Inhibition of a Mitotic Motor Compromises the Formation of Dendrite-like Processes from Neuroblastoma Cells

Microtubules in the axon are uniformly oriented, while microtubules in the dendrite are nonuniformly oriented. We have proposed that these distinct microtubule polarity patterns may arise from a redistribution of molecular motor proteins previously used for mitosis of the developing neuroblast. To address this issue, we performed studies on neuroblastoma cells that undergo mitosis but also generate short processes during interphase. Some of these processes are similar to axons with regard to their morphology and microtubule polarity pattern, while others are similar to dendrites. Treatment with cAMP or retinoic acid inhibits cell division, with the former promoting the development of the axon-like processes and the latter promoting the development of the dendrite-like processes. During mitosis, the kinesin-related motor termed CHO1/MKLP1 is localized within the spindle midzone where it is thought to transport microtubules of opposite orientation relative to one another. During process formation, CHO1/ MKLP1 becomes concentrated within the dendrite-like processes but is excluded from the axon-like processes. The levels of CHO1/MKLP1 increase in the presence of retinoic acid but decrease in the presence of cAMP, consistent with a role for the protein in dendritic differentiation. Moreover, treatment of the cultures with antisense oligonucleotides to CHO1/MKLP1 compromises the formation of the dendrite-like processes. We speculate that a redistribution of CHO1/MKLP1 is required for the formation of dendrite-like processes, presumably by establishing their characteristic nonuniform microtubule polarity pattern.     

Nonuniform Microtubular Polarity Established by CHO1/MKLP1 Motor Protein Is Necessary for Process Formation of Podocytes

Podocytes are unique cells that are decisively involved in glomerular filtration. They are equipped with a complex process system consisting of major processes and foot processes whose function is insufficiently understood (Mundel, P., and W. Kriz. 1995. Anat. Embryol. 192:385–397). The major processes of podocytes contain a microtubular cytoskeleton. Taking advantage of a recently established cell culture system for podocytes with preserved ability to form processes (Mundel, P., J. Reiser, A. Zúñiga Mejía Borja, H. Pavenstädt, G.R. Davidson, W. Kriz, and R. Zeller. 1997b. Exp. Cell Res. 36:248–258), we studied the functional significance of the microtubular system in major processes. The following data were obtained: (a) Microtubules (MTs) in podocytes show a nonuniform polarity as revealed by hook-decoration. (b) CHO1/ MKLP1, a kinesin-like motor protein, is associated with MTs in podocytes. (c) Treatment of differentiating podocytes with CHO1/MKLP1 antisense oligonucleotides abolished the formation of processes and the nonuniform polarity of MTs. (d) During the recovery from taxol treatment, taxol-stabilized (nocodazole- resistant) MT fragments were distributed in the cell periphery along newly assembled nocodazole-sensitive MTs. A similar distribution pattern of CHO1/MKLP1 was found under these circumstances, indicating its association with MTs. (e) In the recovery phase after complete depolymerization, MTs reassembled exclusively at centrosomes. Taken together, these findings lead to the conclusion that the nonuniform MT polarity in podocytes established by CHO1/MKLP1 is necessary for process formation.     

Tubulin transport in neurons

A question of broad importance in cellular neurobiology has been, how is microtubule cytoskeleton of the axon organized? It is of particular interest because of the history of conflicting results concerning the form in which tubulin is transported in the axon. While many studies indicate a stationary nature of axonal microtubules, a recent series of experiments reports that microtubules are recruited into axons of neurons grown in the presence of a microtubule-inhibitor, vinblastine (Baas, P.W., and F.J. Ahmad. 1993.J. Cell Biol. 120:1427-1437: Ahmad F.J., and P.W. Baas. 1995. J. Cell Sci, 108:2761-2769; Sharp, D.J., W. Yu, and P.W. Baas. 1995. J. Cell Biol, 130:93-103; Yu, W., and P.W. Baas. 1995. J. Neurosci. 15:6827-6833.). Since vinblastine stabilizes bulk microtubule-dynamics in vitro, it was concluded that preformed microtubules moved into newly grown axons. By visualizing the polymerization of injected fluorescent tubulin, we show that substantial microtubule polymerization occurs in neurons grown at reported vinblastine concentrations. Vinblastine inhibits, in a concentration-dependent manner, both neurite outgrowth and microtubule assembly. More importantly, the neuron growth conditions of low vinblastine concentration allowed us to visualize the footprints of the tubulin wave as it polymerized and depolymerized during its slow axonal transport. In contrast, depolymerization resistant fluorescent microtubules did not move when injected in neurons. We show that tubulin subunits, not microtubules, are the primary form of tubulin transport in neurons.     

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.     

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 microtubule polarity orientation during the development of hippocampal neurons in culture

Microtubules in the dendrites of cultured hippocampal neurons are of nonuniform polarity orientation. About half of the microtubules have their plus ends oriented distal to the cell body, and the other half have their minus ends distal; in contrast, microtubules in the axon are of uniform polarity orientation, all having their plus ends distal (Baas, P.W., J.S. Deitch, M. M. Black, and G. A. Banker. 1988. Proc. Natl. Acad. Sci. USA. 85:8335-8339). Here we describe the developmental changes that give rise to the distinct microtubule patterns of axons and dendrites. Cultured hippocampal neurons initially extend several short processes, any one of which can apparently become the axon (Dotti, C. G., and G. A. Banker. 1987. Nature [Lond.]. 330:477-479). A few days after the axon has begun its rapid growth, the remaining processes differentiate into dendrites (Dotti, C. G., C. A. Sullivan, and G. A. Banker. 1988. J. Neurosci. 8:1454-1468). The polarity orientation of the microtubules in all of the initial processes is uniform, with plus ends distal to the cell body, even through most of these processes will become dendrites. This uniform microtubule polarity orientation is maintained in the axon at all stages of its growth. The polarity orientation of the microtubules in the other processes remains uniform until they begin to grow and acquire the morphological characteristics of dendrites. It is during this period that microtubules with minus ends distal to the cell body first appear in these processes. The proportion of minus end-distal microtubules gradually increases until, by 7 d in culture, about equal numbers of dendritic microtubules are oriented in each direction. Thus, the establishment of regional differences in microtubule polarity orientation occurs after the initial polarization of the neuron and is temporally correlated with the differentiation of the dendrites.     

A Highlights from MBoC Selection: Global Up-Regulation of Microtubule Dynamics and Polarity Reversal during Regeneration of an Axon from a Dendrite

Axon regeneration is crucial for recovery after trauma to the nervous system. For neurons to recover from complete axon removal they must respecify a dendrite as an axon: a complete reversal of polarity. We show that Drosophila neurons in vivo can convert a dendrite to a regenerating axon and that this process involves rebuilding the entire neuronal microtubule cytoskeleton. Two major microtubule rearrangements are specifically induced by axon and not dendrite removal: 1) 10-fold up-regulation of the number of growing microtubules and 2) microtubule polarity reversal. After one dendrite reverses its microtubules, it initiates tip growth and takes on morphological and molecular characteristics of an axon. Only neurons with a single dendrite that reverses polarity are able to initiate tip growth, and normal microtubule plus-end dynamics are required to initiate this growth. In addition, we find that JNK signaling is required for both the up-regulation of microtubule dynamics and microtubule polarity reversal initiated by axon injury. We conclude that regulation of microtubule dynamics and polarity in response to JNK signaling is key to initiating regeneration of an axon from a dendrite.     

Wnt signaling establishes the microtubule polarity in neurons through regulation of Kinesin-13

Neuronal polarization is facilitated by the formation of axons with parallel arrays of plus-end-out and dendrites with the nonuniform orientation of microtubules. In C. elegans, the posterior lateral microtubule (PLM) neuron is bipolar with its two processes growing along the anterior–posterior axis under the guidance of Wnt signaling. Here we found that loss of the Kinesin-13 family microtubule-depolymerizing enzyme KLP-7 led to the ectopic extension of axon-like processes from the PLM cell body. Live imaging of the microtubules and axonal transport revealed mixed polarity of the microtubules in the short posterior process, which is dependent on both KLP-7 and the minus-end binding protein PTRN-1. KLP-7 is positively regulated in the posterior process by planar cell polarity components of Wnt involving rho-1/rock to induce mixed polarity of microtubules, whereas it is negatively regulated in the anterior process by the unc-73/ced-10 cascade to establish a uniform microtubule polarity. Our work elucidates how evolutionarily conserved Wnt signaling establishes the microtubule polarity in neurons through Kinesin-13.     

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

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

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.     

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.     

PAR‐1 promotes microtubule breakdown during dendrite pruning in Drosophila

Pruning of unspecific neurites is an important mechanism during neuronal morphogenesis. Drosophila sensory neurons prune their dendrites during metamorphosis. Pruning dendrites are severed in their proximal regions. Prior to severing, dendritic microtubules undergo local disassembly, and dendrites thin extensively through local endocytosis. Microtubule disassembly requires a katanin homologue, but the signals initiating microtubule breakdown are not known. Here, we show that the kinase PAR‐1 is required for pruning and dendritic microtubule breakdown. Our data show that neurons lacking PAR‐1 fail to break down dendritic microtubules, and PAR‐1 is required for an increase in neuronal microtubule dynamics at the onset of metamorphosis. Mammalian PAR‐1 is a known Tau kinase, and genetic interactions suggest that PAR‐1 promotes microtubule breakdown largely via inhibition of Tau also in Drosophila. Finally, PAR‐1 is also required for dendritic thinning, suggesting that microtubule breakdown might precede ensuing plasma membrane alterations. Our results shed light on the signaling cascades and epistatic relationships involved in neurite destabilization during dendrite pruning.     

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 nucleation and organization in dendrites

Dendrite branching is an essential process for building complex nervous systems. It determines the number, distribution and integration of inputs into a neuron, and is regulated to create the diverse dendrite arbor branching patterns characteristic of different neuron types. The microtubule cytoskeleton is critical to provide structure and exert force during dendrite branching. It also supports the functional requirements of dendrites, reflected by differential microtubule architectural organization between neuron types, illustrated here for sensory neurons. Both anterograde and retrograde microtubule polymerization occur within growing dendrites, and recent studies indicate that branching is enhanced by anterograde microtubule polymerization events in nascent branches. The polarities of microtubule polymerization events are regulated by the position and orientation of microtubule nucleation events in the dendrite arbor. Golgi outposts are a primary microtubule nucleation center in dendrites and share common nucleation machinery with the centrosome. In addition, pre-existing dendrite microtubules may act as nucleation sites. We discuss how balancing the activities of distinct nucleation machineries within the growing dendrite can alter microtubule polymerization polarity and dendrite branching, and how regulating this balance can generate neuron type-specific morphologies.     

Expression of the mitotic kinesin Kif15 in postmitotic neurons: Implications for neuronal migration and development

Kif15 is a kinesin-related protein whose mitotic homologues are believed to crosslink and immobilize spindle microtubules. We have obtained rodent sequences of Kif15, and have studied their expression and distribution in the developing nervous system. Kif15 is indeed expressed in actively dividing fibroblasts, but is also expressed in terminally postmitotic neurons. In mitotic cells, Kif15 localizes to spindle poles and microtubules during prometaphase to early anaphase, but then to the actin-based cleavage furrow during cytokinesis. In interphase fibroblasts, Kif15 localizes to actin bundles but not to microtubules. In cultured neurons, Kif15 localizes to microtubules but shows no apparent co-localization with actin. Localization of Kif15 to microtubules is particularly good when the microtubules are bundled, and there is a notable enrichment of Kif15 in the microtubule bundles that occupy stalled growth cones and dendrites. Studies on developing rodent brain show a pronounced enrichment of Kif15 in migratory neurons compared to other neurons. Notably, migratory neurons have a cage-like configuration of microtubules around their nucleus that is linked to the microtubule array within the leading process, such that the entire array moves in unison as the cell migrates. Since the capacity of microtubules to move independently of one another is restricted in all of these cases, we propose that Kif15 opposes the capacity of other motors to generate independent microtubule movements within key regions of developing neurons.     

Clasp-mediated microtubule bundling regulates persistent motility and contact repulsion in Drosophila macrophages in vivo

Drosophila melanogaster macrophages are highly migratory cells that lend themselves beautifully to high resolution in vivo imaging experiments. By expressing fluorescent probes to reveal actin and microtubules, we can observe the dynamic interplay of these two cytoskeletal networks as macrophages migrate and interact with one another within a living organism. We show that before an episode of persistent motility, whether responding to developmental guidance or wound cues, macrophages assemble a polarized array of microtubules that bundle into a compass-like arm that appears to anticipate the direction of migration. Whenever cells collide with one another, their microtubule arms transiently align just before cell–cell repulsion, and we show that forcing depolymerization of microtubules by expression of Spastin leads to their defective polarity and failure to contact inhibit from one another. The same is true in orbit/clasp mutants, indicating a pivotal role for this microtubule-binding protein in the assembly and/or functioning of the microtubule arm during polarized migration and contact repulsion.     

Signaling mechanisms underlying reversible,activity-dependent dendrite formation

Neuronal activity and neurotrophins play a central role in the formation, maintenance, and plasticity of dendritic arbors. Here, we show that neuronal activity, mediated by electrical stimulation, KCl depolarization, or cholinergic receptor activation, promotes reversible dendrite formation in sympathetic neurons and that this effect is enhanced by NGF. Activity-dependent dendrite formation is accompanied by increased association of HMW MAP2 with microtubules and increased microtubule stability. Inhibition of either CaMKII or the MEK-ERK pathway, both of which phosphorylate MAP2, inhibits dendrite formation, but inhibition of both pathways simultaneously is required for dendrites to retract. These data indicate that neuronal activity signals via CamKII and the ERKs to regulate MAP2:microtubule interactions and hence reversible dendrite stability, and to provide a mechanism whereby activity and neurotrophins converge intracellularly to dynamically regulate dendritic morphology.     

Plasticity of polarization: changing dendrites into axons in neurons integrated in neuronal circuits

Developing neurons can change axonal and dendritic fate upon axonal lesion, but it is unclear whether neurons retain such plasticity when they are synaptically interconnected. To address whether polarity is reversible in mature neurons, we cut the axon of GFP-labeled hippocampal neurons in dissociated and organotypic cultures and found that a new axon arose from a mature dendrite. The regenerative response correlated with the length of the remaining stump: proximal axotomies (<35 microm) led to the transformation of a dendrite into an axon (identity change), whereas distal cuts (>35 microm) induced axon regrowth, similar to what is seen in young neurons. Searching for a putative landmark in the distal axon that could determine axon identity, we focused on the stability of microtubules, which regulate initial neuronal polarization during early development. We found that functionally polarized neurons contain a distinctively high proportion of stable microtubules in the distal axon. Moreover, pharmacological stabilization of microtubules was sufficient to induce the formation of multiple axons out of differentiated dendrites. Our data argue that mature neurons integrated in functional networks remain flexible in their polarity and that mechanisms acting during initial axon selection can be reactivated to induce axon growth out of functionally mature dendrites.     

Establishment of polarity during organization of the acentrosomal plant cortical microtubule array

The plant cortical microtubule array is a unique acentrosomal array that is essential for plant morphogenesis. To understand how this array is organized, we exploited the microtubule (+)-end tracking activity of two Arabidopsis EB1 proteins in combination with FRAP (fluorescence recovery after photobleaching) experiments of GFP-tubulin to examine the relationship between cortical microtubule array organization and polarity. Significantly, our observations show that the majority of cortical microtubules in ordered arrays, within a particular cell, face the same direction in both Arabidopsis plants and cultured tobacco cells. We determined that this polar microtubule coalignment is at least partially due to a selective stabilization of microtubules, and not due to a change in microtubule polymerization rates. Finally, we show that polar microtubule coalignment occurs in conjunction with parallel grouping of cortical microtubules and that cortical array polarity is progressively enhanced during array organization. These observations reveal a novel aspect of plant cortical microtubule array organization and suggest that selective stabilization of dynamic cortical microtubules plays a predominant role in the self-organization of cortical arrays.     

A Highlights from MBoC Selection: γ-Tubulin controls neuronal microtubule polarity independently of Golgi outposts

Neurons have highly polarized arrangements of microtubules, but it is incompletely understood how microtubule polarity is controlled in either axons or dendrites. To explore whether microtubule nucleation by γ-tubulin might contribute to polarity, we analyzed neuronal microtubules in Drosophila containing gain- or loss-of-function alleles of γ-tubulin. Both increased and decreased activity of γ-tubulin, the core microtubule nucleation protein, altered microtubule polarity in axons and dendrites, suggesting a close link between regulation of nucleation and polarity. To test whether nucleation might locally regulate polarity in axons and dendrites, we examined the distribution of γ-tubulin. Consistent with local nucleation, tagged and endogenous γ-tubulins were found in specific positions in dendrites and axons. Because the Golgi complex can house nucleation sites, we explored whether microtubule nucleation might occur at dendritic Golgi outposts. However, distinct Golgi outposts were not present in all dendrites that required regulated nucleation for polarity. Moreover, when we dragged the Golgi out of dendrites with an activated kinesin, γ-tubulin remained in dendrites. We conclude that regulated microtubule nucleation controls neuronal microtubule polarity but that the Golgi complex is not directly involved in housing nucleation sites.