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Cytoplasmic dynein motors are localized to the cytoplasmic face of the er component of es in rat sertoli cells

University of British Columbia

In vitro motility assays indicate that minus-end directed microtubule dependant motors are attached to specialized junction plaques (ectoplasmic specializations) that occur in regions of Sertoli cells attached to spermatids. These plaques are characterized by a layer of hexagonally arrayed actin filaments situated between and linked to a cistern of ER and the plasma membrane. In this study, I use immuno-EM and actin-severing (gelsolin) experiments to test the hypothesis that cytoplasmic dynein is localized to the ER. For immuno-EM, perfusion fixed rat testis were probed for the intermediate chain of cytoplasmic dynein (IC74) before (mechanically fragmented tissue) or after (intact tissue) embedding and compared with controls. Labeling was clearly evident along, although not restricted to, the cytoplasmic face of the ER. For the gelsolin experiments testicular fractions enriched for spermatid/junction complexes were incubated with and without the gelsolin enzyme, which was used to artificially disassemble the junction plaque and release the ER, centrifuged, and the supernatants compared by western blot analysis for GRP94 (a marker for ER), IC74 (Pfister and Babco), and DHC1a (Asai). All three probes reacted more strongly with appropriate bands from gelsolin treated supernatants than with corresponding bands from controls. The data are consistent with the conclusion that a cytoplasmic dynein is associated with ectoplasmic specializations and that the motor is anchored to the cytoplasmic face of the ER component of the plaque. Because Sertoli cell microtubules have their minus-end located apically, a cytoplasmic dynein may be the motor associated with moving spermatids to the apex of the epithelium prior to sperm release.

Thesis/Dissertation

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2009-07-06

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http://hdl.handle.net/2429/10268

Anatomy, Cell Biology and Physiology

University of British Columbia

UBCV

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