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

Post-translational control of occludin membrane assembly in mouse trophectoderm: a mechanism to regulate timing of tight junction biogenesis and blastocyst formation
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 the blastocoelic cavity. This developmental transition is dependent upon the differentiation and transport function of the trophectoderm epithelium which forms the wall of the blastocyst and exhibits functional intercellular tight junctions (TJs) to maintain epithelial integrity during blastocoele expansion. To investigate mechanisms regulating the timing of blastocyst formation, we have examined the dynamics of expression of occludin, an integral membrane protein of the TJ. Confocal microscopy of intact embryos and synchronised cell clusters revealed that occludin first assembles at the apicolateral membrane contact site between nascent trophectoderm cells usually during the early 32-cell stage, just prior to the time of blastocoele cavitation. 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 stages. Occludin membrane assembly is dependent upon prior E-cadherin-mediated cell-cell adhesion and is sensitive to brefeldin A, an inhibitor of Golgi-to-membrane transport. Occludin is delivered to the TJ site in association with the TJ plaque protein, ZO-1(&agr;)+, which we have shown previously is newly transcribed and translated during late cleavage. Immediately after assembly and before cavitation, occludin localised at the TJ site switches from a Triton X-100-soluble to -insoluble form indicative of actin cytoskeletal and/or membrane anchorage. Occludin mRNA and protein are detectable throughout cleavage by RT-PCR and immunoblotting, respectively, indicating that timing of membrane assembly is not controlled by expression alone. Rather, we have identified changes in the pattern of different occludin forms expressed during cleavage which, using phosphatase treatment of embryo lysates, include post-translational modifications. We propose that the phosphorylation of one form of occludin (band 2, 65-67 kDa) during late cleavage, which leads to its exclusive conversion from a Triton X-100-soluble to -insoluble pool, may regulate occludin association with ZO-1(&agr;)+ and membrane assembly, and thereby act to control completion of TJ biogenesis and the timing of blastocyst formation.
1477-9129
831-840
Sheth, B.
2ca6ed58-a992-47b7-b3a5-3c5df82aada7
Moran, B.
bf15d3aa-f887-4029-8702-d5bf0ff0d4bf
Anderson, J.M.
06a6a589-e95e-42e3-a3c4-9db963fddc97
Fleming, T.P.
2abf761a-e5a1-4fa7-a2c8-12e32d5d4c03
Sheth, B.
2ca6ed58-a992-47b7-b3a5-3c5df82aada7
Moran, B.
bf15d3aa-f887-4029-8702-d5bf0ff0d4bf
Anderson, J.M.
06a6a589-e95e-42e3-a3c4-9db963fddc97
Fleming, T.P.
2abf761a-e5a1-4fa7-a2c8-12e32d5d4c03

Sheth, B., Moran, B., Anderson, J.M. and Fleming, T.P. (2000) Post-translational control of occludin membrane assembly in mouse trophectoderm: a mechanism to regulate timing of tight junction biogenesis and blastocyst formation. Development, 127 (4), 831-840.

Record type: Article

Abstract

The mouse blastocyst forms during the 32-cell stage with the emergence of the blastocoelic cavity. This developmental transition is dependent upon the differentiation and transport function of the trophectoderm epithelium which forms the wall of the blastocyst and exhibits functional intercellular tight junctions (TJs) to maintain epithelial integrity during blastocoele expansion. To investigate mechanisms regulating the timing of blastocyst formation, we have examined the dynamics of expression of occludin, an integral membrane protein of the TJ. Confocal microscopy of intact embryos and synchronised cell clusters revealed that occludin first assembles at the apicolateral membrane contact site between nascent trophectoderm cells usually during the early 32-cell stage, just prior to the time of blastocoele cavitation. 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 stages. Occludin membrane assembly is dependent upon prior E-cadherin-mediated cell-cell adhesion and is sensitive to brefeldin A, an inhibitor of Golgi-to-membrane transport. Occludin is delivered to the TJ site in association with the TJ plaque protein, ZO-1(&agr;)+, which we have shown previously is newly transcribed and translated during late cleavage. Immediately after assembly and before cavitation, occludin localised at the TJ site switches from a Triton X-100-soluble to -insoluble form indicative of actin cytoskeletal and/or membrane anchorage. Occludin mRNA and protein are detectable throughout cleavage by RT-PCR and immunoblotting, respectively, indicating that timing of membrane assembly is not controlled by expression alone. Rather, we have identified changes in the pattern of different occludin forms expressed during cleavage which, using phosphatase treatment of embryo lysates, include post-translational modifications. We propose that the phosphorylation of one form of occludin (band 2, 65-67 kDa) during late cleavage, which leads to its exclusive conversion from a Triton X-100-soluble to -insoluble pool, may regulate occludin association with ZO-1(&agr;)+ and membrane assembly, and thereby act to control completion of TJ biogenesis and the timing of blastocyst formation.

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Published date: 1 February 2000

Identifiers

Local EPrints ID: 56319
URI: https://eprints.soton.ac.uk/id/eprint/56319
ISSN: 1477-9129
PURE UUID: f3741f03-af56-49f2-be14-482b7a8fa756

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Date deposited: 07 Aug 2008
Last modified: 17 Jul 2017 14:31

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