Recovery of sliding ability in arm depleted flagellar axonemes after recombination with extracted dynein I

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Abstract
We compared sliding velocity between outer doublet tubules in demembranated axonemes of sea-urchin (Pseudocentrotus depressus) sperm flagella with that of arm-depleted axonemes recombined with extracted dynein 1. The outer arm-depleted axonemes after extraction with 0·5 M NaCl had a velocity of 6·9 ± 1·0 μm/s, while the intact axonemes had a velocity of 14·3 ± 1·5 μm/s in the presence of 1 mM ATP and 2 μg/ml trypsin at 25 °C. The sliding velocity was closely related to the number of remaining outer arms following the NaCl-extraction process. When the outer arm-depleted axonemes were recombined with dynein 1, the sliding velocity increased to 11·3 ± 1·3μm/s. Electron microscopy confirmed the recovery of 94 % of outer arms in the axonemes. After extraction with Tris-EDTA solution for 10 min, the axonemes lost their sliding ability completely, even in the presence of ATP and trypsin. Such axonemes lacked most of both inner and outer arms, although sometimes the basal segment of the arms appeared to remain. When the exogenous dynein 1 fraction extracted from other axonemes was added, the axonemes could extrude tubules, and both types of arms reappeared clearly and distinctly in the axonemes. The recombined axonemes with one-fold stoichiometric excess of dynein 1 recovered 58 % of the total number of arms and had a velocity of 7·4 ± 1·6 μm/s. Those with 2-fold stoichiometric excess had a velocity of 11·0 ± 1·5 μm/s, up to 82% of the arms in these axonemes being restored. These results indicated that the exogenous dynein 1 fraction derived from the outer arms restored sliding ability to arm-depleted axonemes, recombining with the outer doublet tubules as inner and outer arms, and that the sliding velocity had a close relationship to the total number of arms in the axonemes, irrespective of their being inner or outer arms.