Abstract Neurofilaments are abundant space-filling cytoskeletal polymers that are transported into and along axons. During postnatal development, these polymers accumulate in myelinated axons causing an expansion of axon caliber, which is necessary for rapid electrical transmission. Studies on cultured nerve cells have shown that axonal neurofilaments move rapidly and intermittently along microtubule tracks in both anterograde and retrograde directions. However, it is unclear whether neurofilament transport is also bidirectional in vivo . Here, we describe a pulse-spread fluorescence photoactivation method to address this in peripheral nerves dissected from hThy1-paGFP-NFM transgenic mice, which express a photoactivatable fluorescent neurofilament protein. Neurofilaments were photoactivated in short segments of myelinated axons in tibial nerves at 2, 4, 8, and 16 weeks of age. The proximal and distal spread of the fluorescence due to the movement of the fluorescent neurofilaments was measured over time. We show that the directional bias and velocity of neurofilament transport can be calculated from these measurements. The directional bias was ∼60% anterograde and 40% retrograde and did not change significantly with age or distance along the nerve. The net velocity decreased with age and distance along the nerve, which is consistent with previous studies using radioisotopic pulse labeling. This decrease in velocity was caused by a decrease in both anterograde and retrograde movement. Thus, neurofilament transport is bidirectional in vivo , with a significant fraction of the filaments moving retrogradely in both juvenile and adult mice.
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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.
more » « less- NSF-PAR ID:
- 10046914
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Cytoskeleton
- Volume:
- 75
- Issue:
- 1
- ISSN:
- 1949-3584
- Page Range / eLocation ID:
- p. 22-41
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract 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.
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null (Ed.)Abstract. During the Program for Research on Oxidants: PHotochemistry, Emissions, and Transport (PROPHET) campaign from 21 July to 3 August 2016,field experiments on leaf-level trace gas exchange of nitric oxide (NO), nitrogen dioxide (NO2), and ozone (O3) were conducted for thefirst time on the native American tree species Pinus strobus (eastern white pine), Acer rubrum (redmaple), Populus grandidentata (bigtooth aspen), and Quercus rubra (red oak) in a temperate hardwood forest inMichigan, USA. We measured the leaf-level trace gas exchange rates andinvestigated the existence of an NO2 compensation point, hypothesizedbased on a comparison of a previously observed average diurnal cycle ofNOx (NO2+NO) concentrations with that simulated using amulti-layer canopy exchange model. Known amounts of trace gases wereintroduced into a tree branch enclosure and a paired blank referenceenclosure. The trace gas concentrations before and after the enclosures weremeasured, as well as the enclosed leaf area (single-sided) and gas flow rate to obtain the trace gas fluxes with respect to leaf surface. There was nodetectable NO uptake for all tree types. The foliar NO2 and O3uptake largely followed a diurnal cycle, correlating with that of the leafstomatal conductance. NO2 and O3 fluxes were driven by theirconcentration gradient from ambient to leaf internal space. The NO2 loss rate at the leaf surface, equivalently the foliar NO2 deposition velocity toward the leaf surface, ranged from 0 to 3.6 mm s−1 for bigtooth aspen and from 0 to 0.76 mm s−1 for red oak, both of which are∼90 % of the expected values based on the stomatalconductance of water. The deposition velocities for red maple and white pineranged from 0.3 to 1.6 and from 0.01 to 1.1 mm s−1, respectively, and were lower than predicted from the stomatal conductance, implying amesophyll resistance to the uptake. Additionally, for white pine, theextrapolated velocity at zero stomatal conductance was 0.4±0.08 mm s−1, indicating a non-stomatal uptake pathway. The NO2compensation point was ≤60 ppt for all four tree species andindistinguishable from zero at the 95 % confidence level. This agrees withrecent reports for several European and California tree species butcontradicts some earlier experimental results where the compensation pointswere found to be on the order of 1 ppb or higher. Given that the sampledtree types represent 80 %–90 % of the total leaf area at this site, theseresults negate the previously hypothesized important role of a leaf-scaleNO2 compensation point. Consequently, to reconcile these findings,further detailed comparisons between the observed and simulated in- and above-canopy NOx concentrations and the leaf- and canopy-scaleNOx fluxes, using the multi-layer canopy exchange model withconsideration of the leaf-scale NOx deposition velocities as well asstomatal conductances reported here, are recommended.more » « less
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Abstract Background Ankle-targeting resistance training for improving plantarflexion function during walking increases rehabilitation intensity, an important factor for motor recovery after stroke. However, understanding of the effects of resisting plantarflexion during stance on joint kinetics and muscle activity—key outcomes in evaluating its potential value in rehabilitation—remains limited. This initial study uses a unilateral exosuit that resists plantarflexion during mid-late stance in unimpaired individuals to test the hypotheses that when plantarflexion is resisted, individuals would (1) increase plantarflexor ankle torque and muscle activity locally at the resisted ipsilateral ankle, but (2) at higher forces, exhibit a generalized response that also uses the unresisted joints and limb. Further, we expected (3) short-term retention into gait immediately after removal of resistance.
Methods Ten healthy young adults walked at 1.25 m s−1for four 10-min discrete bouts, each comprising baseline, exposure to active exosuit-applied resistance, and post-active sections. In each bout, a different force magnitude was applied based on individual baseline ankle torques. The peak resistance torque applied by the exosuit was 0.13 ± 0.01, 0.19 ± 0.01, 0.26 ± 0.02, and 0.32 ± 0.02 N m kg−1, in the LOW, MED, HIGH, and MAX bouts, respectively.
Results (1) Across all bouts, participants increased peak ipsilateral biological ankle torque by 0.13–0.25 N m kg−1(p < 0.001) during exosuit-applied resistance compared to corresponding baselines. Additionally, ipsilateral soleus activity during stance increased by 5.4–11.3% (p < 0.05) in all but the LOW bout. (2) In the HIGH and MAX bouts, vertical ground reaction force decreased on the ipsilateral limb while increasing on the contralateral limb (p < 0.01). Secondary analysis found that the force magnitude that maximized increases in biological ankle torque without significant changes in limb loading varied by subject. (3) Finally, peak ipsilateral plantarflexion angle increased significantly during post-exposure in the intermediate HIGH resistance bout (p < 0.05), which corresponded to the greatest average increase in soleus activity (p > 0.10).
Conclusions Targeted resistance of ankle plantarflexion during stance by an exosuit consistently increased local ipsilateral plantarflexor effort during active resistance, but force magnitude will be an important parameter to tune for minimizing the involvement of the unresisted joints and limb during training.
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