June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
The second Extracellular Domain of Connexin 50 Mediates Lens Development by Mediating cell-cell Adhesion
Author Affiliations & Notes
  • Jean X Jiang
    Biochemistry, Univ of Texas Hlth Sci Ctr, Helotes, Texas, United States
  • Zhen Li
    Biochemistry, Univ of Texas Hlth Sci Ctr, Helotes, Texas, United States
  • Sumin Gu
    Biochemistry, Univ of Texas Hlth Sci Ctr, Helotes, Texas, United States
  • Footnotes
    Commercial Relationships   Jean Jiang, None; Zhen Li, None; Sumin Gu, None
  • Footnotes
    Support  NIH Grant EY012085
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 2303. doi:
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    • Get Citation

      Jean X Jiang, Zhen Li, Sumin Gu; The second Extracellular Domain of Connexin 50 Mediates Lens Development by Mediating cell-cell Adhesion. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2303.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Besides its function in forming membrane channels, we have shown that connexin (Cx) 50 plays a unique role in cell-cell adhesion. To understand the molecular mechanism of Cx50 in cell adhesion and lens development, we expressed Cx50 and mutants in Cx-null cell lines and embryonic chick lens using retroviral expression.

Methods : By swapping the second extracellular loop domain (E2) between lens connexins, 4 domain-swapped mutants, Cx50/46E2, Cx50/43E2, Cx46/50E2 and Cx43/50E2 were reconstructed into a RCAS(A) retroviral vector. Additionally, 8 site mutants of Cx50 in the E2 domain were generated by site-directed mutagenesis. Chick embryo fibroblast (CEF) cells transfected with these DNA constructs were cultured and condition media were collected and concentrated to generate high titter recombinant retrovirus. An adhesion assay was conducted using CEF cells infected with recombinant RCAS(A) retrovirus. High titter retroviruses containing mutants of Cx50 E2 domain were injected into lens placodes of embryonic day (E) 3 chick lens. Chick lens was collected and tissue sections were prepared for histochemical analysis at E14 and E20.

Results : The expression of Cx50, but not Cx43 or Cx46 promoted cell adhesion and Cx50E2 domain was primarily responsible for cell adhesive function. The cell adhesive function decreased in cells expressing Cx50 in which the E2 domain was swapped with the E2 domain of either Cx43 or Cx46. In contrast, adhesion increased in cells expressing chimeric Cx43 and Cx46 with Cx50(E2) domain. This function is channel independent as the adhesive property occurred in both homotypic with Cx50 expressed in both pairing cells and heterotypic manner with Cx50 expressed in only one pairing cell. Furthermore, mutation of eight amino acid residues in the Cx50E2 domains impaired cell-cell adhesion in a dominant negative manner. Importantly, these mutants did not affect gap junctions, except Cx50R200G, which causes congenital cataract formation in humans. Cx50W188P caused enlargement of the extracellular spaces and distortion of fiber organization at embryonic day (E) 14, while expression of Cx50W188P mutant showed delayed nuclei degeneration at E20.

Conclusions : These results suggest that the E2 domain of Cx50 plays a critical role in cell adhesion and lens development and mutations at the eight non-conserved amino acid residues of Cx50E2 compromise lens development and transparency.

This is a 2020 ARVO Annual Meeting abstract.

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