Jeff Lichtman Lab

Supplemental media for Bishop DL, Misgeld T, Walsh MK, Gan WB, Lichtman JW. Axon Branch Removal at Developing Synapses by Axosome Shedding. Neuron. 2004 44:651-661.  [abstract]
Movies:


1 (1.5 MB, avi)
Corresponds to Figure 2B. Average frame rate is 1 frame per 1.5 min (see clock, which shows hr: min: s).

2 (1 MB, avi)
Corresponds to Figure S1. Average frame rate is 1 frame per 21 min (see clock, which shows hr: min: s).

3 (1.1 MB, avi)
This movie shows a bulb-tipped axon near a singly innervated neuromuscular junction (top) at P8. As the bulb-tipped axon retreats, numerous large fragments are shed. Figure 3B shows a detail of this bulb at higher time resolution; see also Supplemental Movie S6. Average frame rate is 1 frame per 40 min (see clock, which shows hr: min).

4 (2.2 MB, avi)
Corresponds to Figure S2. Average frame rate is 1 frame per 27 s (see clock, which shows hr: min: s).

5 (1.4 MB, avi)
Corresponds to Figure 3A but also shows the axon that is tipped by the bulb. Average frame rate is 1 frame per 14.5 min (see clock, which shows hr: min: s).

6 (10.6 MB, avi)
Corresponds to Figure 3B. The entire bulb is shown to the left; the area that is shown in detail to the left is marked by a red box in the first frames. Compare also Movie 3, which shows the same bulb at lower temporal resolution. Average frame rate is 1 frame per 6 s (see clock, which shows hr: min: s).

7 (25.5 MB, mpeg)
Corresponds to Figure 4. This movie shows an animation of the rendering of the serial electron microscopic images. Initially, the confocal image can be seen, and then the corresponding rendered bulb-tipped axon appears. A 360° view of the bulb is shown with numerous axosomes (brown) nearby. Note the several mushroom-like “proto-axosomes” that protrude from the bulb’s surface.

8 (105.5 MB, avi)
Corresponds to Figure 5. A 360° view of the segregated neuromuscular junction in Figure 5 (muscle surface is shown in red). The animation provides a closeup of the green bulb-tipped axon branch and a view through a semitransparent rendering of this bulb. Numerous mitochondria (yellow), 8290 40 nm vesicles (white), and two vesicle-filled axosomes (brown) can be seen near the bulb.

High-Resolution Figures:


1 (1.75 MB, jpg)

2 (3.33 MB, jpg)

3 (459 K, jpg)

4 (1.5 MB, jpg)

5 (1.9 MB, jpg)

6 (1.1 MB, jpg)

7 (1.5 MB, jpg)

8 (835 K, jpg)

S1 (500 K, jpg)
A P11 acute explant of the mouse triangularis sterni muscle showing considerable shortening of an axon in a nerve fascicle over 6 hr. As the tip of this axon appears to retract (average speed 3.5 µm/hr), a few small remnants (arrow) are left behind. Scale bar, 10 µm. See Movie 2. Upper panel shows lower-magnification view of retreating axon in nerve fascicle; boxed area is shown in panels below.


S2 (1 MB, jpg)
Acute explant at P10 showing a bulb-tipped axon (blue) retreat from a singly innervated neuromuscular junction (axon in green; postsynaptic acetylcholine receptors in red) over 1 hr. The bulb appears to retract over a distance of 8 µm (vertical line), while shedding numerous axosomes (white arrow), which quickly disappear. The bulb and a more proximal swelling along the axon transiently show fissures (filled arrowheads). A “bolus” of material appears to move retrogradely (open arrowheads). See Supplemental Movie S4. Scale bar, 10 µm.

S3 (279 K, jpg)
Schematic drawing of potential mechanisms of axon branch removal. During development, individual branches of motor axons are lost. There were three alternatives that could explain the disappearance of branches. Some previous evidence supported the idea that branches degenerate. In this scenario, axonal branches would suddenly disintegrate over long distances. Alternatively, retraction was suggested as the mechanism of branch removal. In this case, axons would shorten and their contents would be transported in a retrograde direction. The present work, however, suggests another alternative: axosome shedding. We show that the tips of axons that are in the process of being removed are locally dismantled into axosomes, which are internalized by the surrounding glial cell.