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. 2016 Jun 1;57(7):3039-46.
doi: 10.1167/iovs.16-19521.

Connexin 50 Regulates Surface Ball-and-Socket Structures and Fiber Cell Organization

Eddie Wang, Andrew Geng, Ankur M Maniar, Byron W H Mui, Xiaohua Gong

Connexin 50 Regulates Surface Ball-and-Socket Structures and Fiber Cell Organization

Eddie Wang et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: The roles of gap junction protein connexin 50 (Cx50) encoded by Gja8, during lens development are not fully understood. Connexin 50 knockout (KO) lenses have decreased proliferation of epithelial cells and altered fiber cell denucleation. We further investigated the mechanism for cellular defects in Cx50 KO (Gja8-/-) lenses.

Methods: Fiber cell morphology and subcellular distribution of various lens membrane/cytoskeleton proteins from wild-type and Cx50 KO mice were visualized by immunofluorescent staining and confocal microscopy.

Results: We observed multiple morphological defects in the cortical fibers of Cx50 KO lenses, including abnormal fiber cell packing geometry, decreased F-actin enrichment at tricellular vertices, and disrupted ball-and-socket (BS) structures on the long sides of hexagonal fibers. Moreover, only small gap junction plaques consisting of Cx46 (α3 connexin) were detected in cortical fibers and the distributions of the BS-associated beta-dystroglycan and ZO-1 proteins were altered.

Conclusions: Connexin 50 gap junctions are important for BS structure maturation and cortical fiber cell organization. Connexin 50-based gap junction plaques likely form structural domains with an array of membrane/cytoskeletal proteins to stabilize BS. Loss of Cx50-mediated coupling, BS disruption, and altered F-actin in Cx50 KO fibers, thereby contribute to the small lens and mild cataract phenotypes.

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Figures

Figure 1
Figure 1
Fiber organization and F-actin distribution in WT and Cx50 KO lens fibers. Composite images of F-actin (green) and DAPI (blue)-stained cortical cross sections. The WT lens has regularly arranged, parallel columns of hexagonal fiber cells with enriched F-actin at cell vertices (A) and enlarged region in (C). Connexin 50 KO lens fibers are ordered near the surface but have areas with poorer organization approximately 80 μm from the periphery (B) and enlarged region in (D). Staining with F-actin also becomes more uniform around the cell boundaries. Scale bars: 5 μm.
Figure 2
Figure 2
The distribution of Cx46 in WT and Cx50 KO lenses. (A) Drawing of a hexagonal-shaped fiber cell to clarify the orientation of the imaging planes in (B) to (D). (B) Cross section of WT fibers (top) shows Cx46 (green) staining is enriched on BS and corresponds to weak areas of WGA (magenta) staining (arrowheads). Cross section of Cx50 KO fibers (bottom) shows punctate staining on the membrane, but no enriched areas corresponding to BS. Images of a plane going through the broad sides of WT and Cx50 KO fibers (C) and onto the broad side surface of WT and Cx50 KO fibers (D) confirm lack of BS enriched with Cx46 staining in Cx50 KO fibers compared with WT fibers. Adjacent cells in (D) were changed to grayscale for clarity. (B) and (C) collected approximately 75 to 125 μm from the lens surface. Scale bars: 5 μm.
Figure 3
Figure 3
Ball-and-socket structures with Cx50 gap junctions are unchanged in Cx46 KO (Gja3−/−) fibers. (A) Connexin 50 is enriched on the BS structures of WT lens fibers. (B) Colabeling of F-actin and Cx50 shows presence of enriched F-actin in tricellular vertices and BS structures with Cx50 gap junctions in a Cx46 KO (Gja3−/−) section. Scale bars: 5 μm.
Figure 4
Figure 4
Distribution of ZO-1 in WT and Cx50 KO lenses. In the most peripheral fibers of WT (A) and KO (B) lenses, ZO-1 localizes to the epithelial-fiber interface and to tricellular vertices. However, in KO lenses, ZO-1 shows irregular distribution along fiber cell boundaries and in fiber nuclei. (C) Colabeling of ZO-1 (green) and WGA (magenta) shows that they are each enriched on BS in fibers near the denucleation zone. (D) Zonula occludens-1 is diffuse in Cx50 KO fibers at a similar depth. Scale bars: 5 μm.
Figure 5
Figure 5
Distribution of βDys in WT and Cx50 KO lenses. (A) Beta-dystroglycan staining is strong on the membranes of epithelial cells, weak along fiber membranes, and strong on BS in WT lenses. (B) Beta-dystroglycan staining is similar in KO lenses except for the absence from BS and positive staining in fiber nuclei. Approximately 150 μm in from the lens capsule, βDys clearly labels BS in WT sections (C) but no specific localization is seen in KO sections (D). Scale bars: 5 μm.
Figure 6
Figure 6
A conceptual model for the loss of BS structures between WT and Cx50 KO lenses. A BS is a specialized interlocking membrane domain that several proteins partition to in WT mice. Connexin C-terminals interact with unknown PDZ-domain–containing proteins (PDZ?) and their downstream binding partners. An extracellular binding partner for dystroglycan in the lens has yet to be identified. In Cx50 KO (ΔGja8) lenses, BS are lost, Cx46-containing plaques are smaller in size, and at least a portion of the BS-associating proteins, ZO-1 and βDys, end up in fiber nuclei. Cytosolic, downstream partners for βDys and ZO-1 are also unknown. Ball-and-socket structure and gap junctions and proteins not drawn to scale.
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