Wenqian Yu

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

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

The microtubule-severing proteins spastin and katanin participate differently in the formation of axonal branches.

Neurons express two different microtubule-severing proteins, namely P60-katanin and spastin. Here, we performed studies on cultured neurons to ascertain whether these two proteins participate differently in axonal branch formation. P60-katanin is more highly expressed in the neuron, but spastin is more concentrated at sites of branch formation. Overexpression of spastin dramatically enhances the formation of branches, whereas overexpression of P60-katanin does not. The excess spastin results in large numbers of short microtubules, whereas the excess P60-katanin results in short microtubules intermingled with longer microtubules. We hypothesized that these different microtubule-severing patterns may be due to the presence of molecules such as tau on the microtubules that more strongly shield them from being severed by P60-katanin than by spastin. Consistent with this hypothesis, we found that axons depleted of tau show a greater propensity to branch, and that this is true whether or not the axons are also depleted of spastin. We propose that there are two modes by which microtubule severing is orchestrated during axonal branch formation, one based on the local concentration of spastin at branch sites and the other based on local detachment from microtubules of molecules such as tau that regulate the severing properties of P60-katanin.

Changes in microtubule number and length during axon differentiation

Hippocampal neurons in culture initially extend several minor processes that are approximately 20 microns in length. The first minor process to grow approximately 10 microns longer than the others will continue to grow rapidly and become the axon (Goslin and Banker, 1989). We sought to define changes in the microtubule (MT) array that occur during axon differentiation. In theory, axon differentiation could involve an increase in MT number, MT length, or some combination of both. To address this issue, we first serially reconstructed the entire MT array of a minor process from a cell whose axon had not yet differentiated. This minor process contained 182 MTs that ranged in length from 0.14 to 20.09 microns. The average MT length was 3.87 +/- 3.83 microns, and the total MT length was 704 microns. We then reconstructed the MT arrays of a minor process and the 56 microns axon from a cell that had undergone axon differentiation. The minor process contained 157 MTs that ranged in length from 0.24 to 17.95 microns. The average MT length was 3.91 +/- 4.84 microns, and the total MT length was 600 microns. The axon contained 1430 MTs that ranged in length from 0.05 to 40.14 microns. The average MT length was 4.02 +/- 5.28 microns, and the total MT length was 5750 microns. These data indicate that a shift occurs toward shorter as well as longer MTs, but that virtually no change in average MT length occurs during axon differentiation. Thus, elongation of existing MTs cannot account for the major expansion of the MT array that occurs as a minor process becomes an axon. In contrast, the number of MTs increases by approximately 10-fold as a minor process differentiates and grows into an axon of the length we analyzed. Based on these data, we conclude that the MT array of a minor process is substantially expanded as it differentiates into an axon, and that the principal mechanism by which this expansion occurs is the copious addition of new MTs.

Tau Protects Microtubules in the Axon from Severing by Katanin

Microtubules in the axon are more resistant to severing by katanin than microtubules elsewhere in the neuron. We have hypothesized that this is because of the presence of tau on axonal microtubules. When katanin is overexpressed in fibroblasts, the microtubules are severed into short pieces, but this phenomenon is suppressed by the coexpression of tau. Protection against severing is also afforded by microtubule-associated protein 2 (MAP2), which has a tau-like microtubule-binding domain, but not by MAP1b, which has a different microtubule-binding domain. The microtubule-binding domain of tau is required for the protection, but within itself, provides less protection than the entire molecule. When tau (but not MAP2 or MAP1b) is experimentally depleted from neurons, the microtubules in the axon lose their characteristic resistance to katanin. These results, which validate our hypothesis, also suggest a potential explanation for why axonal microtubules deteriorate in neuropathies involving the dissociation of tau from the microtubules.

Axonal Growth Is Sensitive to the Levels of Katanin, a Protein That Severs Microtubules

Katanin is a heterodimeric enzyme that severs microtubules from the centrosome so that they can move into the axon. Katanin is broadly distributed in the neuron, and therefore presumably also severs microtubules elsewhere. Such severing would generate multiple short microtubules from longer microtubules, resulting in more microtubule ends available for assembly and interaction with other structures. In addition, shorter microtubules are thought to move more rapidly and undergo organizational changes more readily than longer microtubules. In dividing cells, the levels of P60-katanin (the subunit with severing properties) increase as the cell transitions from interphase to mitosis. This suggests that katanin is regulated in part by its absolute levels, given that katanin activity is high during mitosis. In the rodent brain, neurons vary significantly in katanin levels, depending on their developmental stage. Levels are high during rapid phases of axonal growth but diminish as axons reach their targets. Similarly, in neuronal cultures, katanin levels are high when axons are allowed to grow avidly but drop when the axons are presented with target cells that cause them to stop growing. Expression of a dominant-negative P60-katanin construct in cultured neurons inhibits microtubule severing and is deleterious to axonal growth. Overexpression of wild-type P60-katanin results in excess microtubule severing and is also deleterious to axonal growth, but this only occurs in some neurons. Other neurons are relatively unaffected by overexpression. Collectively, these observations indicate that axonal growth is sensitive to the levels of P60-katanin, but that other factors contribute to modulating this sensitivity.

Regulation of Microtubule Severing by Katanin Subunits during Neuronal Development

Katanin, the microtubule-severing protein, consists of a subunit termed P60 that breaks the lattice of the microtubule and another subunit termed P80, the functions of which are not well understood. Data presented here show that the ratio of P60 to P80 varies markedly in different tissues, at different phases of development, and regionally within the neuron. P80 is more concentrated in the cell body and less variable during development, whereas P60 often shows concentrations in the distal tips of processes as well as dramatic spikes in expression at certain developmental stages. Overexpression of P60 at various stages in the differentiation of cultured hippocampal neurons results in substantial loss of microtubule mass and a diminution in total process length. In comparison, overexpression of P80, which is thought to augment the severing of microtubules by P60, results in a milder loss of microtubule mass and diminution in process length. At the developmental stage corresponding to axogenesis, overexpression of P60 decreases the total number of processes extended by the neuron, whereas overexpression of P80 produces the opposite result, suggesting that the effects on neuronal morphology are dependent on the degree of microtubule severing and loss of polymer. The microtubules that occupy the axon are notably more resistant to depolymerization in response to excess P60 or P80 than microtubules elsewhere in the neuron, suggesting that regional differences in the susceptibility of microtubules to severing proteins may be a critical factor in the generation and maintenance of neuronal polarity.

Microtubule Reconfiguration during Axonal Retraction Induced by Nitric Oxide

Axonal retraction is induced by different types of physiological cues and is responsible for the elimination of mistargeted axons. There is broad agreement that alterations in the cytoskeleton underlie axonal retraction. The prevailing view is that axonal retraction involves a wholesale depolymerization of microtubules and microfilaments. However, axons retracting physiologically display a very different morphology than axons induced to retract by experimental depolymerization of microtubules. Experimental depolymerization of microfilaments actually prevents retraction rather than causing it. We have proposed an alternative hypothesis, namely that axonal retraction involves a backward retreat of cytoskeletal elements rather than their wholesale depolymerization. In the present study, we sought to test this hypothesis with regard to microtubules. When a donor of nitric oxide was applied to cultured chick sensory neurons, the majority of axons retracted dramatically within 30–60 min. Retracting axons were characterized by an enlarged distal region, a thin trailing remnant, and sinusoidal bends along the shaft. Quantitative immunofluorescence analyses showed no detectable loss of microtubule mass during retraction, even with regard to the most labile microtubules. Instead, microtubules were reconfigured into coiling and sinusoidal bundles to accommodate the shortening of the axon. Stabilization of microtubules by taxol did not prevent the retraction, even at concentrations of the drug that actually caused microtubule levels to increase. The retractions induced by nitric oxide were remarkably similar to those observed when motor proteins are manipulated, suggesting that these retractions may result from alterations in the activities of the motors that configure microtubules.

Septin-Driven Coordination of Actin and Microtubule Remodeling Regulates the Collateral Branching of Axons

Axon branching is fundamental to the development of the peripheral and central nervous system. Branches that sprout from the axon shaft are termed collateral or interstitial branches. Collateral branching of axons requires the formation of filopodia from actin microfilaments (F-actin) and their engorgement with microtubules (MTs) that splay from the axon shaft. The mechanisms that drive and coordinate the remodeling of actin and MTs during branch morphogenesis are poorly understood. Septins comprise a family of GTP-binding proteins that oligomerize into higher-order structures, which associate with membranes and the actin and microtubule cytoskeleton. Here, we show that collateral branching of axons requires SEPT6 and SEPT7, two interacting septins. In the axons of sensory neurons, both SEPT6 and SEPT7 accumulate at incipient sites of filopodia formation. We show that SEPT6 localizes to axonal patches of F-actin and increases the recruitment of cortactin, a regulator of Arp2/3-mediated actin polymerization, triggering the emergence of filopodia. Conversely, SEPT7 promotes the entry of axonal MTs into filopodia, enabling the formation of collateral branches. Surprisingly, septins provide a novel mechanism for the collateral branching of axons by coordinating the remodeling of the actin and microtubule cytoskeleton.

Kinesin-12, a Mitotic Microtubule-Associated Motor Protein, Impacts Axonal Growth, Navigation, and Branching

Kinesin-12 (also called Kif15) is a mitotic motor protein that continues to be expressed in developing neurons. Depletion of kinesin-12 causes axons to grow faster, more than doubles the frequency of microtubule transport in both directions in the axon, prevents growth cones from turning properly, and enhances the invasion of microtubules into filopodia. These results are remarkably similar to those obtained in previous studies in which neurons were depleted of kinesin-5 (also called Eg5 or Kif11), another mitotic motor protein that continues to be expressed in developing neurons. However, there are also notable differences in the phenotypes obtained with depleting each of these motors. Depleting kinesin-12 decreases axonal branching and growth cone size, whereas inhibiting kinesin-5 increases these parameters. In addition, depleting kinesin-12 diminishes the appearance of growth-cone-like waves along the length of the axon, an effect not observed with depletion of kinesin-5. Finally, depletion of kinesin-12 abolishes the “waggling” behavior of microtubules that occurs as they assemble along actin bundles within filopodia, whereas inhibition of kinesin-5 does not. Interestingly, and perhaps relevant to these differences in phenotype, in biochemical studies, kinesin-12 coimmunoprecipitates with actin but kinesin-5 does not. Collectively, these findings support a scenario whereby kinesin-12 shares functions with kinesin-5 related to microtubule–microtubule interactions, but kinesin-12 has other functions not shared by kinesin-5 that are related to the ability of kinesin-12 to interact with actin.

Basic Fibroblast Growth Factor Elicits Formation of Interstitial Axonal Branches via Enhanced Severing of Microtubules

This article demonstrates that the augmentation of axonal branching induced by bFGF is explicable on the basis of an enhancement of microtubule-severing, and that three different proteins related to microtubule-severing are affected by treatment of neurons with bFGF.