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Aplysia neuron after axotomy

Supplement movies for the Journal of Comparative Neurology

These files are in AVI format. 

S1. (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. 

Supplement 1

S2. (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. 

Supplement 2

S3. (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 frames/second. 

Supplement 3

S4. (related to Fig. 4A). RH237.

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 axotomy. 

Supplement 4

 

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 and S6.

Prior to 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.

Supplement 5

Supplement 6

Supplement 7

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