An official website of the United States government
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.
more »
« less
Kymograph analysis with high temporal resolution reveals new features of neurofilament transport kinetics
Abstract 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
- 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
More Like this
-
-
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.more » « less
-
Humans typically walk at low speeds and run at higher speeds. Previous studies of transitions between walking and running were mostly on treadmills, but real-world locomotion allows more flexibility. Here, we study overground locomotion over long distances (800 or 2400 m) under time constraints, simulating everyday scenarios like traveling to an appointment. Unlike on treadmills, participants can vary both speed and gait during this task. Gait transition in this overground task occurs over a broad ‘gait transition regime’ spanning average speeds from 1.9 to 3.0 m s−1. In this regime, people use mixtures of walking and running on each travel bout: mostly walking at low average speeds (around 1.9 m s−1) and mostly running at high average speeds (3.0 m s−1). The walk–run fraction changes gradually between these speed limits and is 50% at about 2.5 m s−1. Within each walk–run mixture, walking is slower than running, with an unused gap between the two gait speeds. These gait mixtures and their speed dependence are predicted by energy optimality. These findings extend earlier results for shorter distances, showing that similar energetic principles govern longer, more physically and cognitively demanding tasks. Our results highlight the role of whole-task energy minimization including transients in shaping human locomotion and gait choice.more » « less
-
Mogilner, Alex (Ed.)Neurofilaments are abundant space-filling cytoskeletal polymers in axons that are transported along microtubule tracks. Neurofilament transport is accelerated at nodes of Ranvier, where axons are locally constricted. Strikingly, these constrictions are accompanied by sharp decreases in neurofilament number, no decreases in microtubule number, and increases in the packing density of these polymers, which collectively bring nodal neurofilaments closer to their microtubule tracks. We hypothesize that this leads to an increase in the proportion of time that the filaments spend moving and that this can explain the local acceleration. To test this, we developed a stochastic model of neurofilament transport that tracks their number, kinetic state, and proximity to nearby microtubules in space and time. The model assumes that the probability of a neurofilament moving is dependent on its distance from the nearest available microtubule track. Taking into account experimentally reported numbers and densities for neurofilaments and microtubules in nodes and internodes, we show that the model is sufficient to explain the local acceleration of neurofilaments within nodes of Ranvier. This suggests that proximity to microtubule tracks may be a key regulator of neurofilament transport in axons, which has implications for the mechanism of neurofilament accumulation in development and disease.more » « less
-
Abstract This study investigated the high‐intensity focused ultrasound (HIFU)‐mediated propulsion of mesoporous silica nanoparticles (MSNs) and microspheres (MSMs). Nanoparticles are heavily sought as vehicles for drug delivery, but their transport through tissue is often restricted. Here, MSNs and MSMs are hydrophobically modified and coated with phospholipids to facilitate inertial cavitation to promote propulsion under HIFU. Modified nanoparticles show significantly enhanced cavitation and propulsion, achieving a maximum displacement of 250 µm (≈2500 body length) and speed of ≈1600 µm s−1(16 000 body length s−1), compared to unmodified nanoparticles (2 µm, 20 body length, 60 µm s−1, 600 body length). In contrast, microparticles demonstrate comparable cavitation responses. Modified microparticles reached a maximum speed of 4000 µm s−1(800 body length s−1) and displacement of 230 µm (46 body length), and unmodified microparticles achieved 2000 µm s−1(400 body length s−1) and 75 µm (15 body length). In all HIFU‐responsive samples, displacement and speed decreased with successive pulses, implying that particles fatigue with continued pulsing. Analyses of particle trajectories and rotational diffusion times suggest that cavitation occurs uniformly on particle surfaces rather than at specific sites. These principles are important for the design of future drug‐delivery vehicles capable of ultrasound‐triggered motion.more » « less
