptides targeting the binding domain to disrupt this high-affinity protein-protein interaction was assayed, and a short peptide sequence was shown to bind and compete with viral protein binding to dynein. Given the complexity and number of proteins involved in cellular transport, the prevention of this viral-DLC8 interaction might not be relevant for successful viral infection. Thus, we tested the capacity of these peptides to interfere with viral infection by disrupting dynein interaction with viral p54. Using this approach, we report on short peptides that inhibit viral growth.

Bidirectional Ca2+-dependent Control Of Mitochondrial Dynamics By The Mitochondrial Rhogtpase, Miro

Mitochondrial motility (mito-motility) is regulated by cytoplasmic [Ca2+] ([Ca2+]c) oscillations in a homeostatic manner to optimize the mitochondrial contribution to intracellullar Ca2+ buffering, ATP production and signal transduction. To control mitochondrial movements, Ca2+ targets a yet unidentified cytoplasmic factor that does not seem to be a microtubular motor or a kinase/phosphatase.We here studied the dependence of Ca2+-sensitive mito-motility on Miro (mitochondrial Rho GTPases), an integral mitochondrial membrane protein that has two Ca2+-binding site of EF-hands. Mitochondrial matrix targeted YFP expressing H9c2 cardiac myoblasts were co-transfected with Miro plasmid DNA or siRNA, and then loaded with fura-2 (5 μM) to monitor the mito-motility and [Ca2+]c simultaneously.(1) At resting [Ca2+]c (< 40 nM), mito-motility was enhanced by Miro overexpression irrespective of the presence of the EF-hands and was suppressed when Miro was depleted by siRNA.(2) When [Ca2+]c was increased (0.1 - 2 μM) directly or by agonist stimulation, the Ca2+-induced arrest of mito-motility was also promoted by Miro overexpression and was suppressed when either the Miros were depleted or their EF-hand was mutated.(3) Miro also enhanced the fusion state of the mitochondria at resting [Ca2+]c but promoted mitochondrial fragmentation at high [Ca2+]c. The Miro effects on mitochondrial morphology seem to involve Dynamin related protein 1 (Drp1).Thus, Miro serves as a [Ca2+]c-sensitive bidirectional regulator of both the motility and fusion-fission dynamics of mitochondria.

Bidirectional Ca 2+ -dependent control of mitochondrial dynamics by the Miro GTPase

Calcium oscillations suppress mitochondrial movements along the microtubules to support on-demand distribution of mitochondria. To activate this mechanism, Ca(2+) targets a yet unidentified cytoplasmic factor that does not seem to be a microtubular motor or a kinase/phosphatase. Here, we have studied the dependence of mitochondrial dynamics on the Miro GTPases that reside in the mitochondria and contain two EF-hand Ca(2+)-binding domains, in H9c2 cells and primary neurons. At resting cytoplasmic [Ca(2+)] ([Ca(2+)](c)), movements of the mitochondria were enhanced by Miro overexpression irrespective of the presence of the EF-hands. The Ca(2+)-induced arrest of mitochondrial motility was also promoted by Miro overexpression and was suppressed when either the Miro were depleted or their EF-hand was mutated. Miro also enhanced the fusion state of the mitochondria at resting [Ca(2+)](c) but promoted mitochondrial fragmentation at high [Ca(2+)](c). These effects of Miro on mitochondrial morphology seem to involve Drp1 suppression and activation, respectively. In primary neurons, Miro also caused an increase in dendritic mitochondrial mass and enhanced mitochondrial calcium signaling. Thus, Miro proteins serve as a [Ca(2+)](c)-sensitive switch and bifunctional regulator for both the motility and fusion-fission dynamics of the mitochondria.

Flagellar membrane trafficking in kinetoplastids

Trypanosomes are flagellated protozoa belonging to the order Kinetoplastidae. Three trypanosomes, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania are the causative agents of the human diseases: African sleeping sickness, Chagas disease, and leishmaniasis, respectively. These organisms have complex life cycles involving bloodsucking insects and vertebrates; humans become infected when infested insects feed on them. The trypanosome contains several unique organelles, one of the most important and conspicuous of which is the flagellum (Fig. 1). The trypanosome flagellum is a unique multifunctional organelle that plays critical roles in motility, chemotaxis, cell signaling, and host cell invasion (Landfear and Ignatushchenko 2001; Gull 2003; Hill 2003). In addition to containing a classical axonemal microtubular motor, the trypanosome flagellum also includes a paracrystalline structure called the paraflagellar rod (PFR), positioned alongside the axoneme. The flagellum emerges from the cell body from an invagination known as the flagellar pocket. As the cell body is constrained by a tight corset of cross-linked subpellicular microtubules (Fig. 1b), all vesicular trafficking into and out of the cell occurs at the non-microtubule-bound flagellar pocket, as does the trafficking of flagellar membrane-associated proteins between the cytoplasm and the flagellum (Bloodgood 1990; McConville et al. 2002). Because no new protein synthesis takes place in the flagellum, all protein components must be imported to the flagellum from the cell body, also via the flagellar pocket. To enter the flagellum, a protein must first pass through a cytoskeletal “neck” region within the flagellar pocket known as the flagellar pore complex (Rosenbaum and Witman 2002; Gull 2003). This complex, which contains structures including the basal body and transition zone filaments, is believed to act as a “stopper” for the flagellum, providing a selective barrier for protein entry (Vickerman and Preston 1976; Dentler and Adams 1992). Regulated flagellar protein import creates a flagellar microenvironment that is unique from that of the rest of the organism, where some of the protein and lipid components differ in composition from those in the cell body (Bloodgood 1990; Landfear and Ignatushchenko 2001). Because of differences in composition and function, the surface membrane of a trypanosome is divided into three distinct but contiguous domains: the pellicular (cell body) membrane, flagellar membrane, and flagellar pocket membrane (Balber 1990; Landfear and Ignatushchenko 2001; Gull 2003). As the flagellum is not a “closed” membrane-bound organelle like the mitochondrion or a lysosome, it has this far Parasitol Res DOI 10.1007/s00436-006-0329-2

African swine fever virus protein p54 interacts with the microtubular motor complex through direct binding to light-chain dynein.

Dynein is a minus-end-directed microtubule-associated motor protein involved in cargo transport in the cytoplasm. African swine fever virus (ASFV), a large DNA virus, hijacks the microtubule motor complex cellular transport machinery during virus infection of the cell through direct binding of virus protein p54 to the light chain of cytoplasmic dynein (LC8). Interaction of p54 and LC8 occurs both in vitro and in cells, and the two proteins colocalize at the microtubular organizing center during viral infection. p50/dynamitin, a dominant-negative inhibitor of dynein-dynactin function, impeded ASFV infection, suggesting an essential role for dynein during virus infection. A 13-amino-acid domain of p54 was sufficient for binding to LC8, an SQT motif within this domain being critical for this binding. Direct binding of a viral structural protein to LC8, a small molecule of the dynein motor complex, could constitute a molecular mechanism for microtubule-mediated virus transport.

Molecular mechanisms of microtubular organelle assembly in Tetrahymena.

Thanks to recent technological advances, the ciliate Tetrahymena thermophila has emerged as an attractive model organism for studies on the assembly of microtubular organelles in a single cell. Tetrahymena assembles 17 types of distinct microtubules, which are localized in cilia, cell cortex, nuclei, and the endoplasm. These diverse microtubules have distinct morphologies, stabilities, and associations with specific Microtubule-Associated Proteins. For example, kinesin-111, a microtubular motor protein, is required for assembly of cilia and is preferentially targeted to microtubules of actively assembled, immature cilia. It is unlikely that the unique properties of individual microtubules are derived from the utilization of diverse tubulin genes, because Tetrahymena expresses only a single isotype of alpha- and two isotypes of 1-tubulin. However, Tetrahymena tubulins are modified secondarily by a host of posttranslational mechanisms. Each microtubule organelle type displays a unique set of secondary tubulin modifications. The results of systematic in vivo mutational analyses of modification sites indicate a divergence in significance among post-translational mechanisms affecting either alpha- or beta-tubulin. Both acetylation and polyglycylation of alpha-tubulin are not essential and their complete elimination does not change the cell's phenotype in an appreciable way. However, the multiple polyglycylation sites on 1-tubulin are essential for survival, and their partial elimination dramatically affects cell motility, growth and morphology. Thus, both high-precision targeting of molecular motors to individual organelles as well as organelle-specific tubulin modifications contribute to the creation of diverse microtubules in a single cytoplasm of Tetrahymena.

Dynein-related polypeptides in pollen and pollen tubes

Microtubules in pollen tubes are evident within the vegetative and generative cell cytoplasm. This observation led to the formulation of several hypotheses regarding the role of microtubules in cytoplasmic movement and the migration of the vegetative nucleus/generative cell along the pollen tube. The study of microtubular motor proteins in pollen tubes followed the discovery and characterization of an immunoreactive homolog of mammalian kinesin in tobacco pollen tubes. Recent identification of dynein-related polypeptides in pollen tubes ofNicotiana tabacum and pollen ofGinkgo biloba is a significant step in the definition of the role of microtubule function within pollen and pollen tubes.

Electrodynamics of microtubular motors: the building blocks of a new model

Microtubules are ubiquitous components of the cytoskeleton. They participate in many motility processes ranging from intracellular transport or chromosome movement during mitosis to ciliary and flagellar beating. The biophysical mechanism inherent in the generation and control of movement in all these motility phenomena has not yet been entirely elucidated. The authors propose a new model based on a charge transfer mechanism capable of shedding a new light on the molecular foundations of all motility processes. Electron transfer along the microtubular lattice is responsible for activation and control of all microtubule-associated ATPases (i.e. force generating enzymes). Microtubules are thus shown to be the basic motors of cell dynamics. The model is first applied to intracellular transport and ciliary and flagellar beating. Through two additional examples, the authors show the heuristic capabilities of the suggested hypothesis. The application of charge transfer control to the Protozoan Euglena gracilis leads to a plausible model capable of accounting for its phototactic response mechanism. Furthermore, the model allows a new interpretation of the electrophysiological response in vertebrate photoreceptors.

Cytoplasmic microtubular motors

Numerous observations over the past 20 years have shown that microtubules are involved in many processes of intracellular organelle transport. These conclusions are based mainly on experiments showing that the destruction of microtubules inhibits organelle movement and that the direction of this movement coincides with the microtubule distribution in the cells (reviewed by Schliwa. In Cell and Muscle Motility, Vol. 5, edited by Shaw, Plenum, 1984, pp 1-81).