Reversal of rapidly transported protein and organelles at an axonal lesion

The time required for both rapid axonally transported organelles (vesicles and tubulo-vesicular structures) and proteins to undergo anterograde to retrograde reversal at a crush site was examined using sciatic nerve preparations obtained from Xenopus laevis. The transport and reversal of a pulse of newly synthesized 35S-labeled proteins was studied with a position-sensitive detector of ionizing radiation. Organelle transport and reversal were studied using video microscopy. Both protein and organelle reversal were assessed in two bathing media: a physiological saline and a medium that was compatible with the intracellular environment (internal medium). The time required for protein transport to reverse at a ligature was determined as a function of the time interval between the application of the ligature and the arrival of the pulse at the ligature (lesion time). In physiological saline, reversal times were greatest, about 3.5 h, when the lesion time was 1 h or less and decreased to approximately 1.5 h for lesion times of 4-12 h. When corrected for the approximately 2 mm length of degeneration caused by the saline, the results were similar to those obtained in internal medium and indicated a minimal reversal time for proteins of about 2 h. Organelle transport was examined close to narrow lesions in single myelinated axons. That the organelles moving away from the lesion represented organelles that had undergone reversed transport was suggested by observation of the reversal of individual organelles, and by a correlation between the flux of organelles towards and away from the lesion. Analysis of organelle flux within and adjacent to a segment of axon isolated by two lesions indicated that 70-80% of organelles moving away from a lesion represented reversed transport. Observations in internal medium were consistent with a reversal time of < 15 min, and in physiological saline < 30 min. The substantially smaller reversal time for organelle transport as compared to protein transport is consistent either with the existence of two types of organelles with different reversal times and hence different reversal mechanisms, or with the possibility that during reversal proteins are off-loaded from carrier organelles and subsequently up-loaded to different organelles.