Figure 12. Potential mechanisms of neurofilament folding.

Schematic diagram depicting how forces acting on neurofilaments could explain the diversity of neurofilament folding behaviors. The neurofilaments are represented as horizontal black lines. For the flipping events, one end of the filament is marked with an arrowhead to facilitate tracking of filament orientation. Proximal is left and distal is right throughout. Anterograde and retrograde motors are represented in blue and red, respectively, and the movement of these motors is represented with blue and red arrows. The gray dots represent hypothetical stationary obstacles around which filaments could wrap. The gray squares represent hypothetical stationary objects to which filaments could be tethered. (a) A pinch fold could represent a “buckling” of a filament at the site of motor attachment if one end of the filament is tethered. The tethering site is depicted here as being at the distal end of the filament, but it could be at any site along the filament distal to the site of motor attachment. (b) A pinch fold could also arise if motors of opposing directionality attach to a filament and move towards each other. (c) At least some pinch folds appeared to be generated by a motor pulling the filament from the middle against some obstacle, since pinch folds often evolved into hairpin folds (g). (d) Motors could also act indirectly to generate folding, such as via a membranous organelle that links transiently to the filament as it moved past it. (e) Hairpin folds could form if a retrograde motor engaged with the distal end of a filament to form a distal hairpin (shown here) or an anterograde motor engaged with the proximal end to form a proximal hairpin (not shown). (f) In some cases, the apex of the hairpin bend remained fixed in place during the evolution of the hairpin fold, implying that the filament wrapped around some obstacle in the axon. (g) A pinch fold in the interior of a filament could evolve into a double hairpin fold by a motor pulling the filament around an obstacle. (h) Some filaments changed their direction of movement while simultaneously flipping their proximal/distal orientation. In this example a retrograde motor binds to the distal end of an anterogradely moving filament, reversing the orientation and direction of movement of the filament. Note that the leading end of the filament (black arrowhead) remains the same. (i) Flipping also occurred without a change of directionality. In this case, an anterograde motor binds to the trailing end of an anterogradely moving filament and then pulls the trailing end forwards so that what was the leading end (black arrowhead) is now the trailing end (gray arrowhead). (j,k) The majority of filaments moved in a fully outstretched configuration which implies that motors were bound to their leading ends. (l) Given their flexibility, the movement of filaments in a fully outstretched configuration during reversals implies that motors of opposing directionality can engage with opposite ends of the same filament. The speed of these reversals suggests that these motors could be bound simultaneously (as shown here). (m, n, o) The rare movement of filaments in a hairpin configuration indicates that motors can also bind along the length of the filament, not just at the filament ends. The two arms of the hairpin are equal in length if the motor binds in the middle of the filament (m) and unequal in length if it binds closer to one end than the other (n). In some cases, filaments were observed to transition from the former to the latter or vice versa (o). Overall, these folding behaviors suggest that both anterograde and retrograde motors can engage directly or indirectly with neurofilaments at multiple sites all along their length but with a preference for an association with the filament ends. [Color figure can be viewed at wileyonlinelibrary.com]