Mark M. Black

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 basis of polarity in neurons

It has been recognized since the very early studies on the cytology of vertebrate nervous systems that neurons produce two fundamentally different types of neurite, the axon and the dendrite. Contemporary studies using electron microscopy have defined in detail the many structural differences between axons and dendrites. Perhaps the best known of these differences concerns ribosomes and Golgi elements, which are present in dendrites, but are absent from axons. In this article, we present a possible explanation for this compartmentalization which is based on current understanding of organelle transport in cells and our recent demonstration of a fundamental difference in the organization of the microtubule-based transport systems that convey organelles into the axon or into the dendrite.

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 ∼200–400 mm/d. SCa transports microtubules and neurofilaments at average rates of ∼0.2–1 mm/d, whereas SCb translocates ∼200 diverse proteins critical for axonal growth, regeneration, and synaptic function at average rates of ∼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, α-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 α-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 intitally 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.

Acute Inactivation of Tau Has No Effect on Dynamics of Microtubules in Growing Axons of Cultured Sympathetic Neurons

Tau is a developmentally regulated microtubule (MT)-associated protein in neurons that has been implicated in neuronal morphogenesis. On the basis of test tube studies, tau has been proposed to function in axon growth by stabilizing MTs and thereby promoting MT assembly. We have tested this hypothesis by examining the effects of acute inactivation of tau on axonal MTs. Tau was inactivated by microinjecting purified antibodies against recombinant tau into neurons before they extended axons. The injected antibodies quantitatively precipitated tau into aggregates in the soma. With these conditions the neurons elaborate normal-appearing axons, and MTs extend throughout the axons and into the growth cones, but the axons and their MTs are depleted of tau. The immunodepletion of tau had no detectable effect on several parameters of the dynamics of axonal MTs. Depletion of tau also was not accompanied by a reorganization of other major MT-associated proteins or actin filaments in these neurons. Thus, neurons effectively depleted of tau can extend axons that resemble those of control cells, and the axons contain normal-appearing MT arrays with normal dynamic behavior. These observations are exactly the opposite of those expected on the basis of the hypothesis that the stability of axonal MTs is a direct function of their content of tau, indicating that tau in growing axons of cultured sympathetic neurons is not specialized to promote microtubule assembly and stability.

Solubility properties of neuronal tubulin: Evidence for labile and stable microtubules

The solubility properties of tubulin and microtubules in pure cultures of sympathetic neurons were examined by electron microscopic and biochemical techniques. For morphological analyses, neurons were extracted with Triton X-100 in the presence or absence of 1 mM CaCl2, and the resulting detergent-extracted residues were examined for microtubules. In parallel experiments, the solubility of tubulin was determined under various solution conditions. Detergent-extracted residues of neurons prepared without Ca2+ contained many microtubules. Neurite residues prepared in the presence of Ca2+ also contained microtubules, but at substantially lower numbers than in residues prepared without Ca2+. The biochemical data parallel the morphological observations. Following detergent-extraction under microtubule stabilizing conditions, 30% of the tubulin was detergent-soluble (i.e. unpolymerized), while 70% was detergent-insoluble (i.e. polymerized). A more detailed examination of the solubility properties of tubulin indicated that 62% was detergent-insoluble but soluble in buffers containing mM CaCl2, while 5-8% was detergent and Ca2+-insoluble. A variety of control experiments indicated that non-specific adsorption of tubulin onto detergent-insoluble components of the cultures, assembly of tubulin onto pre-existing microtubules, and incomplete extraction of tubulin from cells contributed minimally to the levels of Ca2+-soluble and insoluble tubulin obtained with the extraction conditions used. These results indicate that (a) the majority of neuronal tubulin is assembled into microtubules which disassemble upon treatment with Ca2+ and (b) a portion of the neuronal tubulin is assembled into microtubules which show the unusual property of Ca2+-stability.

Phosphorylation of neurofilament proteins in intact neurons: demonstration of phosphorylation in cell bodies and axons

The principal subunits of neurofilaments (NFs) of immature cultured sympathetic neurons have apparent Mr of 68,000 and 145,000; a 200,000 Mr subunit is also present, but at comparatively low levels. These subunits are referred to as the low (NFL), middle (NFM), and high (NFH) Mr subunits, respectively. We studied the phosphorylation of NFL and NFM in these neurons in order to characterize the NFL and NFM isoforms generated by this important posttranslational modification. NFL resolved into a single spot in 2-dimensional gels, although 2 spots were occasionally observed. NFM typically resolved into 3 variants, termed NFM a, b, and c, in order of increasing mobility, but as many as 6 variants were detected in some gels. NFL and, to a much greater degree, NFM became labeled following incubation of intact neurons with 32P-PO4. Although all 3 major NFM variants became labeled, NFM a was the most heavily labeled, followed by NFM b, and then NFM c. Two observations suggest that the generation of these 3 NFM variants is due to their phosphorylation. First, treatment of NFs with phosphatase prior to analysis reduced NFM to a single spot or band that comigrated with NFM c; NFM a and b were completely eliminated. However, NFM c was not fully dephosphorylated because it still reacted with a monoclonal antibody (mAb) specific for a phosphate-dependent epitope on NFM. Second, NFM was recognized by 4 mAbs to distinctly different phosphorylated epitopes of NFM, which suggested that at least 4 distinct sites on NFM can be phosphorylated in cultured neurons. Explant cultures were used to study the phosphorylation of NFL and NFM in cell bodies and axons. In these cultures, a central cell body mass (CBM) 0.5 mm in diameter contains all of the cell bodies, while peripheral to the CBM is a halo of pure axons that extends for 4–6 mm. These cultures were incubated with 32P-PO4 and CBM and axon regions were analyzed separately. NFL became phosphorylated to a greater extent in the CBM than in axons. NFM also became labeled in the CBM and axons, although the relative labeling of NFM a, b, and c in these regions differed considerably from each other and also from the pattern observed in whole neurons (cell bodies plus neurites, see above).(ABSTRACT TRUNCATED AT 400 WORDS)

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 ∼2 μm/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 ∼200 diverse proteins from the cell body to the axon and axon tip at average rates of ∼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: α-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.