Variation in ultrastructure of mucoid coat and shell membrane secretion of a dasyurid marsupial

In the dasyurid marsupial Sminthopsis crassicaudata, the shell membrane of cleaving embryos has a compact granular structure but becomes fibrous around blastocysts. Polyclonal antibodies were raised against the extracellular coats, mucoid and shell membrane, of oocytes and early embryos. Immunogold cytochemistry resulted in labelling of secretory granules in the epithelia of both the ampulla and isthmus of the oviduct, although the secretory granules of these two regions differed in their ultrastructural appearance. Those in the ampulla were heterogeneous with areas of varying electron density, whereas those in the isthmus were electron dense and homogeneous. Shell membrane precursors were found in secretory granules in the epithelia of the uterotubal junction and endometrial glands and were electron lucent.

Structures linking microfilament bundles to the membrane at focal contacts

We used quick-freeze, deep-etch, rotary replication and immunogold cytochemistry to identify a new structure at focal contacts. In Xenopus fibroblasts, elongated aggregates of particles project from the membrane to contact bundles of actin microfilaments. Before terminating, a single bundle of microfilaments interacts with several aggregates that appear intermittently over a distance of several microns. Aggregates are enriched in proteins believed to mediate actin-membrane interactions at focal contacts, including beta 1-integrin, vinculin, and talin, but they appear to contain less alpha-actinin and filamin. We also identified a second, smaller class of aggregates of membrane particles that contained beta 1-integrin but not vinculin or talin and that were not associated with actin microfilaments. Our results indicate that vinculin, talin, and beta 1-integrin are assembled into distinctive structures that mediate multiple lateral interactions between microfilaments and the membrane at focal contacts.