The Mobility of Neurofilaments in Mature Myelinated Axons of Adult Mice.

Studies in cultured neurons have shown that neurofilaments are cargoes of axonal transport that move rapidly but intermittently along microtubule tracks. However, the extent to which axonal neurofilaments move in vivo has been controversial. Some researchers have proposed that most axonally transported neurofilaments are deposited into a persistently stationary network and that only a small proportion of axonal neurofilaments are transported in mature axons. Here we use the fluorescence photoactivation pulse-escape technique to test this hypothesis in intact peripheral nerves of adult male hThy1-paGFP-NFM mice, which express low levels of mouse neurofilament protein M tagged with photoactivatable GFP. Neurofilaments were photoactivated in short segments of large, myelinated axons, and the mobility of these fluorescently tagged polymers was determined by analyzing the kinetics of their departure. Our results show that >80% of the fluorescence departed the window within 3 h after activation, indicating a highly mobile neurofilament population. The movement was blocked by glycolytic inhibitors, confirming that it was an active transport process. Thus, we find no evidence for a substantial stationary neurofilament population. By extrapolation of the decay kinetics, we predict that 99% of the neurofilaments would have exited the activation window after 10 h. These data support a dynamic view of the neuronal cytoskeleton in which neurofilaments cycle repeatedly between moving and pausing states throughout their journey along the axon, even in mature myelinated axons. The filaments spend a large proportion of their time pausing, but on a timescale of hours, most of them move.

Dynamic catch-bonding generates the large stall forces of cytoplasmic dynein.

Cytoplasmic dynein is an important molecular motor involved in the transport of vesicular and macromolecular cargo along microtubules in cells, often in conjunction with kinesin motors. Dynein is larger and more complex than kinesin and the mechanism and regulation of its movement is currently the subject of intense research. While it was believed for a long time that dynein motors are relatively weak in terms of the force they can generate, recent studies have shown that interactions with regulatory proteins confer large stall forces comparable to those of kinesin. This paper reports on a theoretical study which suggests that these large stall forces may be the result of an emergent, ATP-dependent, bistability resulting in a dynamic catch-bonding behavior that can cause the motor to switch between high and low load-force states.

Local Acceleration of Neurofilament Transport at Nodes of Ranvier.

Myelinated axons are constricted at nodes of Ranvier. These constrictions are important physiologically because they increase the speed of saltatory nerve conduction, but they also represent potential bottlenecks for the movement of axonally transported cargoes. One type of cargo are neurofilaments, which are abundant space-filling cytoskeletal polymers that function to increase axon caliber. Neurofilaments move bidirectionally along axons, alternating between rapid movements and prolonged pauses. Strikingly, axon constriction at nodes is accompanied by a reduction in neurofilament number that can be as much as 10-fold in the largest axons. To investigate how neurofilaments navigate these constrictions, we developed a transgenic mouse strain that expresses a photoactivatable fluorescent neurofilament protein in neurons. We used the pulse-escape fluorescence photoactivation technique to analyze neurofilament transport in mature myelinated axons of tibial nerves from male and female mice of this strain ex vivo Fluorescent neurofilaments departed the activated region more rapidly in nodes than in flanking internodes, indicating that neurofilament transport is faster in nodes. By computational modeling, we showed that this nodal acceleration can be explained largely by a local increase in the duty cycle of neurofilament transport (i.e., the proportion of the time that the neurofilaments spend moving). We propose that this transient acceleration functions to maintain a constant neurofilament flux across nodal constrictions, much as the current increases where a river narrows its banks. In this way, neurofilaments are prevented from piling up in the flanking internodes, ensuring a stable neurofilament distribution and uniform axonal morphology across these physiologically important axonal domains.SIGNIFICANCE STATEMENT Myelinated axons are constricted at nodes of Ranvier, resulting in a marked local decrease in neurofilament number. These constrictions are important physiologically because they increase the efficiency of saltatory nerve conduction, but they also represent potential bottlenecks for the axonal transport of neurofilaments, which move along axons in a rapid intermittent manner. Imaging of neurofilament transport in mature myelinated axons ex vivo reveals that neurofilament polymers navigate these nodal axonal constrictions by accelerating transiently, much as the current increases where a river narrows its banks. This local acceleration is necessary to ensure a stable axonal morphology across nodal constrictions, which may explain the vulnerability of nodes of Ranvier to neurofilament accumulations in animal models of neurotoxic neuropathies and neurodegenerative diseases.

Axonal neurofilaments exhibit frequent and complex folding behaviors.

Neurofilaments are flexible cytoskeletal polymers that are capable of folding and unfolding between their bouts of bidirectional movement along axons. Here we present a detailed characterization of this behavior in cultured neurons using kymograph analysis with approximately 30 ms temporal resolution. We analyzed 781 filaments ranging from 0.6-42 µm in length. We observed complex behaviors including pinch folds, hairpin folds, orientation changes (flips), and occasional severing and annealing events. On average, the filaments spent approximately 40% of their time in some sort of folded configuration. A small proportion of filaments (4%) moved while folded, but most (96%) moved in an outstretched configuration. Collectively, our observations suggest that motors may interact with neurofilaments at multiple points along their length, but preferentially at their ends. In addition, the prevalence of neurofilament folding and the tendency of neurofilaments to straighten out when they move, suggest that an important function of the movement of these polymers in axons may be to maintain them in an outstretched and longitudinally co-aligned configuration. Thus, neurofilament movement may function as much to organize these polymers as to move them, and this could explain why they spend so much time engaged in apparently unproductive bidirectional movement.

Kymograph analysis with high temporal resolution reveals new features of neurofilament transport kinetics.

We have used kymograph analysis combined with edge detection and an automated computational algorithm to analyze the axonal transport kinetics of neurofilament polymers in cultured neurons at 30 ms temporal resolution. We generated 301 kymographs from 136 movies and analyzed 726 filaments ranging from 0.6 to 42 µm in length, representing ∼37,000 distinct moving and pausing events. We found that the movement is even more intermittent than previously reported and that the filaments undergo frequent, often transient, reversals which suggest that they can engage simultaneously with both anterograde and retrograde motors. Average anterograde and retrograde bout velocities (0.9 and 1.2 µm s-1 , respectively) were faster than previously reported, with maximum sustained bout velocities of up to 6.6 and 7.8 µm s-1 , respectively. Average run lengths (∼1.1 µm) and run times (∼1.4 s) were in the range reported for molecular motor processivity in vitro, suggesting that the runs could represent the individual processive bouts of the neurofilament motors. Notably, we found no decrease in run velocity, run length or run time with increasing filament length, which suggests that either the drag on the moving filaments is negligible or that longer filaments recruit more motors.

Live-cell imaging of neurofilament transport in cultured neurons.

Neurofilaments, which are the intermediate filaments of nerve cells, are space-filling cytoskeletal polymers that contribute to the growth of axonal caliber. In addition to their structural role, neurofilaments are cargos of axonal transport that move along microtubule tracks in a rapid, intermittent, and bidirectional manner. Though they measure just 10nm in diameter, which is well below the diffraction limit of optical microscopes, these polymers can reach 100 µm or more in length and are often packed densely, just tens of nanometers apart. These properties of neurofilaments present unique challenges for studies on their movement. In this article, we describe several live-cell fluorescence imaging strategies that we have developed to image neurofilament transport in axons of cultured neurons on short and long timescales. Together, these methods form a powerful set of complementary tools with which to study the axonal transport of these unique intracellular cargos.

Local regulation of neurofilament transport by myelinating cells.

Axons in the vertebrate nervous system only expand beyond ∼ 1 µm in diameter if they become myelinated. This expansion is due in large part to the accumulation of space-filling cytoskeletal polymers called neurofilaments, which are cargoes of axonal transport. One possible mechanism for this accumulation is a decrease in the rate of neurofilament transport. To test this hypothesis, we used a fluorescence photoactivation pulse-escape technique to compare the kinetics of neurofilament transport in contiguous myelinated and unmyelinated segments of axons in long-term myelinating cocultures established from the dorsal root ganglia of embryonic rats. The myelinated segments contained more neurofilaments and had a larger cross-sectional area than the contiguous unmyelinated segments, and this correlated with a local slowing of neurofilament transport. By computational modeling of the pulse-escape kinetics, we found that this slowing of neurofilament transport could be explained by an increase in the proportion of the time that the neurofilaments spent pausing and that this increase in pausing was sufficient to explain the observed neurofilament accumulation. Thus we propose that myelinating cells can regulate the neurofilament content and morphology of axons locally by modulating the kinetics of neurofilament transport.

Rapid activity-induced transcription of Arc and other IEGs relies on poised RNA polymerase II.

Transcription of immediate early genes (IEGs) in neurons is highly sensitive to neuronal activity, but the mechanism underlying these early transcription events is largely unknown. We found that several IEGs, such as Arc (also known as Arg3.1), are poised for near-instantaneous transcription by the stalling of RNA polymerase II (Pol II) just downstream of the transcription start site in rat neurons. Depletion through RNA interference of negative elongation factor, a mediator of Pol II stalling, reduced the Pol II occupancy of the Arc promoter and compromised the rapid induction of Arc and other IEGs. In contrast, reduction of Pol II stalling did not prevent transcription of IEGs that were expressed later and largely lacked promoter-proximal Pol II stalling. Together, our data strongly indicate that the rapid induction of neuronal IEGs requires poised Pol II and suggest a role for this mechanism in a wide variety of transcription-dependent processes, including learning and memory.

A preliminary mitochondrial genome phylogeny of Orthoptera (Insecta) and approaches to maximizing phylogenetic signal found within mitochondrial genome data.

The phylogenetic utility of mitochondrial genomes (mtgenomes) is examined using the framework of a preliminary phylogeny of Orthoptera. This study presents five newly sequenced genomes from four orthopteran families. While all ensiferan and polyneopteran taxa retain the ancestral gene order, all caeliferan lineages including the newly sequenced caeliferan species contain a tRNA rearrangement from the insect ground plan tRNA(Lys)(K)-tRNA(Asp)(D) swapping to tRNA(Asp) (D)-tRNA(Lys) (K) confirming that this rearrangement is a possible molecular synapomorphy for this suborder. The phylogenetic signal in mtgenomes is rigorously examined under the analytical regimens of parsimony, maximum likelihood and Bayesian inference, along with how gene inclusion/exclusion, data recoding, gap coding, and different partitioning schemes influence the phylogenetic reconstruction. When all available data are analyzed simultaneously, the monophyly of Orthoptera and its two suborders, Caelifera and Ensifera, are consistently recovered in the context of our taxon sampling, regardless of the optimality criteria. When protein-coding genes are analyzed as a single partition, nearly identical topology to the combined analyses is recovered, suggesting that much of the signals of the mtgenome come from the protein-coding genes. Transfer and ribosomal RNAs perform poorly when analyzed individually, but contribute signal when analyzed in combination with the protein-coding genes. Inclusion of third codon position of the protein-coding genes does not negatively affect the phylogenetic reconstruction when all genes are analyzed together, whereas recoding of the protein-coding genes into amino acid sequences introduces artificial resolution. Over-partitioning in a Bayesian framework appears to have a negative effect in achieving convergence. Our findings suggest that the best phylogenetic inferences are made when all available nucleotide data from the mtgenome are analyzed simultaneously, and that the mtgenome data can resolve over a wide time scale from the Permian (approximately 260 MYA) to the Tertiary (approximately 50 MYA).