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-1 and Dynein are the primary motors for fast transport of mitochondria in Drosophila motor axons

To address questions about mechanisms of filament-based organelle transport, a system was developed to image and track mitochondria in an intact Drosophila nervous system. Mutant analyses suggest that the primary motors for mitochondrial movement in larval motor axons are kinesin-1 (anterograde) and cytoplasmic dynein (retrograde), and interestingly that kinesin-1 is critical for retrograde transport by dynein. During transport, there was little evidence that force production by the two opposing motors was competitive, suggesting a mechanism for alternate coordination. Tests of the possible coordination factor P150(Glued) suggested that it indeed influenced both motors on axonal mitochondria, but there was no evidence that its function was critical for the motor coordination mechanism. Observation of organelle-filled axonal swellings ("organelle jams" or "clogs") caused by kinesin and dynein mutations showed that mitochondria could move vigorously within and pass through them, indicating that they were not the simple steric transport blockades suggested previously. We speculate that axonal swellings may instead reflect sites of autophagocytosis of senescent mitochondria that are stranded in axons by retrograde transport failure; a protective process aimed at suppressing cell death signals and neurodegeneration.     

Kinesin-1 and Cytoplasmic Dynein Act Sequentially to Move the Meiotic Spindle to the Oocyte Cortex in Caenorhabditis elegans

During female meiosis in animals, the meiotic spindle is attached to the egg cortex by one pole during anaphase to allow selective disposal of half the chromosomes in a polar body. In Caenorhabditis elegans, this anaphase spindle position is achieved sequentially through kinesin-1–dependent early translocation followed by anaphase-promoting complex (APC)-dependent spindle rotation. Partial depletion of cytoplasmic dynein heavy chain by RNA interference blocked spindle rotation without affecting early translocation. Dynein depletion also blocked the APC-dependent late translocation that occurs in kinesin-1–depleted embryos. Time-lapse imaging of green fluorescent protein-tagged dynein heavy chain as well as immunofluorescence with dynein-specific antibodies revealed that dynein starts to accumulate at spindle poles just before the initiation of rotation or late translocation. Accumulation of dynein at poles was kinesin-1 independent and APC dependent, just like dynein driven spindle movements. This represents a case of kinesin-1/dynein coordination in which these two motors of opposite polarity act sequentially and independently on a cargo to move it in the same direction.     

Posttranslational Modifications of Tubulin and the Polarized Transport of Kinesin-1 in Neurons

Polarized transport by microtubule-based motors is critical for neuronal development and function. Selective translocation of the Kinesin-1 motor domain is the earliest known marker of axonal identity, occurring before morphological differentiation. Thus, Kinesin-1–mediated transport may contribute to axonal specification. We tested whether posttranslational modifications of tubulin influence the ability of Kinesin-1 motors to distinguish microtubule tracks during neuronal development. We detected no difference in microtubule stability between axons and minor neurites in polarized stage 3 hippocampal neurons. In contrast, microtubule modifications were enriched in a subset of neurites in unpolarized stage 2 cells and the developing axon in polarized stage 3 cells. This enrichment correlated with the selective accumulation of constitutively active Kinesin-1 motors. Increasing tubulin acetylation, without altering the levels of other tubulin modifications, did not alter the selectivity of Kinesin-1 accumulation in polarized cells. However, globally enhancing tubulin acetylation, detyrosination, and polyglutamylation by Taxol treatment or inhibition of glycogen synthase kinase 3β decreased the selectivity of Kinesin-1 translocation and led to the formation of multiple axons. Although microtubule acetylation enhances the motility of Kinesin-1, the preferential translocation of Kinesin-1 on axonal microtubules in polarized neuronal cells is not determined by acetylation alone but is probably specified by a combination of tubulin modifications.     

Bidirectional Transport by Molecular Motors: Enhanced Processivity and Response to External Forces

Intracellular transport along cytoskeletal filaments is often mediated by two teams of molecular motors that pull on the same cargo and move in opposite directions along the filaments. We have recently shown theoretically that this bidirectional transport can be understood as a stochastic tug-of-war between the two motor teams. Here, we further develop our theory to investigate the experimentally accessible dynamic behavior of cargos transported by strong motors such as kinesin-1 or cytoplasmic dynein. By studying the run and binding times of such a cargo, we show that the properties of biological motors, such as the large ratio of stall/detachment force and the small ratio of superstall backward/forward velocity, are favorable for bidirectional cargo transport, leading to fast motion and enhanced diffusion. In addition, cargo processivity is shown to be strongly enhanced by transport via several molecular motors even if these motors are engaged in a tug-of-war. Finally, we study the motility of a bidirectional cargo under force. Frictional forces arising, e.g., from the viscous cytoplasm, lead to peaks in the velocity distribution, while external forces as exerted, e.g., by an optical trap, lead to hysteresis effects. Our results, in particular our explicit expressions for the cargo binding time and the distance of the peaks in the velocity relation under friction, are directly accessible to in vitro as well as in vivo experiments.     

Miro-1 Links Mitochondria and Microtubule Dynein Motors To Control Lymphocyte Migration and Polarity

The recruitment of leukocytes to sites of inflammation is crucial for a functional immune response. In the present work, we explored the role of mitochondria in lymphocyte adhesion, polarity, and migration. We show that during adhesion to the activated endothelium under physiological flow conditions, lymphocyte mitochondria redistribute to the adhesion zone together with the microtubule-organizing center (MTOC) in an integrin-dependent manner. Mitochondrial redistribution and efficient lymphocyte adhesion to the endothelium require the function of Miro-1, an adaptor molecule that couples mitochondria to microtubules. Our data demonstrate that Miro-1 associates with the dynein complex. Moreover, mitochondria accumulate around the MTOC in response to the chemokine CXCL12/SDF-1α; this redistribution is regulated by Miro-1. CXCL12-dependent cell polarization and migration are reduced in Miro-1-silenced cells, due to impaired myosin II activation at the cell uropod and diminished actin polymerization. These data point to a key role of Miro-1 in the control of lymphocyte adhesion and migration through the regulation of mitochondrial redistribution.     

A cAMP/PKA/Kinesin-1 Axis Promotes the Axonal Transport of Mitochondria in Aging Drosophila Neurons

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ARL8 Relieves SKIP Autoinhibition to Enable Coupling of Lysosomes to Kinesin-1

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Epithelial Fluid Transport: Protruding Macromolecules and Space Charges Can Bring about Electro-Osmotic Coupling at the Tight Junctions

The purpose of the present work is to investigate whether the idea of epithelial fluid transport based on electro-osmotic coupling at the level of the leaky tight junction (TJ) can be further supported by a plausible theoretical model. We develop a model for fluid transport across epithelial layers based on electro-osmotic coupling at leaky tight junctions (TJ) possessing protruding macromolecules and fixed electrical charges. The model embodies systems of electro-hydrodynamic equations for the intercellular pathway, namely the Brinkman and the Poisson-Boltzmann differential equations applied to the TJ. We obtain analytical solutions for a system of these two equations, and are able to derive expressions for the fluid velocity profile and the electrostatic potential. We illustrate the model by employing geometrical parameters and experimental data from the corneal endothelium, for which we have previously reported evidence for a central role for electro-osmosis in translayer fluid transport. Our results suggest that electro-osmotic coupling at the TJ can account for fluid transport by the corneal endothelium. We conclude that electro-osmotic coupling at the tight junctions could represent one of the basic mechanisms driving fluid transport across some leaky epithelia, a process that remains unexplained.     

Myosin-V, Kinesin-1, and Kinesin-3 cooperate in hyphal growth of the fungus Ustilago maydis

Long-distance transport is crucial for polar-growing cells, such as neurons and fungal hyphae. Kinesins and myosins participate in this process, but their functional interplay is poorly understood. Here, we investigate the role of kinesin motors in hyphal growth of the plant pathogen Ustilago maydis. Although the microtubule plus-ends are directed to the hyphal tip, of all 10 kinesins analyzed, only conventional kinesin (Kinesin-1) and Unc104/Kif1A-like kinesin (Kinesin-3) were up-regulated in hyphae and they are essential for extended hyphal growth. deltakin1 and deltakin3 mutant hyphae grew irregular and remained short, but they were still able to grow polarized. No additional phenotype was detected in deltakin1rkin3 double mutants, but polarity was lost in deltamyo5rkin1 and deltamyo5rkin3 mutant cells, suggesting that kinesins and class V myosin cooperate in hyphal growth. Consistent with such a role in secretion, fusion proteins of green fluorescent protein and Kinesin-1, Myosin-V, and Kinesin-3 accumulate in the apex of hyphae, a region where secretory vesicles cluster to form the fungal Spitzenk?rper. Quantitative assays revealed a role of Kin3 in secretion of acid phosphatase, whereas Kin1 was not involved. Our data demonstrate that just two kinesins and at least one myosin support hyphal growth.     

Cell Cycle-Dependent Microtubule-Based Dynamic Transport of Cytoplasmic Dynein in Mammalian Cells

Background

Cytoplasmic dynein complex is a large multi-subunit microtubule (MT)-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood.

Methodology/Principal Findings

Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP)-tagged 74-kDa intermediate chain (IC74). IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs), suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed.

Conclusions and Significance

These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein.     

Cytoplasmic Dynein and Dynactin Are Required for the Transport of Microtubules into the Axon

Previous work from our laboratory suggested that microtubules are released from the neuronal centrosome and then transported into the axon (Ahmad, F.J., and P.W. Baas. 1995. J. Cell Sci. 108: 2761–2769). In these studies, cultured sympathetic neurons were treated with nocodazole to depolymerize most of their microtubule polymer, rinsed free of the drug for a few minutes to permit a burst of microtubule assembly from the centrosome, and then exposed to nanomolar levels of vinblastine to suppress further microtubule assembly from occurring. Over time, the microtubules appeared first near the centrosome, then dispersed throughout the cytoplasm, and finally concentrated beneath the periphery of the cell body and within developing axons. In the present study, we microinjected fluorescent tubulin into the neurons at the time of the vinblastine treatment. Fluorescent tubulin was not detected in the microtubules over the time frame of the experiment, confirming that the redistribution of microtubules observed with the experimental regime reflects microtubule transport rather than microtubule assembly. To determine whether cytoplasmic dynein is the motor protein that drives this transport, we experimentally increased the levels of the dynamitin subunit of dynactin within the neurons. Dynactin, a complex of proteins that mediates the interaction of cytoplasmic dynein and its cargo, dissociates under these conditions, resulting in a cessation of all functions of the motor tested to date (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132: 617–633). In the presence of excess dynamitin, the microtubules did not show the outward progression but instead remained near the centrosome or dispersed throughout the cytoplasm. On the basis of these results, we conclude that cytoplasmic dynein and dynactin are essential for the transport of microtubules from the centrosome into the axon.     

Structural and Functional Characterization of Paramecium Dynein: Initial Studies

ABSTRACT Dynein arms and isolated dynein from Paramecium tetraurelia ciliary axonemes are comparable in structure, direction of force generation, and microtubule translocation ability to other dyneins. In situ arms have dimensions and substructure similar to those of Tetrahymena. Based on spoke arrangement in intact axonemes, arms translocate axonemal microtubules in sliding such that active dynein arms are (-) end directed motors and the doublet to which the body and cape of the arms binds (N) translocates the adjacent doublet (N+1) upward. After salt extraction, based on ATPase activity, paramecium dynein is found as a 22S and a 14S species. the 22S dynein is a three-headed molecule that has unfolded from the in situ dimensions; the 14S dynein is single headed. Both dyneins can be photocleaved by UV light (350 nm) in the presence of Mg^2-, ATP and vanadate; the photocleavage pattern of 22S dynein differs from that seen with Tetrahymena. Both isolated dyneins translocate taxol-stabilized, bovine brain microtubules in vitro. Under standard conditions, 22S dynein, like comparable dyneins from other organisms, translocates at velocities that are about three times faster than 14S dynein.     

Neurofilaments and Orthograde Transport Are Reduced in Ventral Root Axons of Transgenic Mice that Express Human SOD1 with a G93A Mutation

Mice engineered to express a transgene encoding a human Cu/Zn superoxide dismutase (SOD1) with a Gly⁹³ → Ala (G93A) mutation found in patients who succumb to familial amyotrophic lateral sclerosis (FALS) develop a rapidly progressive and fatal motor neuron disease (MND) similar to amyotrophic lateral sclerosis (ALS). Hallmark ALS lesions such as fragmentation of the Golgi apparatus and neurofilament (NF)-rich inclusions in surviving spinal cord motor neurons as well as the selective degeneration of this population of neurons were also observed in these animals. Since the mechanism whereby mutations in SOD1 lead to MND remains enigmatic, we asked whether NF inclusions in motor neurons compromise axonal transport during the onset and progression of MND in a line of mice that contained ∼30% fewer copies of the transgene than the original G93A (Gurney et al., 1994). The onset of MND was delayed in these mice compared to the original G93A mice, but they developed the same neuropathologic abnormalities seen in the original G93A mice, albeit at a later time point with fewer vacuoles and more NF inclusions. Quantitative Western blot analyses showed a progressive decrease in the level of NF proteins in the L5 ventral roots of G93A mice and a concomitant reduction in axon caliber with the onset of motor weakness. By ∼200 d, both fast and slow axonal transports were impaired in the ventral roots of these mice coincidental with the appearance of NF inclusions and vacuoles in the axons and perikarya of vulnerable motor neurons. This is the first demonstration of impaired axonal transport in a mouse model of ALS, and we infer that similar impairments occur in authentic ALS. Based on the temporal correlation of these impairments with the onset of motor weakness and the appearance of NF inclusions and vacuoles in vulnerable motor neurons, the latter lesions may be the proximal cause of motor neuron dysfunction and degeneration in the G93A mice and in FALS patients with SOD1 mutations.Neurofilaments (NFs)¹ comprise the major class of neuron-specific intermediate filaments and are the most abundant cytoskeletal components found in large myelinated axons (for reviews see Nixon, 1993; Fuchs and Weber, 1994). NFs are heteropolymers formed by three subunits known as the high (NFH; 110 kD), middle (NFM; 95 kD), and low (NFL; 62 kD) molecular weight NF proteins, all of which are synthesized in neuronal perikarya and transported in the SCa phase (i.e., slow component a) of axonal transport at ∼0.2 to 1.2 mm/d (Lasek and Hoffman, 1976). Other cytoskeletal components (e.g., actin, tubulin) are transported three to four times faster in the SCb of slow axonal transport (Hoffman and Lasek, 1980).The carboxy termini or tail domains of NFH and NFM harbor tandem repeats of lysine-serine-proline (KSP) motifs, and the serine in this motif may be phosphorylated under both physiological and pathological conditions (Jones and Williams, 1982; Julien and Mushynski, 1982; Black and Lee, 1988; Lee et al., 1988a ,b; Clark and Lee, 1991; Giasson and Mushynski, 1996). Normally, the serines in these motifs become highly phosphorylated only after NFH and NFM have been transported into axons where this phosphorylation regulates the caliber of axons (de Waegh et al., 1992; Cole et al., 1994; Tu et al., 1995). In a variety of human neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson''s disease (PD), as well as in neurotoxin-induced neuropathies, such as those that result from exposure to aluminum and acrylamide (Troncoso et al., 1985; Yase, 1988; Johnson and Jope, 1988; Johnson et al., 1990; Strong, 1994), NF inclusions typically form in the perikarya and processes of neurons, and these inclusions contain highly phosphorylated NFH and NFM (Tu et al., 1997a ,b). The major functions of NFs are to provide mechanical support, especially in large myelinated axons, and to regulate axonal caliber. Thus, disruption of the NF network has been hypothesized to play a mechanistic role in the degeneration of selectively vulnerable neurons that accumulate inclusions in a subset of human neurodegenerative disease (Lee et al., 1994; Julien, 1995). Significantly, this hypothesis has been supported by several recent studies of a number of different lines of transgenic mice that develop an ALS-like phenotype, including prominent NF inclusions in motor neurons that subsequently degenerate (Côté et al., 1993; Xu et al., 1993; Eyer and Peterson, 1994; Lee et al., 1994; Tu et al., 1997a ). Since only variations in the number of KSP motifs in NFH have been observed in some ALS patients (Figlewicz et al., 1994), other factors may lead to disruption of the NF network in neurodegenerative disorders. For example, mutations in the Cu/Zn superoxide dismutase gene (SOD1), which occurs in ∼20% of familial ALS (FALS) kindreds, lead to perturbations of the NF network (for recent review see Tu et al., 1997b ). FALS (as well as sporadic ALS) is a motor neuron disease (MND) characterized by progressive motor weakness due to the selective degeneration of motor neurons, many of which accumulate NF inclusions before their demise (Schmidt et al., 1987; Hill et al., 1991; Hirano, 1991; Tu et al., 1997b ). Transgenic mice that express one of four different mutant forms of the human SOD1 gene recapitulate many of the hallmarks of FALS including a fatal, progressive motor neuron weakness, the selective loss of motor neurons (Gurney et al., 1994; Ripps et al., 1995; Wong et al., 1995; Bruijn et al., 1997), fragmentation of the Golgi apparatus (Mourelatos et al., 1996), and the accumulation of NF inclusions in motor neurons that are vulnerable to degenerate (Dal Canto and Gurney, 1994, 1995, 1997; Tu et al., 1996). The precise mechanisms that lead to the selective degeneration of neurons in authentic ALS as well as in transgenic mouse models of this disorder remain enigmatic, but there is evidence to suggest that NF inclusions may impede axonal transport and thereby contribute to the degeneration of affected neurons (Collard et al., 1995). Alternatively, other studies suggest that NF inclusions may compromise the viability of affected neurons by sequestering vital organelles (Tu et al., 1997a ). Thus, the present study exploited a classic experimental paradigm to determine if axonal transport in the ventral roots was impaired in transgenic mice that expresses human SOD1 with a Gly⁹³→ Ala mutation (G93A) and develop an ALS-like phenotype.