Effect of vesicle traps on traffic jam formation in fast axonal transport

The purpose of this paper is to develop a model for simulation of the formation of organelle traps in fast axonal transport. Such traps may form in the regions of microtubule polar mismatching. Depending on the orientation of microtubules pointing toward the trap region, these traps can accumulate either plus-end or minus-end oriented vesicles. The model predicts that the maximum concentrations of organelles occur at the boundaries of the trap regions; the overall concentration of organelles in the axon with traps is greatly increased compared to that in a healthy axon, which is expected to contribute to mechanical damages of the axon. The organelle traps induce hindrance to organelle transport down the axon; the total organelle flux down the axon with traps is found to be significantly reduced compared to that in a healthy axon.     

Tau-induced traffic jams reflect organelles accumulation at points of microtubule polar mismatching

It is currently accepted that tau overexpression leads to impaired organelle transport and thus to neuronal degeneration. Nevertheless, the underlying mechanisms that lead to impaired organelle transport are not entirely clear. Using cultured Aplysia neurons and online confocal imaging of human tau, microtubules (MTs), the plus-end tracking protein – end-binding protein 3, retrogradely and anterogradely transported organelles, we found that overexpression of tau generates the hallmarks of human tau pathogenesis. Nevertheless, in contrast to earlier reports, we found that the tau-induced impairment of organelle transport is because of polar reorientation of the MTs along the axon or their displacement to submembrane domains. 'Traffic jams' reflect the accumulation of organelles at points of MT polar discontinuations or polar mismatching rather than because of MT depolymerization. Our findings offer a new mechanistic explanation for earlier observations, which established that tau overexpression leads to impaired retrograde and anterograde organelle transport, while the MT skeleton appeared intact.     

Modelling organelle transport after traumatic axonal injury

This paper is motivated by recent experimental research (Tang-Schomer et al. 2012) on the formation of periodic varicosities in axons after traumatic brain injury (TBI). TBI leads to the formation of undulated distortions in the axons due to their dynamic deformation. These distortions result in the breakage of some microtubules (MTs) near the peaks of undulations. The breakage is followed by catastrophic MT depolymerisation around the broken ends. Although after relaxation axons regain their straight geometry, the structure of the axon after TBI is characterised by the presence of periodic regions where the density of MTs has been decreased due to depolymerisation. We modelled organelle transport in an axon segment with such a damaged MT structure and investigated how this structure affects the distributions of organelle concentrations and fluxes. The modelling results suggest that organelles accumulate at the boundaries of the region where the density of MTs has been decreased by depolymerisation. According to the model, the presence of such damaged regions decreases the organelle flux by only about 12%. This provides evidence that axon degradation after TBI may be caused by organelle accumulation rather than by starvation due to insufficient organelle flux.     

Method of modelling intracellular transport in branching neurites: application to axons and dendrites of Drosophila sensory neurons

This paper develops a method of calculating the transport of intracellular organelles in neurons with branching neurites which is based on the Smith–Simmons equations of motor-assisted transport. The method is aimed at understanding the effects of microtubule (MT) polarity orientation in branching neurites on transport of organelles at the fundamental level. The method is applied to calculating the organelle transport in axons and dendrites of Drosophila neurons, using the map of MT orientation in such neurons developed by Stone et al. (Mol Biol Cell 19:4122–4129, 2008). The proximal dendrite is assumed to branch and form two distal dendrites. Two different MT polarity arrangements in a proximal dendrite are considered, and implications of these MT arrangements on organelle transport are analysed. It is demonstrated that the MT arrangement found in Drosophila dendrites (MTs have their minus ends out in a proximal dendrite) results in much more efficient motor-driven transport than the structure with a mixed MT orientation in proximal dendrites.     

Modeling organelle transport in branching dendrites with a variable cross-sectional area

The purpose of this paper is to develop a method for calculating organelle transport in dendrites with a non-uniform cross-sectional area that depends on the distance from the neuron soma. The model is based on modified Smith–Simmons equations governing molecular motor-assisted organelle transport. The developed method is then applied to simulating organelle transport in branching dendrites with two particular microtubule (MT) orientations reported from experiments. It is found that the rate of organelle transport toward a dendrite’s growth cone heavily depends on the MT orientation, and since there is experimental evidence that the MT orientation in a particular region of a dendrite may depend on the dendrite’s developmental stage, the obtained results suggest that a rearrangement of the MT structure may depend on the amount of organelles needed at the growth cone.     

Modelling transport of layered double hydroxide nanoparticles in axons and dendrites of cortical neurons

This paper develops a model of nanoparticle transport in neurons. It is assumed that nanoparticles are transported inside endocytic vesicles by a combined effect of dynein-driven transport and diffusion. It is further assumed that in axons nanoparticles are internalised only at axon terminals, whereas in dendrites nanoparticles can enter through the entire plasma membrane. This causes differences in transport of nanoparticles in axons and dendrites; these differences are investigated in this paper. Another difference is microtubule (MT) orientation in axons and dendrites; in axons, all MTs have their plus-ends oriented towards the axon terminal; in a proximal region of a dendrite, MTs have mixed orientation, whereas in a distal dendritic region the MT orientation is similar to that in an axon. It is shown that if molecular-motor-driven transport were powered by dynein alone, such MT orientation in a dendrite would result in a region of nanoparticle accumulation located at the border between the proximal and distal dendritic regions.     

Modelling transport of layered double hydroxide nanoparticles in axons and dendrites of cortical neurons

This paper develops a model of nanoparticle transport in neurons. It is assumed that nanoparticles are transported inside endocytic vesicles by a combined effect of dynein-driven transport and diffusion. It is further assumed that in axons nanoparticles are internalised only at axon terminals, whereas in dendrites nanoparticles can enter through the entire plasma membrane. This causes differences in transport of nanoparticles in axons and dendrites; these differences are investigated in this paper. Another difference is microtubule (MT) orientation in axons and dendrites; in axons, all MTs have their plus-ends oriented towards the axon terminal; in a proximal region of a dendrite, MTs have mixed orientation, whereas in a distal dendritic region the MT orientation is similar to that in an axon. It is shown that if molecular-motor-driven transport were powered by dynein alone, such MT orientation in a dendrite would result in a region of nanoparticle accumulation located at the border between the proximal and distal dendritic regions.     

Quantitative analysis of microtubule transport in growing nerve processes

In neurons, tubulin is synthesized primarily in the cell body, whereas the molecular machinery for neurite extension and elaboration of microtubule (MT) array is localized to the growth cone region. This unique functional and biochemical compartmentalization of neuronal cells requires transport mechanisms for the delivery of newly synthesized tubulin and other cytoplasmic components from the cell body to the growing axon. According to the polymer transport model, tubulin is transported along the axon as a polymer. Because the majority of axonal MTs are stationary at any given moment, it has been assumed that only a small fraction of MTs translocates along the axon by saltatory movement reminiscent of the fast axonal transport. Such intermittent "stop and go" MT transport has been difficult to detect or to exclude by using direct video microscopy methods. In this study, we measured the translocation of MT plus ends in the axonal shaft by expressing GFP-EB1 in Xenopus embryo neurons in culture. Formal quantitative analysis of MT assembly/disassembly indicated that none of the MTs in the axonal shaft were rapidly transported. Our results suggest that transport of axonal MTs is not required for delivery of newly synthesized tubulin to the growing nerve processes.     

Modeling anterograde and retrograde transport of short mobile microtubules from the site of axonal branch formation

This theoretical research is motivated by a recent model of microtubule (MT) transport put forward by Baas and Mozgova (Cytoskeleton 69:416–425, 2012). According to their model, in an axon all plus-end-distal mobile MTs move anterogradely while all minus-end-distal mobile MTs move retrogradely. Retrograde MT transport thus represents a mechanism by which minus-end-distal MTs are removed from the axon. We suggested equations that implement Baas and Mozgova’s model. We employed these equations to simulate transport of short mobile MTs from a region (such as the site of axonal branch formation) where MT severing activity results in generation of a large number of short MTs of both orientations. We obtained the exact and approximate transient solutions of these equations utilizing the Laplace transform technique. We applied the obtained solutions to calculate the average rates of anterograde and retrograde transport of short MTs.     

Modeling transport of a pulse of radiolabeled organelles in a Drosophila unipolar motor neuron

Based on published experimental evidence, this paper develops a model for the transport of a pulse of radiolabeled organelles in a unipolar Drosophila motor neuron. In particular, since published data indicate that no microtubules (MTs) travel from the primary neurite into the dendrite, it is investigated how organelles are transported into the dendrite. Analytical solutions describing concentrations of kinesin- and dynein-driven organelles in the primary neurite, axon, and dendrite are obtained. The effects of increasing the width of the pulse and increasing the rate of organelle transition rate from the kinesin-driven to the dynein-driven state are investigated.     

A live-cell marker to visualize the dynamics of stable microtubules throughout the cell cycle

The microtubule (MT) cytoskeleton underlies processes such as intracellular transport and cell division. Immunolabeling for posttranslational modifications of tubulin has revealed the presence of different MT subsets, which are believed to differ in stability and function. Whereas dynamic MTs can readily be studied using live-cell plus-end markers, the dynamics of stable MTs have remained obscure due to a lack of tools to directly visualize these MTs in living cells. Here, we present StableMARK (Stable Microtubule-Associated Rigor-Kinesin), a live-cell marker to visualize stable MTs with high spatiotemporal resolution. We demonstrate that a rigor mutant of Kinesin-1 selectively binds to stable MTs without affecting MT organization and organelle transport. These MTs are long-lived, undergo continuous remodeling, and often do not depolymerize upon laser-based severing. Using this marker, we could visualize the spatiotemporal regulation of MT stability before, during, and after cell division. Thus, this live-cell marker enables the exploration of different MT subsets and how they contribute to cellular organization and transport.     

Microtubule organization requires cell cycle-dependent nucleation at dispersed cytoplasmic sites: polar and perinuclear microtubule organizing centers in the plant pathogen Ustilago maydis

Growth of most eukaryotic cells requires directed transport along microtubules (MTs) that are nucleated at nuclear-associated microtubule organizing centers (MTOCs), such as the centrosome and the fungal spindle pole body (SPB). Herein, we show that the pathogenic fungus Ustilago maydis uses different MT nucleation sites to rearrange MTs during the cell cycle. In vivo observation of green fluorescent protein-MTs and MT plus-ends, tagged by a fluorescent EB1 homologue, provided evidence for antipolar MT orientation and dispersed cytoplasmic MT nucleating centers in unbudded cells. On budding gamma-tubulin containing MTOCs formed at the bud neck, and MTs reorganized with >85% of all minus-ends being focused toward the growth region. Experimentally induced lateral budding resulted in MTs that curved out of the bud, again supporting the notion that polar growth requires polar MT nucleation. Depletion or overexpression of Tub2, the gamma-tubulin from U. maydis, affected MT number in interphase cells. The SPB was inactive in G2 phase but continuously recruited gamma-tubulin until it started to nucleate mitotic MTs. Taken together, our data suggest that MT reorganization in U. maydis depends on cell cycle-specific nucleation at dispersed cytoplasmic sites, at a polar MTOC and the SPB.     

Myosin II activity facilitates microtubule bundling in the neuronal growth cone neck

The cell biological processes underlying axon growth and guidance are still not well understood. An outstanding question is how a new segment of the axon shaft is formed in the wake of neuronal growth cone advance. For this to occur, the highly dynamic, splayed-out microtubule (MT) arrays characteristic of the growth cone must be consolidated (bundled together) to form the core of the axon shaft. MT-associated proteins stabilize bundled MTs, but how individual MTs are brought together for initial bundling is unknown. Here, we show that laterally moving actin arcs, which are myosin II-driven contractile structures, interact with growing MTs and transport them from the sides of the growth cone into the central domain. Upon Myosin II inhibition, the movement of actin filaments and MTs immediately stopped and MTs unbundled. Thus, Myosin II-dependent compressive force is necessary for normal MT bundling in the growth cone neck.     

Role of cytoplasmic dynein in the axonal transport of microtubules and neurofilaments

Recent studies have shown that the transport of microtubules (MTs) and neurofilaments (NFs) within the axon is rapid, infrequent, asynchronous, and bidirectional. Here, we used RNA interference to investigate the role of cytoplasmic dynein in powering these transport events. To reveal transport of MTs and NFs, we expressed EGFP-tagged tubulin or NF proteins in cultured rat sympathetic neurons and performed live-cell imaging of the fluorescent cytoskeletal elements in photobleached regions of the axon. The occurrence of anterograde MT and retrograde NF movements was significantly diminished in neurons that had been depleted of dynein heavy chain, whereas the occurrence of retrograde MT and anterograde NF movements was unaffected. These results support a cargo model for NF transport and a sliding filament model for MT transport.     

Tuning microtubule-based transport through filamentous MAPs: the problem of dynein

We recently proposed that regulating the single-to-multiple motor transition was a likely strategy for regulating kinesin-based transport in vivo . In this study, we use an in vitro bead assay coupled with an optical trap to investigate how this proposed regulatory mechanism affects dynein-based transport. We show that tau's regulation of kinesin function can proceed without interfering with dynein-based transport. Surprisingly, at extremely high tau levels – where kinesin cannot bind microtubules (MTs) – dynein can still contact MTs. The difference between tau's effects on kinesin- and dynein-based motility suggests that tau can be used to tune relative amounts of plus-end and minus-end-directed transport. As in the case of kinesin, we find that the 3RS isoform of tau is a more potent inhibitor of dynein binding to MTs. We show that this isoform-specific effect is not because of steric interference of tau's projection domains but rather because of tau's interactions with the motor at the MT surface. Nonetheless, we do observe a modest steric interference effect of tau away from the MT and discuss the potential implications of this for molecular motor structure.     

The transport properties of axonal microtubules establish their polarity orientation

It is well established that axonal microtubules (MTs) are uniformly oriented with their plus ends distal to the neuronal cell body (Heidemann, S. R., J. M. Landers, and M. A. Hamborg. 1981. J. Cell Biol. 91:661-665). However, the mechanisms by which these MTs achieve their uniform polarity orientation are unknown. Current models for axon growth differ with regard to the contributions of MT assembly and transport to the organization and elaboration of the axonal MT array. Do the transport properties or assembly properties of axonal MTs determine their polarity orientation? To distinguish between these possibilities, we wished to study the initiation and outgrowth of axons under conditions that would arrest MT assembly while maintaining substantial levels of preexisting polymer in the cell body that could still be transported into the axon. We found that we could accomplish this by culturing rat sympathetic neurons in the presence of nanomolar levels of vinblastine. In concentrations of the drug up to and including 100 nM, the neurons actively extend axons. The vinblastine- axons are shorter than control axons, but clearly contain MTs. To quantify the effects of the drug on MT mass, we compared the levels of polymer throughout the cell bodies and axons of neurons cultured overnight in the presence of 0, 16, and 50 nM vinblastine with the levels of MT polymer in freshly plated neurons before axon outgrowth. Without drug, the total levels of polymer increase by roughly twofold. At 16 nM vinblastine, the levels of polymer are roughly equal to the levels in freshly plated neurons, while at 50 nM, the levels of polymer are reduced by about half this amount. Thus, 16 nM vinblastine acts as a "kinetic stabilizer" of MTs, while 50 nM results in some net MT disassembly. At both drug concentrations, there is a progressive increase in the levels of MT polymer in the axons as they grow, and a corresponding depletion of polymer from the cell body. These results indicate that highly efficient mechanisms exist in the neuron to transport preassembled MTs from the cell body into the axon. These mechanisms are active even at the expense of the cell body, and even under conditions that promote some MT disassembly in the neuron. MT polarity analyses indicate that the MTs within the vinblastine-axons, like those in control axons, are uniformly plus-end-distal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Switching of membrane organelles between cytoskeletal transport systems is determined by regulation of the microtubule-based transport

Intracellular transport of membrane organelles occurs along microtubules (MTs) and actin filaments (AFs). Although transport along each type of the cytoskeletal tracks is well characterized, the switching between the two types of transport is poorly understood because it cannot be observed directly in living cells. To gain insight into the regulation of the switching of membrane organelles between the two major transport systems, we developed a novel approach that combines live cell imaging with computational modeling. Using this approach, we measured the parameters that determine how fast membrane organelles switch back and forth between MTs and AFs (the switching rate constants) and compared these parameters during different signaling states. We show that regulation involves a major change in a single parameter: the transferring rate from AFs onto MTs. This result suggests that MT transport is the defining factor whose regulation determines the choice of the cytoskeletal tracks during the transport of membrane organelles.     

A coupled model of fast axonal transport of organelles and slow axonal transport of tau protein

We have developed a model that accounts for the effect of a non-uniform distribution of tau protein along the axon length on fast axonal transport of intracellular organelles. The tau distribution is simulated by using a slow axonal transport model; the numerically predicted tau distributions along the axon length were validated by comparing them with experimentally measured tau distributions reported in the literature. We then developed a fast axonal transport model for organelles that accounts for the reduction of kinesin attachment rate to microtubules by tau. We investigated organelle transport for two situations: (1) a uniform tau distribution and (2) a non-uniform tau distribution predicted by the slow axonal transport model. We found that non-uniform tau distributions observed in healthy axons (an increase in tau concentration towards the axon tip) result in a significant enhancement of organelle transport towards the synapse compared with the uniform tau distribution with the same average amount of tau. This suggests that tau may play the role of being an enhancer of organelle transport.     

Transport of dendritic microtubules establishes their nonuniform polarity orientation

The immature processes that give rise to both axons and dendrites contain microtubules (MTs) that are uniformly oriented with their plus- ends distal to the cell body, and this pattern is preserved in the developing axon. In contrast, developing dendrites gradually acquire nonuniform MT polarity orientation due to the addition of a subpopulation of oppositely oriented MTs (Baas, P. W., M. M. Black, and G. A. Banker. 1989. J. Cell Biol. 109:3085-3094). In theory, these minus-end-distal MTs could be locally nucleated and assembled within the dendrite itself, or could be transported into the dendrite after their nucleation within the cell body. To distinguish between these possibilities, we exposed cultured hippocampal neurons to nanomolar levels of vinblastine after one of the immature processes had developed into the axon but before the others had become dendrites. At these levels, vinblastine acts as a kinetic stabilizer of MTs, inhibiting further assembly while not substantially depolymerizing existing MTs. This treatment did not abolish dendritic differentiation, which occurred in timely fashion over the next two to three days. The resulting dendrites were flatter and shorter than controls, but were identifiable by their ultrastructure, chemical composition, and thickened tapering morphology. The growth of these dendrites was accompanied by a diminution of MTs from the cell body, indicating a net transfer of MTs from one compartment into the other. During this time, minus-end-distal microtubules arose in the experimental dendrites, indicating that new MT assembly is not required for the acquisition of nonuniform MT polarity orientation in the dendrite. Minus-end-distal microtubules predominated in the more proximal region of experimental dendrites, indicating that most of the MTs at this stage of development are transported into the dendrite with their minus-ends leading. These observations indicate that transport of MTs from the cell body is an essential feature of dendritic development, and that this transport establishes the nonuniform polarity orientation of MTs in the dendrite.     

Engineered Tug‐of‐War Between Kinesin and Dynein Controls Direction of Microtubule Based Transport In Vivo

Bidirectional transport of membrane organelles along microtubules (MTs) is driven by plus‐end directed kinesins and minus‐end directed dynein bound to the same cargo. Activities of opposing MT motors produce bidirectional movement of membrane organelles and cytoplasmic particles along MT transport tracks. Directionality of MT‐based transport might be controlled by a protein complex that determines which motor type is active at any given moment of time, or determined by the outcome of a tug‐of‐war between MT motors dragging cargo organelles in opposite directions. However, evidence in support of each mechanisms of regulation is based mostly on the results of theoretical analyses or indirect experimental data. Here, we test whether the direction of movement of membrane organelles in vivo can be controlled by the tug‐of‐war between opposing MT motors alone, by attaching a large number of kinesin‐1 motors to organelles transported by dynein to minus‐ends of MTs. We find that recruitment of kinesin significantly reduces the length and velocity of minus‐end‐directed dynein‐dependent MT runs, leading to a reversal of the overall direction of dynein‐driven organelles in vivo. Therefore, in the absence of external regulators tug‐of‐war between opposing MT motors alone is sufficient to determine the directionality of MT transport in vivo.