Post-translational control of occludin membrane assembly in mouse trophectoderm: a mechanism to regulate timing of tight junction biogenesis and blastocyst formation

The mouse blastocyst forms during the 32-cell stage with the emergence of t he blastocoelic cavity. This developmental transition is dependent upon the differentiation and transport function of the trophectoderm epithelium whi ch forms the wall of the blastocyst and exhibits functional intercellular t ight junctions (TJs) to maintain epithelial integrity during blastocoele ex pansion. To investigate mechanisms regulating the timing of blastocyst form ation, we have examined the dynamics of expression of occludin, an integral membrane protein of the TJ, Confocal microscopy of intact embryos and sync hronised cell clusters revealed that occludin first assembles at the apicol ateral membrane contact site between nascent trophectoderm cells usually du ring the early 32-cell stage, just prior to the time of blastocoele cavitat ion, This is a late event in the assembly of TJ-associated proteins within trophectoderm which, from our previous data, spans from 8- to 32-cell stage s. Occludin membrane assembly is dependent upon prior E-cadherin-mediated c ell-cell adhesion and is sensitive to brefeldin A, an inhibitor of Golgi-to -membrane transport. Occludin is delivered to the TJ site in association wi th the stage This the the blastocoelic cavity, dependent upon TJ plaque pro tein, ZO-1 alpha+, which we have shown previously is newly transcribed and translated during late cleavage, Immediately after assembly and before cavi tation, occludin localised at the TJ site switches from a Triton X-100-solu ble to -insoluble form indicative of actin cytoskeletal and/or membrane anc horage. Occludin mRNA and protein are detectable throughout cleavage by RT- PCR and immunoblotting, respectively, indicating that timing of membrane as sembly is not controlled by expression alone. Rather, we have identified ch anges in the pattern of different occludin forms expressed during cleavage which, using phosphatase treatment of embryo lysates, include post-translat ional modifications. We propose that the phosphorylation of one form of occ ludin (band 2, 65-67 kDa) during late cleavage, which leads to its exclusiv e conversion from a Triton X-100-soluble to -insoluble pool, may regulate o ccludin association with ZO-1 alpha+ and membrane assembly, and thereby act to control completion of TJ biogenesis and the timing of blastocyst format ion.