Dynein and plus-end microtubule-dependent motors are associated with specialized Sertoli cell junction plaques...

The mechanism responsible for spermatid translocation in the mammalian seminiferous epithelium was proposed to be the microtubule-based transport of specialized junction plaques (ectoplasmic specializations) that occur in Sertoli cell regions attached to spermatid heads. These plaques each consist of a cistern of endoplasmic reticulum, a layer of actin filaments and the adjacent plasma membrane. It is predicted that motor proteins function to move the junction plaques, and hence the attached spermatids, first towards the base and then back to the apex of the epithelium, along microtubules. If this hypothesis is true, motor proteins should be associated with the cytoplasmic face of the endoplasmic reticulum component of ectoplasmic specializations. In addition, isolated junction plaques should support microtubule movement both in the plus and minus directions to account for the bidirectional translocation of spermatids in vivo. To determine if cytoplasmic dynein is localized to the endoplasmic reticulum of the plaques, perfusion-fixed rat testes were immunologically probed, at the ultrastructural level, for the intermediate chain of cytoplasmic dynein (IC74). In addition, testicular fractions enriched for spermatid/junction complexes were incubated with and without gelsolin, centrifuged and the supernatants compared, by western blot analysis, for Glucose Regulated Protein 94 (a marker for endoplasmic reticulum) and IC74. At the ultrastructural level, the probe for IC74 clearly labelled material associated with the cytoplasmic face of the endoplasmic reticulum component of the junction plaques. In the gelsolin experiments, both probes reacted more strongly with appropriate bands from the gelsolin-treated supernatants than with corresponding bands from controls. To determine if the junction plaques support microtubule transport in both directions, polarity-labelled microtubules were bound to isolated spermatid/junction complexes and then assessed for motility in the presence of ATP and testicular cytosol (2 mg/ml). Of 25 recorded motility events, 17 were in a direction consistent with a plus-end directed motor being present, and 8 were in the minus-end direction. The results are consistent with the conclusion that the junction plaques have the potential for moving along microtubules in both the plus and minus directions and that both a kinesin-type and a dynein-type motor may be associated with the junction plaques. The data also indicate that cytoplasmic dynein is localized to the cytoplasmic face of the endoplasmic reticulum component of the plaques.