Comparison of active transport in neuronal axons and dendrites

This paper presents a theoretical study, based on modified Smith-Simmons equations, that compares transport of intracellular organelles in two different neurite outgrowths, dendrites and axons. It is demonstrated that the difference in microtubule polarity orientations in dendrites and axons has significant implications on motor-assisted transport in these neurite outgrowths. The developed approach presents a qualitative theoretical basis for understanding important questions such as why axons exhibit almost an unlimited grows potential in vitro while dendrites remain relatively short. It is shown that the difference in a microtubule polarity arrangement between axons and dendrites may be a regulatory mechanism for limiting dendritic growth. Other biological implications of the developed theory as well as other possible reasons for the difference in microtubule structure between axons and dendrites are discussed.     

A macroscopic model of traffic jams in axons

The purpose of this paper is to develop a minimal macroscopic model capable of explaining the formation of traffic jams in fast axonal transport. The model accounts for the decrease of the number density of positively (and negatively) oriented microtubules near the location of the traffic jam due to formation of microtubule swirls; the model also accounts for the reduction of the effective velocity of organelle transport in the traffic jam region due to organelles falling off microtubule tracks more often in the swirl region. The model is based on molecular-motor-assisted transport equations and the hydrodynamic model of traffic jams in highway traffic. Parametric analyses of the model’s predictions for various values of viscosity of the traffic flow, variance of the velocity distribution, diffusivity of microtubule-bound and free organelles, rate constants for binding to and detachment from microtubules, relaxation time, and average motor velocities of the retrograde and anterograde transport, are carried out.     

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.     

Modeling bidirectional transport of quantum dot nanoparticles in membrane nanotubes

This paper develops a model of transport of quantum dot (QD) nanoparticles in membrane nanotubes (MNTs). It is assumed that QDs are transported inside intracellular organelles (called here nanoparticle-loaded vesicles, NLVs) that are propelled by either kinesin or dynein molecular motors while moving on microtubules (MTs). A vesicle may have both types of motors attached to it, but the motors are assumed to work in a cooperative fashion, meaning that at a given time the vesicle is moved by either kinesin or dynein motors. The motors are assumed not to work against each other, when one type of motors is pulling the vesicle, the other type is inactive. From time to time the motors may switch their roles: passive motors can become active motors and vice versa, resulting in the change of the vesicle’s direction of motion. It is further assumed that QDs can escape NLVs and become free QDs, which are then transported by diffusion. Free QDs can be internalized by NLVs. The effects of two possible types of MT orientation in MNTs are investigated: when all MTs have a uniform polarity orientation, with their plus-ends directed toward one of the cells connected by an MNT, and when MTs have a mixed polarity orientation, with half of MTs having their plus-ends directed toward one of the cells and the other half having their plus-ends directed toward the other cell. Computational results are presented for three cases. The first case is when organelles are as likely to be transported by kinesin motors as by dynein motors. The second case is when organelles are more likely to be transported by kinesin motors than by dynein motors, and the third case is when NLVs do not associate with dynein motors at all.     

Absorption by a moving spherical organelle in a heterogeneous cytoplasm: implications for the role of trafficking in a symplast

An organelle which absorbs (or secretes) a particular factor will find its mass transfer rate diffusion-limited if it is stationary with respect to its ambient cytoplasm; but organellar motion will raise that limit as a non-decreasing function of the Peclet number P. It is shown analytically that (i) no Whitehead paradox need be encountered in the creeping flow regime and (ii) the flux of the factor will be an even function of the Peclet number, P. By a novel analytic solution method, the flux is shown numerically to increase as P2 for P < or = 1. For P > or = 10, a quasi-planar approximating geometry yields analytically a flux which increases as P1/3. These two solutions overlap smoothly in the range 1 < or = P > or = 10. For P approximately 1, convection should increase the mass flux by roughly 100%.     

Slow potential changes due to transport number effects in cells with unstirred membrane invaginations or dendrites

Summary Many neurones are extremely invaginated and possess branching processes, axons and dendrites. In general, they are surrounded by a restricted diffusion space. Many of these cells exhibit large, slow potential changes during the passage of current across their membranes. Whenever currents cross membranes separating aqueous solutions, differences in transport numbers of the major permeant ions give rise to local concentration changes of these ions adjacent to the membranes, which will result in various electrical and osmotic effects. These transport number effects are expected to be enhanced by the presence of membrane invaginations. Dendrites are equivalent to reversed invaginations and there should be significant changes in concentrations of permeant ions within them. In general, the effects of such changes on the electrical response of a cell will be greater when the concentration of a major permeant ion is low. The effects have been modelled in terms of two nondimensional parameters: the invagination transport number parameter and the relative area occupied by the invaginations A. If these two parameters are known, the magnitudes and time course of the slow potential changes can immediately be estimated and the time course converted to real time, if the length of the invaginations (l) and ionic diffusion coefficient (D) within them are also known. Both analytical and numerical solutions have been given and predictions compared. It is shown that in the case of large currents and potentials the analytical solution predictions will underestimate the magnitudes and rates of onset of the voltage responses. The relative magnitude of the transport number effect within the invaginations (or dendrites) and other transport number contributions to slow potential changes have also been assessed and order-of-magnitude values of these are estimated for some biological data.     

On the use of in vivo cargo velocity as a biophysical marker

Molecular motors move many intracellular cargos along microtubules. Recently, it has been hypothesized that in vivo cargo velocity can be used to determine the number of engaged motors. We use theoretical and experimental approaches to investigate these assertions, and find that this hypothesis is inconsistent with previously described motor behavior, surveyed and re-analyzed in this paper. Studying lipid droplet motion in Drosophila embryos, we compare transport in a mutant, Delta(halo), with that in wild-type embryos. The minus-end moving cargos in the mutant appear to be driven by more motors (based on in vivo stall force observations). Periods of minus-end motion are indeed longer than in wild-type embryos but the corresponding velocities are not higher. We conclude that velocity is not a definitive read-out of the number of motors propelling a cargo.     

Processive cytoskeletal motors studied with single-molecule fluorescence techniques

Processive cytoskeletal motors from the myosin, kinesin, and dynein families walk on actin filaments and microtubules to drive cellular transport and organization in eukaryotic cells. These remarkable molecular machines are able to take hundreds of successive steps at speeds of up to several microns per second, allowing them to effectively move vesicles and organelles throughout the cytoplasm. Here, we focus on single-molecule fluorescence techniques and discuss their wide-ranging applications to the field of cytoskeletal motor research. We cover both traditional fluorescence and sub-diffraction imaging of motors, providing examples of how fluorescence data can be used to measure biophysical parameters of motors such as coordination, stepping mechanism, gating, and processivity. We also outline some remaining challenges in the field and suggest future directions.     

Metabolism and axonal transport of polyamines in a single identified neuron of Aplysia californica

Abstract: The metabolism of polyamines was investigated by injecting purified [³H]putrescine directly into the soma of the giant neuron R2 of Aplysia . Injected putrescine was rapidly metabolized to spermidine, spermine, and several catabolites, including GABA and monoacetylputrescine. Identification of these products was by comparison with the authentic compound using ion exchange chromatography. When R2 was injected with amounts of [³H]putrescine determined so that the intracellular content of labeled precursor was less than 6 × 10-⁶ M , metabolism was rapid and occurred via pathways similar to those in mammalian tissues. At concentrations of labeled precursor greater than 2 × 10⁻⁴ M , relatively little putrescine was converted to product. By 4 h after injection, putrescine and its labeled products appeared in R2's axon, where additional metabolism occurred. These results indicated that the enzymes involved in polyamine interconversion are not restricted to R2's cell body, and this suggestion was corroborated by finding ornithine decarboxylase and S -adenosylmethionine decarboxylase activities in Aplysia nerves. The distribution of the polyamines along R2's axon was compared with that of ³H-glycoproteins, with the finding that while the acid-soluble polyamines move by diffusion, labeled polyamines associated with protein are rapidly transported.     

Ferritin forms dynamic oligomers to associate with microtubules in vivo: implication for the role of microtubules in iron metabolism

Ferritin, a ubiquitously distributed iron storage protein, has been reported to interact with microtubules in vitro (Hasan et al., 2005, FEBS journal 272:822-831). Here, we demonstrate that ferritin binds with the microtubules in an oligomeric form and that the microtubule-bound ferritin contains more than two-fold amount of iron compared to the unbound ferritin fraction in vitro. Indirect immunofluorescence microscopy showed that a significant fraction of the ferritin molecules colocalized with the microtubules as oligomers in a wide variety of cell lines. These findings are consistent with the immediate oligomerization of rhodamine-labeled ferritin, microinjected in living human hepatoma cells. Ferritin oligomers were dynamic in the cytoplasm, and an anti-microtubule drug significantly inhibited their intracellular movement. Treatment of cells with an iron donor, ferric ammonium citrate, remarkably increased the number of cells containing ferritin oligomers. On the other hand, when the cells, such as mouse neuroblastoma cells, were deprived of iron, ferritin oligomers were localized in the microtubule dense, neurite shafts, but were disappeared from the microtubule deficient neurite tips. These data indicate that the microtubules provide a scaffold for the cytoplasmic distribution and transport of the iron-rich ferritin and implicate the role of microtubules in iron metabolism.     

Synergism between halide binding and proton transport in a CLC-type exchanger

The Cl-/H+ exchange-transporter CLC-ec1 mediates stoichiometric transmembrane exchange of two Cl- ions for one proton. A conserved tyrosine residue, Y445, coordinates one of the bound Cl- ions visible in the structure of this protein and is located near the intersection of the Cl- and H+ pathways. Mutants of this tyrosine were scrutinized for effects on the coupled transport of Cl- and H+ determined electrophysiologically and on protein structure determined crystallographically. Despite the strong conservation of Y445 in the CLC family, substitution of F or W at this position preserves wild-type transport behavior. Substitution by A, E, or H, however, produces uncoupled proteins with robust Cl- transport but greatly impaired movement of H+. The obligatory 2 Cl-/1 H+ stoichiometry is thus lost in these mutants. The structures of all the mutants are essentially identical to wild-type, but apparent anion occupancy in the Cl- binding region correlates with functional H+ coupling. In particular, as determined by anomalous diffraction in crystals grown in Br-, an electrophysiologically competent Cl- analogue, the well-coupled transporters show strong Br- electron density at the "inner" and "central" Cl- binding sites. However, in the uncoupled mutants, Br- density is absent at the central site, while still present at the inner site. An additional mutant, Y445L, is intermediate in both functional and structural features. This mutant clearly exchanges H+ for Cl-, but at a reduced H+-to-Cl- ratio; likewise, both the central and inner sites are occupied by Br-, but the central site shows lower Br- density than in wild-type (or in Y445F,W). The correlation between proton coupling and central-site occupancy argues that halide binding to the central transport site somehow facilitates movement of H+, a synergism that is not readily understood in terms of alternating-site antiport schemes.     

Modelling sand/mud transport and morphodynamics in the Seine river mouth (France): an attempt using a process-based approach

The mouth of the Seine River estuary (France) has undergone marked morphological evolution over several decades mainly due to engineering works aimed at improving access to Rouen and Le Havre harbours. The intertidal areas are decreasing in size and the lower estuary is accumulating sediment and prograding. In order to understand and better describe the major morphological behaviours of the estuary, a morphodynamic numerical model was developed within the Seine-Aval program. At the end of the 1st part of the research program, a validated fine sediment transport model (3D) was available (Le Hir et al., 2001b). As the present morphological study addresses medium-term issues (a few decades), and because of the need to investigate impacts of local structures or events, we chose to use the so-called “process-based approach” starting from the existing model. First, the existing model was upgraded to account for (suspended) sand transport, and to achieve coupling between morphological changes and sediment transport. Erodability of the sediment accounts for the respective proportions of mud and sand. Simulations starting from an arbitrary surficial sediment cover show that the model is able to reproduce realistic sediment patterns. For example, it is able to change the sediment nature on the intertidal flat near Le Havre from sand to mud. Observed structures of suspended sediment are also reproduced: fine particles mainly follow the turbidity maximum whereas significant concentrations of sand grains in suspension are found where the hydrodynamic stresses are intense. Concerning morphodynamics, simulations with real forcing over one year are discussed. The effect of waves on the bathymetric evolution of the mouth is shown and the sensitivity of morphodynamics to the coupling procedure is tested.     

Model of active transport of ions in cardiac cell

A model of the active transport of ions in a cardiac muscle cell, which takes into account the active transport of Na⁺, K⁺, Ca²⁺, Mg²⁺, HCO₃⁻ and Cl⁻ ions, has been constructed. The model allows independent calculations of the resting potential at the biomembrane and concentrations of basic ions (sodium, potassium, chlorine, magnesium and calcium) in a cell. For the analysis of transport processes in cardiac cell hierarchical algorithm “one ion-one transport system” was offered. The dependence of the resting potential on concentrations of the ions outside a cell has been established. It was shown, that ions of calcium and magnesium, despite their rather small concentration, play an essential role in maintenance of resting potential in cardiac cell. The calculated internal concentrations of ions are in good agreement with the corresponding experimental values.     

Sucrose uptake in isolated phloem of celery is a single saturable transport system

The uptake of different sugars was studied in segments of isolated phloem from petioles of celery (Apium graveolens L.) in order to determine the kinetics and specificity of phloem loading in this highly uniform conductive tissue. The uptake kinetics of sucrose and the sugar alcohol, mannitol, which are both phloem-translocated, indicated presence of a single saturable system, while uptake of non-phloem sugars (glucose and 3-O-methylglucose) exhibited biphasic kinetics with lower uptake rates than those for sucrose and mannitol. The presence of unlabeled mannitol, 3-O-methylglucose and maltose in the incubation solution did not cause inhibition of labeled-sucrose uptake, indicating high carrier specificity and lack of sucrose hydrolysis in vivo. The pH optimum for sucrose uptake was 5–6. Furthermore, a rapid and transient alkalinization of the external media by sucrose indicated a sugar/H⁺-cotransport mechanism. Dual-labeling experiments showed that sucrose influx continued at a constant rate (V _max=15 mol·h^-1·(g FW)^-1), whereas sucrose efflux was low and insensitive to external concentration. Therefore, the saturable uptake kinetics for sucrose did not appear to be the result of an equilibrium between rates of sucrose influx and efflux.Abbreviations 3-OMG 3-O-methylglucose - PCMBS p-chloromercuribenzene sulfonate - SE-CC sieve element-companion cell - VB vascular bundle     

Nucleocytoplasmic shuttling of antigen in mammalian cells conferred by a soluble versus insoluble single-chain antibody fragment equipped with import/export signals

The ectopic expression of antibody fragments within mammalian cells is a challenging approach for interfering with or even blocking the biological function of the intracellular target. For this purpose, single-chain Fv (scFv) fragments are generally preferred. Here, by transfecting several mammalian cell lines, we compared the intracellular behavior of two scFvs (13R4 and 1F4) that strongly differ in their requirement of disulphide bonding for the formation of active molecules in bacteria. The scFv 13R4, which is correctly folded in the bacterial cytoplasm, was solubly expressed in all cell lines tested and was distributed in their cytoplasm and nucleus, as well. In addition, by appending to the 13R4 molecules the SV40 T-antigen nuclear localisation signal (NLS) tag, cytoplasmic-coexpressed antigen was efficiently retargeted to the nucleus. Compared to the scFv 13R4, the scFv 1F4, which needs to be secreted in bacteria for activity, accumulated, even with the NLS tag, as insoluble aggregates within the cytoplasm of the transfected cells, thereby severely disturbing fundamental functions of cell physiology. Furthermore, by replacing the NLS tag with a leucine-rich nuclear export signal (NES), the scFv 13R4 was exclusively located in the cytoplasm, whereas the similarly modified scFv 1F4 still promoted cell death. Coexpression of NES-tagged 13R4 fragments with nuclear antigen promoted its efficient retargeting to the cytoplasm. This dominant effect of the NES tag was also observed after exchange of the nuclear signals between the scFv 13R4 and its antigen. Taken together, the results indicate that scFvs that are active in the cytoplasm of bacteria may behave similarly in mammalian cells and that the requirement of their conserved disulphide bridges for activity is a limiting factor for mediating the nuclear import/export of target in a mammalian cell context. The described shuttling effect of antigen conferred by a soluble scFv may represent the basis of a reliable in vivo assay of effective protein- protein interactions.