Luminal particles within cellular microtubules

The regulation of microtubule dynamics is attributed to microtubule-associated proteins that bind to the microtubule outer surface, but little is known about cellular components that may associate with the internal side of microtubules. We used cryoelectron tomography to investigate in a quantitative manner the three dimensional structure of microtubules in intact mammalian cells. We show that the lumen of microtubules in this native state is filled with discrete, globular particles with a diameter of 7 nm and spacings between 8 and 20 nm in neuronal cells. Cross-sectional views of microtubules confirm the presence of luminal material in vitreous sections of brain tissue. Most of the luminal particles had connections to the microtubule wall, as revealed in tomograms. A higher accumulation of particles was seen near the retracting plus ends of microtubules. The luminal particles were abundant in neurons, but were also observed in other cells, such as astrocytes and stem cells.     

The Translocation Selectivity of the Kinesins that Mediate Neuronal Organelle Transport

Polarized kinesin‐driven transport is crucial for development and maintenance of neuronal polarity. Kinesins are thought to recognize biochemical differences between axonal and dendritic microtubules in order to deliver their cargoes to the appropriate domain. To identify kinesins that mediate polarized transport, we prepared constitutively active versions of all the kinesins implicated in vesicle transport and expressed them in cultured hippocampal neurons. Seven kinesins translocated preferentially to axons and five translocated into both axons and dendrites. None translocated selectively to dendrites. Highly homologous members of the same subfamily displayed distinctly different translocation preferences and were differentially regulated during development. By expressing chimeric kinesins, we identified two microtubule‐binding elements within the motor domain that are important for selective translocation. We also discovered elements in the dimerization domain of kinesin‐2 motors that contribute to their selective translocation. These observations indicate that selective interactions between kinesin motor domains and microtubules can account for polarized transport to the axon, but not for selective dendritic transport.     

Microtubules have opposite orientation in axons and dendrites of Drosophila neurons

In vertebrate neurons, axons have a uniform arrangement of microtubules with plus ends distal to the cell body (plus-end-out), and dendrites have equal numbers of plus- and minus-end-out microtubules. To determine whether microtubule orientation is a conserved feature of axons and dendrites, we analyzed microtubule orientation in invertebrate neurons. Using microtubule plus end dynamics, we mapped microtubule orientation in Drosophila sensory neurons, interneurons, and motor neurons. As expected, all axonal microtubules have plus-end-out orientation. However, in proximal dendrites of all classes of neuron, approximately 90% of dendritic microtubules were oriented with minus ends distal to the cell body. This result suggests that minus-end-out, rather than mixed orientation, microtubules are the signature of the dendritic microtubule cytoskeleton. Surprisingly, our map of microtubule orientation predicts that there are no tracks for direct cargo transport between the cell body and dendrites in unipolar neurons. We confirm this prediction, and validate the completeness of our map, by imaging endosome movements in motor neurons. As predicted by our map, endosomes travel smoothly between the cell body and axon, but they cannot move directly between the cell body and dendrites.     

Rearrangement of microtubule polarity orientation during conversion of dendrites to axons in cultured pyramidal neurons

Axons and dendrites of neurons differ in the polarity orientation of their microtubules. Whereas the polarity orientation of microtubules in axons is uniform, with all plus ends distal, that in dendrites is nonuniform. The mechanisms responsible for establishment and maintenance of microtubule polarity orientation in neuronal processes remain unclear, however. We previously described a culture system in which dendrites of rat cortical neurons convert to axons. In the present study, we examined changes in microtubule polarity orientation in such dendrites. With the use of the hooking procedure and electron microscopy, we found that microtubule polarity orientation changed from nonuniform to uniform, with a plus end-distal arrangement, in dendrites that gave rise to axons during culture of neurons for 24 h. Microtubule polarity orientation remained nonuniform in dendrites that did not elongate. Axon regeneration at the dendritic tip thus triggered the disappearance of minus end-distal microtubules from dendrites. These minus end-distal microtubules also disappeared from dendrites during axon regeneration in the presence of inhibitors of actin polymerization, suggesting that actin-dependent transport of microtubules is not required for this process and implicating a previously unidentified mechanism in the establishment and maintenance of microtubule polarity orientation in neuronal processes.     

Changes in Neurofilament and Microtubule Distribution following Focal Axon Compression

Although a number of cytoskeletal derangements have been described in the setting of traumatic axonal injury (TAI), little is known of early structural changes that may serve to initiate a cascade of further axonal degeneration. Recent work by the authors has examined conformational changes in cytoskeletal constituents of neuronal axons undergoing traumatic axonal injury (TAI) following focal compression through confocal imaging data taken in vitro and in situ. The present study uses electron microscopy to understand and quantify in vitro alterations in the ultrastructural composition of microtubules and neurofilaments within neuronal axons of rats following focal compression. Standard transmission electron microscopy processing methods are used to identify microtubules, while neurofilament identification is performed using antibody labeling through gold nanoparticles. The number, density, and spacing of microtubules and neurofilaments are quantified for specimens in sham Control and Crushed groups with fixation at <1min following load. Our results indicate that the axon caliber dependency known to exist for microtubule and neurofilament metrics extends to axons undergoing TAI, with the exception of neurofilament spacing, which appears to remain constant across all Crushed axon diameters. Confidence interval comparisons between Control and Crushed cytoskeletal measures suggests early changes in the neurofilament spatial distributions within axons undergoing TAI may precede microtubule changes in response to applied loads. This may serve as a trigger for further secondary damage to the axon, representing a key insight into the temporal aspects of cytoskeletal degeneration at the component level, and suggests the rapid removal of neurofilament sidearms as one possible mechanism.     

Hooks and comets: The story of microtubule polarity orientation in the neuron

It is widely believed that signature patterns of microtubule polarity orientation within axons and dendrites underlie compositional and morphological differences that distinguish these neuronal processes from one another. Axons of vertebrate neurons display uniformly plus-end-distal microtubules, whereas their dendrites display non-uniformly oriented microtubules. Recent studies on insect neurons suggest that it is the minus-end-distal microtubules that are the critical feature of the dendritic microtubule array, whether or not they are accompanied by plus-end-distal microtubules. Discussed in this article are the history of these findings, their implications for the regulation of neuronal polarity across the animal kingdom, and potential mechanisms by which neurons establish the distinct microtubule polarity patterns that define axons and dendrites.     

Acute inactivation of MAP1b in growing sympathetic neurons destabilizes axonal microtubules

Microtubule-associated-protein 1b (MAP1b) is abundant in neurons actively extending axons. MAP1b is present on microtubules throughout growing axons, but is preferentially concentrated on microtubule polymer in the distal axon and growth cone. Although MAP1b has been implicated in axon growth and pathfinding, its specific functions are not well understood. Biochemical and transfection studies suggest that MAP1b has microtubule-stabilizing activity, but recent studies with neurons genetically deficient in MAP1b have not confirmed this. We have explored MAP1b functions in growing sympathetic neurons using an acute inactivation approach. Neurons without axons were injected with polyclonal MAP1b antibodies and then stimulated to extend axons. Injected cells were compared to controls in terms of axon growth behavior and several properties of axonal microtubules. The injected antibodies rapidly and quantitatively sequestered MAP1b in the cell body, making it unavailable to perform its normal functions. This immunodepletion of MAP1b had no statistically significant effect on axon growth, the amount of microtubule polymer in the axon, and the relative tyrosinated tubulin content of this polymer, and this was true in sympathetic neurons from rat, wild type mice, and tau knockout mice. Thus, robust axon growth can occur in the absence of MAP1b alone or both MAP1b and tau. However, immunodepletion of MAP1b significantly increased the sensitivity of microtubules in the distal axon and growth cone to nocodazole-induced depolymerization. These results indicate that MAP1b has microtubule-stabilizing activity in growing axons. This stabilizing activity may be required for some axonal functions, but it is not necessary for axon growth.     

Distinct roles of doublecortin modulating the microtubule cytoskeleton

Doublecortin is a neuronal microtubule-stabilising protein, mutations of which cause mental retardation and epilepsy in humans. How doublecortin influences microtubule dynamics, and thereby brain development, is unclear. We show here by video microscopy that purified doublecortin has no effect on the growth rate of microtubules. However, it is a potent anti-catastrophe factor that stabilises microtubules by linking adjacent protofilaments and counteracting their outward bending in depolymerising microtubules. We show that doublecortin-stabilised microtubules are substrates for kinesin translocase motors and for depolymerase kinesins. In addition, doublecortin does not itself oligomerise and does not bind to tubulin heterodimers but does nucleate microtubules. In cells, doublecortin is enriched at the distal ends of neuronal processes and our data raise the possibility that the function of doublecortin in neurons is to drive assembly and stabilisation of non-centrosomal microtubules in these doublecortin-enriched distal zones. These distinct properties combine to give doublecortin a unique function in microtubule regulation, a role that cannot be compensated for by other microtubule-stabilising proteins and nucleating factors.     

Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure

Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo–electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments.     

Effects of dynactin disruption and dynein depletion on axonal microtubules

We investigated potential roles of cytoplasmic dynein in organizing axonal microtubules either by depleting dynein heavy chain from cultured neurons or by experimentally disrupting dynactin. The former was accomplished by siRNA while the latter was accomplished by overexpressing P50-dynamitin. Both methods resulted in a persistent reduction in the frequency of transport of short microtubules. To determine if the long microtubules in the axon also undergo dynein-dependent transport, we ascertained the rates of EGFP-EB3 "comets" observed at the tips of microtubules during assembly. The rates of the comets, in theory, should reflect a combination of the assembly rate and any potential transport of the microtubule. Comets were initially slowed during P50-dynamitin overexpression, but this effect did not persist beyond the first day and was never observed in dynein-depleted axons. In fact, the rates of the comets were slightly faster in dynein-depleted axons. We conclude that the transient effect of P50-dynamitin overexpression reflects a reduction in microtubule polymerization rates. Interestingly, after prolonged dynein depletion, the long microtubules were noticeably misaligned in the distal regions of axons and failed to enter the filopodia of growth cones. These results suggest that the forces generated by cytoplasmic dynein do not transport long microtubules, but may serve to align them with one another and also permit them to invade filopodia.     

Dynein is required for polarized dendritic transport and uniform microtubule orientation in axons

Axons and dendrites differ in both microtubule organization and in the organelles and proteins they contain. Here we show that the microtubule motor dynein has a crucial role in polarized transport and in controlling the orientation of axonal microtubules in Drosophila melanogaster dendritic arborization (da) neurons. Changes in organelle distribution within the dendritic arbors of dynein mutant neurons correlate with a proximal shift in dendritic branch position. Dynein is also necessary for the dendrite-specific localization of Golgi outposts and the ion channel Pickpocket. Axonal microtubules are normally oriented uniformly plus-end-distal; however, without dynein, axons contain both plus- and minus-end distal microtubules. These data suggest that dynein is required for the distinguishing properties of the axon and dendrites: without dynein, dendritic organelles and proteins enter the axon and the axonal microtubules are no longer uniform in polarity.     

Microtubule integrity is essential for apical polarization and epithelial morphogenesis in the thyroid

In this study, we examined the contribution of microtubules to epithelial morphogenesis in primary thyroid cell cultures. Thyroid follicles consist of a single layer of polarized epithelial cells surrounding a closed compartment, the follicular lumen. Freshly isolated porcine thyroid cells aggregate and reorganize to form follicles when grown in primary cultures. Follicular reorganization is principally a morphogenetic process that entails the assembly of biochemically distinct apical and basolateral membrane domains, delimited by tight junctions. The establishment of cell surface polarity during folliculogenesis coincided with the polarized redistribution of microtubules, predominantly in the developing apical poles of cells. Disruption of microtubule integrity using either colchicine or nocodazole caused loss of defined apical membrane domains, tight junctions and follicular lumina. Apical membrane and tight junction markers became randomly distributed at the outer surfaces of aggregates. In contrast, the basolateral surface markers, E-cadherin and Na(+),K(+)-ATPase, remained correctly localized at sites of cell-cell contact and at the free surfaces of cell aggregates. These findings demonstrate that microtubules play a necessary role in thyroid epithelial morphogenesis. Specifically, microtubules are essential to preserve the correct localization of apical membrane components within enclosed cellular aggregates, a situation that is also likely to pertain where lumina must be formed from solid aggregates of epithelial precursors.     

Polarity orientation and assembly process of microtubule bundles in nocodazole-treated, MAP2c-transfected COS cells.

Microtubule bundles reminiscent of those found in neuronal processes are formed in fibroblasts and Sf9 cells that are transfected with the microtubule-associated proteins tau, MAP2, or MAP2c. To analyze the assembly process of these bundles and its relation to the microtubule polarity, we depolymerized the bundles formed in MAP2c-transfected COS cells using nocodazole, and observed the process of assembly of microtubule bundles after removal of the drug in cells microinjected with rhodamine-labeled tubulin. Within minutes of its removal, numerous short microtubule fragments were observed throughout the cytoplasm. These short fragments were randomly oriented and were already bundled. Somewhat longer, but still short bundles, were then found in the peripheral cytoplasm. These bundles became the primordium of the larger bundles, and gradually grew in length and width. The polarity orientation of microtubules in the reformed bundle as determined by "hook" procedure using electron microscope was uniform with the plus end distal to the cell nucleus. The results suggest that some mechanism(s) exists to orient the polarity of microtubules, which are not in direct continuity with the centrosome, during the formation of large bundles. The observed process presents a useful model system for studying the organization of microtubules that are not directly associated with the centrosomes, such as those observed in axons.     

Microtubule-synaptic vesicle associations in cultured rat spinal cord neurons

Summary This paper describes new ultrastructural features of neural processes and of synapses in cultured CNS tissue treated with albumin before fixation using a modification of the technique recently introduced by Gray (1975). Nerve fibre bundles in explants of foetal spinal cord grown in vitro for 15–18 days were transected microsurgically. After transection the cultures were exposed to 20% albumin in distilled water and then fixed in unbuffered osmium tetroxide followed by unbuffered glutaraldehyde.In this material, but not in controls (injured but not exposed to albumin; exposed to albumin without injury) microtubules were found within many axonal varicosities, often situated close to presynaptic membrane specializations. These microtubules were closely associated with vesicles resembling synaptic vesicles, which were occasionally aligned in rows along the microtubules. Similar vesicle-microtubule associations were also found in non-terminal axons. Microtubules were also observed very close to some postsynaptic densities.The possibility that the microtubule-vesicle associations are involved in vesicle movements (along axons and/or within axon terminals) is discussed. A more direct involvement of microtubules in terminals in the mechanism of transmitter release is also considered.The author wishes to thank Dr. A.R. Lieberman for his help and advice, Mr. Derek Fraser and Mr. Peter Felton for their technical assistance, Mr. Stuart Waterman for the photographic prints, and Professor D.W. James for laboratory facilities     

Viscoelasticity of Tau Proteins Leads to Strain Rate-Dependent Breaking of Microtubules during Axonal Stretch Injury: Predictions from a Mathematical Model

The unique viscoelastic nature of axons is thought to underlie selective vulnerability to damage during traumatic brain injury. In particular, dynamic loading of axons has been shown to mechanically break microtubules at the time of injury. However, the mechanism of this rate-dependent response has remained elusive. Here, we present a microstructural model of the axonal cytoskeleton to quantitatively elucidate the interaction between microtubules and tau proteins under mechanical loading. Mirroring the axon ultrastructure, the microtubules were arranged in staggered arrays, cross-linked by tau proteins. We found that the viscoelastic behavior specifically of tau proteins leads to mechanical breaking of microtubules at high strain rates, whereas extension of tau allows for reversible sliding of microtubules without any damage at small strain rates. Based on the stiffness and viscosity of tau proteins inferred from single-molecule force spectroscopy studies, we predict the critical strain rate for microtubule breaking to be in the range 22–44 s⁻¹, in excellent agreement with recent experiments on dynamic loading of micropatterned neuronal cultures. We also identified a characteristic length scale for load transfer that depends on microstructural properties and have derived a phase diagram in the parameter space spanned by loading rate and microtubule length that demarcates those regions where axons can be loaded and unloaded reversibly and those where axons are injured due to breaking of the microtubules.     

Changes in the main submandibular salivary duct of rabbits resulting from ductal ligation

Normal submandibular ducts from rabbits have been examined by mucosubstance histochemistry, transmission and scanning electron microscopy. The results were compared with the appearances of ducts removed 4...6 weeks after ligation. The normal ducts were composed mainly of columnar "light" cells and basal cells but, in addition, some "dark" cells and scattered goblet containing sulphated mucins were always present. The luminal surface of the ductal cells possessed numerous microvilli protruding into the lumen, and a rim of negatively charged mucin was present on this surface of these cells. After ligation the ducts became greatly distended by their fluid contents which remained under pressure until the duct was incised. The epithelial cells were flattened and appeared to contain less cytoplasm per cell; "light" cells, basal cells and "dark" cells were still recognisable. Goblet cells were much more plentiful than in the control ducts and often protruded into the lumen despite the increased intraluminal pressure. The development of a number of ciliated cells had also occurred and they were often situated close to goblet cells. Lymphatic vessels were more prominent around the ligated ducts. Luminal microvilli were less numerous than in the control ducts but the rim of negatively charged mucin on the luminal surface of ductal cells was more conspicuous. Mixed inflammatory cells were present within the lumina of ligated ducts especially in those parts adjacent to the ductal cells. No inflammatory cell has been observed passing through the wall of a main duct and the possibility exists that these cells had entered lumina within the gland and migrated from there to the main duct. The above findings may serve to help our understanding or physiological events in the ducts.     

Viscoelasticity of Tau Proteins Leads to Strain Rate-Dependent Breaking of?Microtubules during Axonal Stretch Injury: Predictions from a Mathematical Model

The unique viscoelastic nature of axons is thought to underlie selective vulnerability to damage during traumatic brain injury. In particular, dynamic loading of axons has been shown to mechanically break microtubules at the time of injury. However, the mechanism of this rate-dependent response has remained elusive. Here, we present a microstructural model of the axonal cytoskeleton to quantitatively elucidate the interaction between microtubules and tau proteins under mechanical loading. Mirroring the axon ultrastructure, the microtubules were arranged in staggered arrays, cross-linked by tau proteins. We found that the viscoelastic behavior specifically of tau proteins leads to mechanical breaking of microtubules at high strain rates, whereas extension of tau allows for reversible sliding of microtubules without any damage at small strain rates. Based on the stiffness and viscosity of tau proteins inferred from single-molecule force spectroscopy studies, we predict the critical strain rate for microtubule breaking to be in the range 22–44 s⁻¹, in excellent agreement with recent experiments on dynamic loading of micropatterned neuronal cultures. We also identified a characteristic length scale for load transfer that depends on microstructural properties and have derived a phase diagram in the parameter space spanned by loading rate and microtubule length that demarcates those regions where axons can be loaded and unloaded reversibly and those where axons are injured due to breaking of the microtubules.     

A composite model for establishing the microtubule arrays of the neuron

Neurons generate two distinct types of processes, termed axons and dendrites, both of which rely on a highly organized array of microtubules for their growth and maintenance. Axonal microtubules are uniformly oriented with their plus ends distal to the cell body, whereas dendritic microtubules are nonuniformly oriented. In neither case are the microtubules attached to the centrosome or any detectable structure that could establish their distinct patterns of polarity orientation. Studies from our laboratory over the past few years have led us to propose the following model for the establishment of the axonal and dendritic microtubule arrays. Microtubules destined for these processes are nucleated at the centrosome within the cell body of the neuron and rapidly released. The released microtubules are then transported into developing axons and dendrites to support their growth. Early in neuronal development, the microtubules are transported with their plus ends leading into immature processes that are the common progenitors of both axons and dendrites. This sets up a uniformly plus-end-distal pattern of polarity orientation, which is preserved in the developing axon. In the case of the dendrite, the plus-end-distal microtubules are joined by another population of microtubules that are transported into these processes with their minus-ends leading. Implicit in this model is that neurons have specialized machinery for regulating the release of microtubules from the centrosome and for transporting them with great specificity.     

Axonal microtubules necessary for generation of sodium current in squid giant axons: I. Pharmacological study on sodium current and restoration of sodium current by microtubule proteins and 260K protein

Effects of the reagents suppressing or supporting axoplasmic microtubule assembly were studied on the Na ionic current of squid giant axons by perfusing the axon internally with the solution containing the reagent. Among the reagents suppressing the assembly, colchicine, vinblastine, podophyllotoxin, sulfhydryl reagents such as DTNB and NEM, and chaotropic anions such as iodide and bromide, were examined. These reagents reduced maximum Na conductance and shifted the voltage dependence of steady-state Na activation in a depolarizing direction along the voltage axis. They also made the voltage dependence less steep, but did not affect sodium inactivation appreciably. Effects on Na ionic current of reagents which support microtubule assembly (Taxol, DMSO, D2O and temperature) were opposite the effects of those agents suppressing assembly. At the same time, we demonstrated that after Na currents were partially reduced, they could be restored by internally perfusing the axon with a solution containing microtubule proteins, 260K proteins and cAMP under conditions favorable for microtubule assembly. For full restoration, it was found that the following conditions were necessary: (1) The microenvironment within the axon is suitable for microtubule assembly. (2) Tubulins incorporated into microtubules are fully tyrosinated at their C-termini. (3) A peripheral protein having a molecular weight of 260,000 daltons (260K protein) is indispensable. These results suggest that axoplasmic microtubules and 260K proteins in the structure underlying the axolemma play a role in generating Na currents in squid giant axons.