Supplement movies for the
Journal of Comparative Neurology
These files are in AVI format.
(related to Fig. 2A.) Microtubules restructuring after axotomy.
A B neuron was cultured for 4
hours, then microinjected with EGFP-α-tubulin mRNA.
Twelve hours after the mRNA injection, the axon was transected and the
distribution of microtubules was confocally imaged at a rate of 1/6 second, 3 µm
above the substrate. Axotomy leads to a retrograde wave of microtubule
disassembly. This is followed by a proximo-distal reassembly process that
progresses for only a short distance. The repolymerization of MTs proceeds along
the sub-membrane domains of the distal zone of the cut axonal end, and is
associated with reduced tubulin fluorescence intensity in the core of the
axoplasm. In parallel, a band of MT-free axoplasm confined by MTs of the
proximal and the distal zone is formed. This transition zone becomes, within
minutes after axotomy, the center of the growth cone. This video clip is
composed of 100 frames displayed at a rate of 10 frames/second.
(related to Fig. 2B) Actin restructuring after axotomy.
A B neuron was cultured for 4
hours, then microinjected with EGFP actin mRNA. Twelve hours after the mRNA
injections the axon was transected and the distribution of actin was confocally
imaged at a rate of 1/10 second, at the substrate level. Prior to axotomy the
EGFP-actin fluorescent is homogeneously distributed in the bulk of the axoplasm.
Fluorescent puncta are observed along the plasma membrane surface facing the
glass substrate. Axotomy leads to dissipation of the actin puncta. Within
minutes after axotomy, actin-rich hot spots gradually reappear from proximal
region towards the most distal tip of the cut axon. With time, laterally
oriented actin bundles form along the edges of the plasma membrane, leading to
the extension of a nascent GC lamellipodium. This video clip is composed of 72
frames displayed at a rate of 10 frames/second.
(related to Fig. 2B). Actin at the leading edge of an extending growth cone
formed after axotomy.
This is a continuation of S2 showing the extension of
the growth cone's lamellipodium. Confocal
images were scanned at a rate of 1/15 second.
The video clip is composed of 28 frames displayed at a rate of 7
S4. (related to Fig. 4A).
Confocal images were scanned at a rate
of 1/6 second. The video clip is composed of 111 frames displayed at a rate of
10 frames/second. Shown is the accumulation of RH237-labeled vesicles after
S5, S6, and S7. Spatial and temporal relationships
between microtubules and actin network restructuring during the transformation
of an axon into a growth cone after axotomy.
A B neuron
was cultured for 4 hours, then microinjected with tetramethyl-rhodamine tubulin
and EGFP-actin mRNA. Twelve hours after the injection, the neuron was transected
and the distribution of actin network (green) and microtubules (red) was imaged.
Figure S5 shows actin, S6 microtubules, and S7 the merged images of videos S5
axotomy the microtubules are oriented in parallel to the longitudinal axis of
the axon and the actin is evenly distributed within the axoplasm. Axotomy leads
to subdivision of the cut end into three zones: the distal (DZ), transition (TZ),
and proximal zones (PZ) (see Figs. 2 and 3). The amorphous tubulin in the DZ
reassembles, within minutes, to form longitudinal MT bundles that occupy
the cortical region of the axoplasm. Thereafter, microtubules polymerize
centripetally from the TZ into the growth cone's lamellipodium (S6).
Actin bundles assemble within minutes of axotomy along the
leading edge of the nascent growth cone’s lamellipodium and maintain this
position for as long as the lamellipodium extends (S5).
The images shown were scanned for 340 minutes, at the following rates:
images 2-8 at a rate of 1/5 minutes, images 9-22 at 1/10 minutes, and images
23-32 at 1/20 minutes. The video
clips are composed of 32 images displayed at a rate of 1/ 4.5 seconds.