Specificity of Gap Junction Formation

Prowse, David M (1993) Specificity of Gap Junction Formation. PhD thesis, University of Glasgow.

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This thesis describes an investigation into the control of specificity of gap junctional intercellular communication, the mechanism which underiies the formation of communication compartments. Ductin, connexins and cadherins are all thought to contribute to gap junction formation, and members of the connexin and cadherin families because of their tissue specific expression may play a role in the control of specificity. Two cell culture systems were used as models for the molecular analysis of junction formation and specificity. The mec+ (defective in junction formation and metabolic cooperation) cell lines (L, MCF-7 and S180) provide a system for expression studies aimed at identifying proteins required for gap junction formation. The epithelial mec- cell line BRL and either of the fibroblast mec+ cell lines BHK or BICR sort out when grown in mixed culture and show a specificity of gap junctional communication, providing a system for the identification of proteins which can break specificity. L and S180 cells express ductin and connexin 43 but do not express the cell adhesion molecules E-, N- or P-cadherin or N-CAM. Stable S180 transfectants expressing E- or P-cadherin protein, have a flatter morphology at confluency than untransfected cells and show increased levels of gap junctional communication, as demonstrated by microinjection of the dye Lucifer Yellow CH. S180 cells therefore appear to have a defect which reduces gap junction formation and this can be restored by expression of two different members of the cadherin family. The transfection of L cells with either E- or P-cadherin causes cells to grow in tight colonies and to show increased calcium dependent cell adhesion, however the transfected cells do not form gap junctions. Western analysis of connexin 43 expression is consistent with transfected S180 cells expressing phosphorylated connexin 43 and transfected L cells non-phosphorylated connexin 43. This suggests that the phosphorylation of connexin 43, which is reported to occur in mec+ cells is important for gap junction formation. The causes of specificity are unknown. The expression and phosphorylation of connexin 43 may be essential for gap junctional communication but it does not account for the specificity of gap junctional communication seen between BRL and BHK or BICR cells, as western analysis of connexin 43 expression is consistent with these cell types expressing phosphorylated connexin 43. The analysis of cell adhesion molecule expression shows that the differential expression of cell adhesion molecules may contribute to specificity. The transfection of BICR but not BHK cells with E- or P-cadherin expression constructs causes marked morphological change and accumulation of exogenous cadherin at points of cell contact. When BICR cells transfected with E-cadherin and BRL cells also transfected with E-cadherin are grown in mixed cultures specificity is broken and dye transfer occurs between the cell types in 50% of injections, (5% in mixed cultures of parental cells). The mechanism by which E-cadherin breaks specificity is unknown. The cell types which show specificity form functional homologous gap junctions and it may be that E-cadherin induced cell-cell adhesion perse increases membrane apposition between different cell types which would not normally occur and this increases the frequency of close membrane apposition required for junction formation. Alternatively E-cadherin induced cell-cell interaction could cause signal transduction which affects proteins Involved in gap junction formation.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: John Pitts
Keywords: Cellular biology
Date of Award: 1993
Depositing User: Enlighten Team
Unique ID: glathesis:1993-75611
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 19:18
Last Modified: 19 Nov 2019 19:18
URI: https://theses.gla.ac.uk/id/eprint/75611

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