Cytoplasmic Dynein Transports Axonal Microtubules in a Polarity-Sorting Manner.

Axonal microtubules are predominantly organized into a plus-end-out pattern. Here, we tested both experimentally and with computational modeling whether a motor-based polarity-sorting mechanism can explain this microtubule pattern. The posited mechanism centers on cytoplasmic dynein transporting plus-end-out and minus-end-out microtubules into and out of the axon, respectively. When cytoplasmic dynein was acutely inhibited, the bi-directional transport of microtubules in the axon was disrupted in both directions, after which minus-end-out microtubules accumulated in the axon over time. Computational modeling revealed that dynein-mediated transport of microtubules can establish and preserve a predominantly plus-end-out microtubule pattern as per the details of the experimental findings, but only if a kinesin motor and a static cross-linker protein are also at play. Consistent with the predictions of the model, partial depletion of TRIM46, a protein that cross-links axonal microtubules in a manner that influences their polarity orientation, leads to an increase in microtubule transport.

Pharmacologically increasing microtubule acetylation corrects stress-exacerbated effects of organophosphates on neurons.

Many veterans of the 1990-1991 Gulf War contracted Gulf War Illness (GWI), a multisymptom disease that primarily affects the nervous system. Here, we treated cultures of human or rat neurons with diisopropyl fluorophosphate (DFP), an analog of sarin, one of the organophosphate (OP) toxicants to which the military veterans were exposed. All observed cellular defects produced by DFP were exacerbated by pretreatment with corticosterone or cortisol, which, in rat and human neurons, respectively, serves in our experiments to mimic the physical stress endured by soldiers during the war. To best mimic the disease, DFP was used below the level needed to inhibit acetylcholinesterase. We observed a diminution in the ratio of acetylated to total tubulin that was correctable by treatment with tubacin, a drug that inhibits HDAC6, the tubulin deacetylase. The reduction in microtubule acetylation was coupled with deficits in microtubule dynamics, which were correctable by HDAC6 inhibition. Deficits in mitochondrial transport and dopamine release were also improved by tubacin. Thus, various negative effects of the toxicant/stress exposures were at least partially correctable by restoring microtubule acetylation to a more normal status. Such an approach may have therapeutic benefit for individuals suffering from GWI or other neurological disorders linked to OP exposure.

Axonal transport: The orderly motion of axonal structures.

Axonal transport is a constitutive process that supplies the axon and axon terminal with materials required to maintain their structure and function. Most materials are supplied via three rate components termed the fast component, slow component a, and slow component b. Each of these delivers a distinct set of materials with distinct transport kinetics. Understanding the basis for how materials sort among these rate components and the mechanisms that generate their distinctive transport kinetics have been long-standing goals in the field. An early view emphasized the relationships between axonally transported cargoes and cytological structures of the axon. In this article, I discuss key observations that led to this view and contemporary studies that have demonstrated its validity and thereby advanced the current understanding of the dynamics of axonal structure.

A novel role for doublecortin and doublecortin-like kinase in regulating growth cone microtubules.

Doublecortin (DCX) and doublecortin-like kinase (DCLK), closely related family members, are microtubule-associated proteins with overlapping functions in both neuronal migration and axonal outgrowth. In growing axons, these proteins appear to have their primary functions in the growth cone. Here, we used siRNA to deplete these proteins from cultured rat sympathetic neurons. Normally, microtubules in the growth cone exhibit a gently curved contour as they extend from the base of the cone toward its periphery. However, following depletion of DCX and DCLK, microtubules throughout the growth cone become much more curvy, with many microtubules exhibiting multiple prominent bends over relatively short distances, creating a configuration that we termed wave-like folds. Microtubules with these folds appeared as if they were buckling in response to powerful forces. Indeed, inhibition of myosin-II, which generates forces on the actin cytoskeleton to push microtubules in the growth cone back toward the axonal shaft, significantly decreases the frequency of these wave-like folds. In addition, in the absence of DCX and DCLK, the depth of microtubule invasion into filopodia is reduced compared with controls, and at a functional level, growth cone responses to substrate guidance cues are altered. Conversely, overexpression of DCX results in microtubules that are straighter than usual, suggesting that higher levels of these proteins can enable an even greater resistance to folding. These findings support a role for DCX and DCLK in enabling microtubules to overcome retrograde actin-based forces, thereby facilitating the ability of the growth cone to carry out its crucial path-finding functions.

The cytoskeleton of the neuron--an essay in celebration of Paul Letourneau's career.

The neuronal cytoskeleton consists of microtubules, actin filaments, neurofilaments, and an array of accessory proteins that regulate and modify these three main filament systems. This essay celebrates the career of Paul Letourneau, a pioneer of the neuronal cytoskeleton, to whom the community owes a debt of gratitude.

Doublecortin associates with microtubules preferentially in regions of the axon displaying actin-rich protrusive structures.

Here we studied doublecortin (DCX) in cultured hippocampal and sympathetic neurons during axonal development. In both types of neurons, DCX is abundant in the growth cone, in which it primarily localizes with microtubules. Its abundance is lowest on microtubules in the neck region of the growth cone and highest on microtubules extending into the actin-rich lamellar regions. Interestingly, the microtubule polymer richest in DCX is also deficient in tau. In hippocampal neurons but not sympathetic neurons, discrete focal patches of microtubules rich in DCX and deficient in tau are present along the axonal shaft. Invariably, these patches have actin-rich protrusions resembling those of growth cones. Many of the DCX/actin filament patches exhibit vigorous protrusive activity and also undergo a proximal-to-distal redistribution within the axon at average rates approximately 2 microm/min and thus closely resemble the growth-cone-like waves described by previous authors. Depletion of DCX using small interfering RNA had little effect on the appearance of the growth cone or on axonal growth in either type of neuron. However, DCX depletion significantly delayed collateral branching in hippocampal neurons and also significantly lowered the frequency of actin-rich patches along hippocampal axons. Branching by sympathetic neurons, which occurs by growth cone splitting, was not impaired by DCX depletion. These findings reveal a functional relationship between the DCX/actin filament patches and collateral branching. Based on the striking resemblance of these patches to growth cones, we discuss the possibility that they reflect a mechanism for locally boosting morphogenetic activity to facilitate axonal growth and collateral branching.

Cytoskeletal requirements in axonal transport of slow component-b.

Slow component-b (SCb) translocates approximately 200 diverse proteins from the cell body to the axon and axon tip at average rates of approximately 2-8 mm/d. Several studies suggest that SCb proteins are cotransported as one or more macromolecular complexes, but the basis for this cotransport is unknown. The identification of actin and myosin in SCb led to the proposal that actin filaments function as a scaffold for the binding of other SCb proteins and that transport of these complexes is powered by myosin: the "microfilament-complex" model. Later, several SCb proteins were also found to bind F-actin, supporting the idea, but despite this, the model has never been directly tested. Here, we test this model by disrupting the cytoskeleton in a live-cell model system wherein we directly visualize transport of SCb cargoes. We focused on three SCb proteins that we previously showed were cotransported in our system: alpha-synuclein, synapsin-I, and glyceraldehyde-3-phosphate dehydrogenase. Disruption of actin filaments with latrunculin had no effect on the velocity or frequency of transport of these three proteins. Furthermore, cotransport of these three SCb proteins continued in actin-depleted axons. We conclude that actin filaments do not function as a scaffold to organize and transport these and possibly other SCb proteins. In contrast, depletion of microtubules led to a dramatic inhibition of vectorial transport of SCb cargoes. These findings do not support the microfilament-complex model, but instead indicate that the transport of protein complexes in SCb is powered by microtubule motors.

Rapid and intermittent cotransport of slow component-b proteins.

After synthesis in neuronal perikarya, proteins destined for synapses and other distant axonal sites are transported in three major groups that differ in average velocity and protein composition: fast component (FC), slow component-a (SCa), and slow component-b (SCb). The FC transports mainly vesicular cargoes at average rates of approximately 200-400 mm/d. SCa transports microtubules and neurofilaments at average rates of approximately 0.2-1 mm/d, whereas SCb translocates approximately 200 diverse proteins critical for axonal growth, regeneration, and synaptic function at average rates of approximately 2-8 mm/d. Several neurodegenerative diseases are characterized by abnormalities in one or more SCb proteins, but little is known about mechanisms underlying SCb compared with FC and SCa. Here, we use live-cell imaging to visualize and quantify the axonal transport of three SCb proteins, alpha-synuclein, synapsin-I, and glyceraldehyde-3-phosphate dehydrogenase in cultured hippocampal neurons, and directly compare their transport to synaptophysin, a prototypical FC protein. All three SCb proteins move rapidly but infrequently with pauses during transit, unlike synaptophysin, which moves much more frequently and persistently. By simultaneously visualizing the transport of proteins at high temporal and spatial resolution, we show that the dynamics of alpha-synuclein transport are distinct from those of synaptophysin but similar to other SCb proteins. Our observations of the cotransport of multiple SCb proteins in single axons suggest that they move as multiprotein complexes. These studies offer novel mechanistic insights into SCb and provide tools for further investigating its role in disease processes.

Antagonistic forces generated by cytoplasmic dynein and myosin-II during growth cone turning and axonal retraction.

Cytoplasmic dynein transports short microtubules down the axon in part by pushing against the actin cytoskeleton. Recent studies have suggested that comparable dynein-driven forces may impinge upon the longer microtubules within the axon. Here, we examined a potential role for these forces on axonal retraction and growth cone turning in neurons partially depleted of dynein heavy chain (DHC) by small interfering RNA. While DHC-depleted axons grew at normal rates, they retracted far more robustly in response to donors of nitric oxide than control axons, and their growth cones failed to efficiently turn in response to substrate borders. Live cell imaging of dynamic microtubule tips showed that microtubules in DHC-depleted growth cones were largely confined to the central zone, with very few extending into filopodia. Even under conditions of suppressed microtubule dynamics, DHC depletion impaired the capacity of microtubules to advance into the peripheral zone of the growth cone, indicating a direct role for dynein-driven forces on the distribution of the microtubules. These effects were all reversed by inhibition of myosin-II forces, which are known to underlie the retrograde flow of actin in the growth cone and the contractility of the cortical actin during axonal retraction. Our results are consistent with a model whereby dynein-driven forces enable microtubules to overcome myosin-II-driven forces, both in the axonal shaft and within the growth cone. These dynein-driven forces oppose the tendency of the axon to retract and permit microtubules to advance into the peripheral zone of the growth cone so that they can invade filopodia.

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.

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.

Transport of neurofilaments in growing axons requires microtubules but not actin filaments.

Neurofilament (NF) polymers are conveyed from cell body to axon tip by slow axonal transport, and disruption of this process is implicated in several neuronal pathologies. This movement occurs in both anterograde and retrograde directions and is characterized by relatively rapid but brief movements of neurofilaments, interrupted by prolonged pauses. The present studies combine pharmacologic treatments that target actin filaments or microtubules with imaging of NF polymer transport in living axons to examine the dependence of neurofilament transport on these cytoskeletal systems. The heavy NF subunit tagged with green fluorescent protein was expressed in cultured sympathetic neurons to visualize NF transport. Depletion of axonal actin filaments by treatment with 5 microM latrunculin for 6 hr had no detectable effect on directionality or transport rate of NFs, but frequency of movement events was reduced from 1/3.1 min of imaging time to 1/4.9 min. Depolymerization of axonal microtubules using either 5 microM vinblastine for 3 hr or 5 microg/ml nocodazole for 4-6 hr profoundly suppressed neurofilament transport. In 92% of treated neurons, NF transport was undetected. These observations indicate that actin filaments are not required for neurofilament transport, although they may have subtle effects on neurofilament movements. In contrast, axonal transport of NFs requires microtubules, suggesting that anterograde and retrograde NF transport is powered by microtubule-based motors.

STOP (stable-tubule-only-polypeptide) is preferentially associated with the stable domain of axonal microtubules.

Axonal microtubules consist of two distinct domains that differ in tyrosinated-tubulin staining. One domain stains weakly for tyrosinated-tubulin, while the other stains strongly, and the transition between these domains is abrupt; the tyrosinated-tubulin-poor domain is at the minus end of the microtubule, and the tyrosinated-tubulin-rich domain extends from the plus end of the tyrosinated-tubulin-poor domain to the end of the microtubule. The tyrosinated-tubulin-poor domain is drug- and cold-stable, whereas the tyrosinated-tubulin-rich domain is drug-labile, but largely cold-stable. STOP (stable-tubule-only-polypeptide) has potent microtubule stabilizing activity, and may contribute to the cold and drug stability of axonal microtubules. To evaluate this possibility, we examined STOP association with the different types of microtubule polymer in cultured sympathetic neurons. By immunofluorescence, STOP is present in the cell body and throughout the axon; axonal staining declines progressively in the distal portion of the axon, and reaches lowest levels in the growth cone. Growth cone microtubules, which are drug and cold labile, do not stain detectably for STOP. To examine individual axonal microtubules for STOP, we used a procedure that causes microtubules to splay out from the main axonal array so that they can be visualized for relatively long distances along their length. Both tyrosinated-tubulin-rich and tyrosinated-tubulin-poor polymer stain for STOP, but STOP is several-fold more concentrated on tyrosinated-tubulin-poor polymer than on tyrosinated-tubulin-rich polymer. These results are consistent with STOP dependent stabilization of axonal microtubules, with the difference between cold-stable polymer versus cold- + drug-stable polymer determined by the amount of STOP on the polymer.