A quantitative study of microtubules in motor and sensory axons

The number, density and distribution of microtubules were compared in the myelinated motor and sensory axons of the spinal roots of lizard (Lacerta muralis). In both motor and sensory axons the average number and density of microtubules were found to be related to the axonal size: the average number of microtubules rose, while the microtubular density decreased with an increase in the cross-sectional area of the axon. More precisely, a linear relationship was observed between the logarithm of the microtubular density and the cross-sectional area of the axon. No significant differences in the microtubular number and density were found between motor and sensory axons of corresponding size. Microtubules were unevenly distributed throughout the cross section of both motor and sensory axons. In particular, a nonaccidental association between microtubules and mitochondria was found in both axon types.     

The role of microtubules in processive kinesin movement

Kinesins are microtubule-based motors that are important for various intracellular transport processes. To understand the mechanism of kinesin movement, X-ray crystallography has been used to study the atomic structures of kinesin. However, as crystal structures of kinesin alone accumulate, it is becoming clear that kinesin structures should also be investigated with the microtubule to understand the contribution of the microtubule track to the nucleotide-induced conformational changes of kinesin. Recently, several high-resolution structures of kinesin with microtubules were obtained using cryo-electron microscopy. Comparison with X-ray crystallographic structures revealed the importance of the microtubule in determining the conformation of kinesin. Together with recent biophysical data, we describe different structural models of processive kinesin movement and provide a framework for future experiments.     

Luminal material in microtubules of frog olfactory axons: structure and distribution

The substructure and distribution of luminal material in microtubules of olfactory axons were studied in the bullfrog, Rana catesbeiana. By using numerous fixation methods, with and without osmium tetroxide, the luminal component was shown not to be an artifact of fixation. The material consists of globular elements 4-5 nm in diameter loosely arranged within the lumen in a discontinuous column. Counts of microtubules showing luminal material were obtained for axons in the proximal and distal ends of the olfactory nerve, and it was found that 16-18% more of the microtubules in the distal regions showed the luminal component. This raises the possibility that the material might be translocated within the microtubule lumen and tends to accumulate as it moves distally toward the axon terminal. In contrast to those of the olfactory axons, microtubules assembled in vitro from frog brain tubulin did not show luminal material. When microtubules in olfactory axons were depolymerized in situ by cold and calcium treatment and then induced to reassemble, most of those that were formed de novo showed empty lumina. Such evidence suggests that the luminal material is not an integral component of the microtubule. The hypothesis is discussed that material may be translocated within the lumina of microtubules. Furthermore, in the case of neuronal microtubules, the possibility is raised that they may serve as conduits for their own wall subunits.     

Differential behavior of photoactivated microtubules in growing axons of mouse and frog neurons

To characterize the behavior of axonal microtubules in vivo, we analyzed the movement of tubulin labeled with caged fluorescein after activation to be fluorescent by irradiation of 365-nm light. When mouse sensory neurons were microinjected with caged fluorescein-labeled tubulin and then a narrow region of the axon was illuminated with a 365-nm microbeam, photoactivated tubulin was stationary regardless of the position of photoactivation. We next introduced caged fluorescein-labeled tubulin into Xenopus embryos and nerve cells isolated from injected embryos were analyzed by photoactivation. In this case, movement of the photoactivated zone toward the axon tip was frequently observed. The photoactivated microtubule segments in the Xenopus axon moved out from their initial position without significant spreading, suggesting that fluorescent microtubules are not sliding as individual filaments, but rather translocating en bloc. Since these observations raised the possibility that the mechanism of nerve growth might differ between two types of neurons, we further characterized the movement of another component of the axon structure, the plasma membrane. Analysis of the position of polystyrene beads adhering to the neurites of Xenopus neurons revealed anterograde movement of the beads at the rate similar to the rate of microtubule movement. In contrast, no movement of the beads relative to the cell body was observed in mouse sensory neurons. These results suggest that the mode of translocation of cytoskeletal polymers and some components of the axon surface differ between two neuron types and that most microtubules are stationary within the axon of mammalian neurons where the surface-related motility of the axon is not observed.     

Membrane marker movement on sympathetic axons in tissue culture

In cultures of dissociated chick sympathetic ganglia, retrograde (somatopetal) movement of concanavalin A receptors in the axolemma can be observed directly. Such movement was visualized by using concanavalin A coated red blood cells (ConA-RBCs) as membrane markers. Forty-eight percent of all ConA-RBCs which bound to sympathetic nerve fibers moved somatopetally at rates ranging from 11-84 μ/hr with a mean and standard error of 49 ± 6 μ/hr (n = 18). On nongrowing nerve fibers, the ConA- RBCs within 60 μ of the cell body showed retrograde movement, while on elongating neurites only those markers within 30 μ showed such movement. The rate of retrograde ConA-RBC movement appeared to increase with distance from the cell soma. The binding of ConA-RBCs to sympathetic neurites was specific for concanavalin A receptors since pretreatment with either concanavalin A or α-methylglucopyranoside prevented this binding. Untreated polystyrene beads (1.1 μ) which bound nonspecifically to the neurite membrane also showed retrograde movement. These beads moved somatopetally at rates similar to those of the ConA-RBCs but did so regardless of their initial distance from the soma. These data suggest that retrograde movement of surface elements might be a general property of cultured sympathetic nerve fibers.     

Cultured cell extracts support organelle movement on microtubules in vitro

Directed movements of organelles have been observed in a variety of cultured cells. To study the regulation and molecular basis of intracellular organelle motility, we have prepared extracts from cultured chick embryo fibroblasts (CEF cells) which support the movement of membraneous organelles along microtubules. The velocity, frequency and characteristics of organelle movements in vitro were similar to those within intact cells. Organelles and extract-coated anionic beads moved predominantly (80%) toward the minus ends of microtubules that had been regrown from centrosomes, corresponding to retrograde translocation. Similar microtubule-dependent organelle movements were observed in extracts prepared from other cultured cells (African green monkey kidney and 3T3 cells). Organelle motility was ATP and microtubule dependent. The frequency of organelle movement was inhibited by acidic (pH less than 7) or alkaline (pH greater than 8) solutions, high ionic strength ([ KCl] = 0.1 M), and the chelation of free magnesium ions. Treatment of the extracts with adenylyl imidodiphosphate (AMP-PNP, 7 mM), sodium orthovanadate (vanadate; Na3VO4, 20 microM), or N-ethylmaleimide (NEM, 2 mM) blocked all organelle motility. The decoration of microtubules with organelles was observed in the presence of AMP-PNP or vanadate. Motility was not affected by cytochalasin D (2 microM) or cAMP (1 mM). Kinesin (Mr = 116,000), an anterograde microtubule-based motor, was partially purified from the CEF extract by microtubule affinity purification in the presence of AMP-PNP, and was able to drive the movement of microtubule on glass coverslips. A similar preparation made in the presence of vanadate contained a different subset of proteins and did not support motility. These results demonstrate that intracellular organelle motility can be reproduced in vitro and provide the basis for investigating the roles of individual molecular components involved in the organelle motor complex.     

Newly assembled microtubules are concentrated in the proximal and distal regions of growing axons

We have investigated the sites of microtubule (MT) assembly in neurons during axon growth by taking advantage of the relationship between the proportion of tyrosinated alpha-tubulin (tyr-tubulin) in MTs and their age. Specifically, young (newly assembled) MTs contain more tyr-tubulin than older (more long-lived) MTs. To quantify the relative proportion of tyr-tubulin in MTs, cultured rat sympathetic neurons were permeabilized under conditions that stabilize existing MTs and remove unassembled tubulin. The MTs were then double-stained with antibodies to tyr-tubulin (as a measure of the amount of tyr-tubulin in MTs) and to beta-tubulin (as a measure of total MT mass), using immunofluorescence procedures. Cells were imaged with a cooled charge-coupled device camera and the relative proportion of tyr-tubulin in the MTs was quantified by computing the ratio of the tyr-tubulin fluorescence to the beta-tubulin fluorescence using a novel application of digital image processing and analysis techniques. The amount of tyr-tubulin in the MTs was highest in the cell body and at the growth cone; peak ratios in these two regions were approximately 10-fold higher than for the axon shaft. Moving out from the cell body into the axon, the tyr-tubulin content declined over an average distance of 40 microns to reach a constant low value within the axon shaft and then rose again more distally, over an average distance of 110 microns, to reach a peak at the growth cone (average axon length = 358 microns). These observations indicate that newly assembled MTs are concentrated in the proximal and distal regions of growing axons and therefore that the cell body and growth cone are the most active sites of MT assembly dynamics in neurons that are actively extending axons.     

Microtubule-associated protein 2 within axons of spinal motor neurons: associations with microtubules and neurofilaments in normal and beta,beta'-iminodipropionitrile-treated axons

We have examined the distribution of microtubule-associated protein 2 (MAP2) in the lumbar segment of spinal cord, ventral and dorsal roots, and dorsal root ganglia of control and beta,beta'-iminodipropionitrile- treated rats. The peroxidase-antiperoxidase technique was used for light and electron microscopic immunohistochemical studies with two monoclonal antibodies directed against different epitopes of Chinese hamster brain MAP2, designated AP9 and AP13. MAP2 immunoreactivity was present in axons of spinal motor neurons, but was not detected in axons of white matter tracts of spinal cord and in the majority of axons of the dorsal root. A gradient of staining intensity among dendrites, cell bodies, and axons of spinal motor neurons was present, with dendrites staining most intensely and axons the least. While dendrites and cell bodies of all neurons in the spinal cord were intensely positive, neurons of the dorsal root ganglia were variably stained. The axons of labeled dorsal root ganglion cells were intensely labeled up to their bifurcation; beyond this point, while only occasional central processes in dorsal roots were weakly stained, the majority of peripheral processes in spinal nerves were positive. beta,beta'- Iminodipropionitrile produced segregation of microtubules and membranous organelles from neurofilaments in the peripheral nervous system portion and accumulation of neurofilaments in the central nervous system portion of spinal motor axons. While both anti-MAP2 hybridoma antibodies co-localized with microtubules in the central nervous system portion, only one co-localized with microtubules in the peripheral nervous system portion of spinal motor axons, while the other antibody co-localized with neurofilaments and did not stain the central region of the axon which contained microtubules. These findings suggest that (a) MAP2 is present in axons of spinal motor neurons, albeit in a lower concentration or in a different form than is present in dendrites, and (b) the MAP2 in axons interacts with both microtubules and neurofilaments.     

Single microtubules from squid axoplasm support bidirectional movement of organelles

Single filaments, dissociated from the extruded axoplasm of the squid giant axon and visualized by video-enhanced differential interference contrast microscopy, transport organelles bidirectionally. Organelles moving in the same or opposite directions along the same filament can pass each other without colliding, indicating that each transport filament has several tracks for organelle movement. In order to characterize transport filaments, organelle movements were first examined by video microscopy, and then the same filaments were examined by electron microscopy after rapid-freezing, freeze-drying, and rotary-shadowing. Transport filaments that supported bidirectional movement of organelles are 22 nm to 27 nm in diameter and have a substructure indicative of a single microtubule. Immunofluorescence showed that virtually all transport filaments contain tubulin. These results show that single microtubules can serve as a substratum for organelle movement, and suggest that an interaction between organelles and microtubules is the basis of fast axonal transport.     

Retrograde but not anterograde bead movement in intact axons requires dynein

Dynein and kinesin have been implicated as the molecular motors that are responsible for the fast transport of axonal membranous organelles and vesicles. Experiments performed in vitro with partially reconstituted preparations have led to the hypothesis that kinesin moves organelles in the anterograde direction and dynein moves them in the retrograde direction. However, the molecular basis of transport directionality remains unclear. In the experiments described here, carboxylated fluorescent beads were injected into living Mauthner axons of lamprey and the beads were observed to move in both the anterograde and retrograde directions. The bead movement in both directions required intact microtubules, occurred at velocities approaching organelle fast transport in vivo, and was inhibited by vanadate at concentrations that inhibit organelle fast transport. When living axons were injected with micromolar concentrations of vanadate and irradiated at 365 nm prior to bead injections, a treatment that results in the V1 photolysis of dynein, the retrograde movement of the beads was specifically abolished. Neither the ultraviolet irradiation alone nor the vanadate alone produced the retrograde-specific inhibition. These results support the hypothesis that dynein is required for retrograde, but not anterograde, transport in vivo. © 1995 John Wiley & Sons, Inc.     

Vaccinia virus utilizes microtubules for movement to the cell surface

Vaccinia virus (VV) egress has been studied using confocal, video, and electron microscopy. Previously, intracellular-enveloped virus (IEV) particles were proposed to induce the polymerization of actin tails, which propel IEV particles to the cell surface. However, data presented support an alternative model in which microtubules transport virions to the cell surface and actin tails form beneath cell-associated enveloped virus (CEV) particles at the cell surface. Thus, VV is unique in using both microtubules and actin filaments for egress. The following data support this proposal. (a) Microscopy detected actin tails at the surface but not the center of cells. (b) VV mutants lacking the A33R, A34R, or A36R proteins are unable to induce actin tail formation but produce CEV and extracellular-enveloped virus. (c) CEV formation is inhibited by nocodazole but not cytochalasin D or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP1). (d) IEV particles tagged with the enhanced green fluorescent protein fused to the VV B5R protein moved inside cells at 60 microm/min. This movement was stop-start, was along defined pathways, and was inhibited reversibly by nocodazole. This velocity was 20-fold greater than VV movement on actin tails and consonant with the rate of movement of organelles along microtubules.     

VIP induces the elongation of dendrites and axons in cultured hippocampal neurons: role of microtubules

In neurons from rat hippocampus, VIP induces the elongation of dendrites. In the present study, we have investigated in cultured hippocampal neurons whether VIP changed the actin and tubulin cytoskeleton in dendrites. VIP caused the elongation of dendrites and induced the outgrowth of microtubules, so that they extended up to the tips. In contrast, VIP reduced the F-actin content measured as total pixel after phalloidin staining in dendritic tips. These results suggest that VIP causes dendrite elongation by facilitating the outgrowth of microtubules into the newly formed extensions.     

Drosophila pod-1 crosslinks both actin and microtubules and controls the targeting of axons

Actin and microtubules (MTs) are tightly coordinated during neuronal growth cone navigation and are dynamically regulated in response to guidance cues; however, little is known about the underlying molecular mechanisms. Here, we characterize Drosophila pod-1 (dpod1) and show that purified Dpod1 can crosslink both actin and MTs. In cultured S2 cells, Dpod1 colocalizes with lamellar actin and MTs, and overexpression remodels the cytoskeleton to promote dynamic neurite-like actin-dependent projections. Consistent with these observations, Dpod1 localizes to the tips of growing axons, regions where actin and MTs interact, and is especially abundant at navigational choice points. In either the absence or overabundance of Dpod1, growth cone targeting but not outgrowth is disrupted. Taken together, these results reveal novel activities for pod-1 and show that proper levels of Dpod1, an actin/MT crosslinker, must be maintained in the growth cone for correct axon guidance.     

The role of kinesin and other soluble factors in organelle movement along microtubules

Kinesin is a force-generating ATPase that drives the sliding movement of microtubules on glass coverslips and the movement of plastic beads along microtubules. Although kinesin is suspected to participate in microtubule-based organelle transport, the exact role it plays in this process is unclear. To address this question, we have developed a quantitative assay that allows us to determine the ability of soluble factors to promote organelle movement. Salt-washed organelles from squid axoplasm exhibited a nearly undetectable level of movement on purified microtubules. Their frequency of movement could be increased greater than 20-fold by the addition of a high speed axoplasmic supernatant. Immunoadsorption of kinesin from this supernatant decreased the frequency of organelle movement by more than 70%; organelle movements in both directions were markedly reduced. Surprisingly, antibody purified kinesin did not promote organelle movement either by itself or when it was added back to the kinesin-depleted supernatant. This result suggested that other soluble factors necessary for organelle movement were removed along with kinesin during immunoadsorption of the supernatant. A high level of organelle motor activity was recovered in a high salt eluate of the immunoadsorbent that contained only little kinesin. On the basis of these results we propose that organelle movement on microtubules involves other soluble axoplasmic factors in addition to kinesin.     

Cell-to-cell movement of TMV RNA is temperature-dependent and corresponds to the association of movement protein with microtubules

The movement protein (MP) of tobacco mosaic virus (TMV) is essential for spread of the viral RNA genome from cell to cell. During infection, the MP associates with microtubules, and it has been proposed that the cytoskeleton transports the viral ribonucleoprotein complex from ER sites of synthesis to plasmodesmata through which infection spreads into adjacent cells. However, microtubule association of MP was observed in cells undergoing late infection rather than in cells undergoing early infection at the leading edge of expanding infection sites where virus RNA cell-to-cell spread occurs. Therefore, alternative roles for microtubules in virus infection have been proposed, including a role in MP degradation. To further investigate the role of microtubules in virus pathogenesis, we tested the efficiency of cell-to-cell spread of infection and microtubule association of the MP in response to changes in temperature. We show that the subcellular distribution of MP is temperature-dependent and that a higher efficiency of intercellular transport of virus RNA at elevated temperatures corresponds to an increased association of MP with microtubules early in infection.