Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells

Abstract

The eye lens consists of layers of tightly packed fiber cells, forming a transparent and avascular organ that is important for focusing light onto the retina. A microcirculation system, facilitated by a network of gap junction channels composed of connexins 46 and 50 (Cx46 and Cx50), is hypothesized to maintain and nourish lens fiber cells. We measured lens impedance in mice lacking tropomodulin 1 (Tmod1, an actin pointed-end capping protein), CP49 (a lens-specific intermediate filament protein), or both Tmod1 and CP49. We were surprised to find that simultaneous loss of Tmod1 and CP49, which disrupts cytoskeletal networks in lens fiber cells, results in increased gap junction coupling resistance, hydrostatic pressure, and sodium concentration. Protein levels of Cx46 and Cx50 in Tmod1(-/-);CP49(-/-) double-knockout (DKO) lenses were unchanged, and electron microscopy revealed normal gap junctions. However, immunostaining and quantitative analysis of three-dimensional confocal images showed that Cx46 gap junction plaques are smaller and more dispersed in DKO differentiating fiber cells. The localization and sizes of Cx50 gap junction plaques in DKO fibers were unaffected, suggesting that Cx46 and Cx50 form homomeric channels. We also demonstrate that gap junction plaques rest in lacunae of the membrane-associated actin-spectrin network, suggesting that disruption of the actin-spectrin network in DKO fibers may interfere with gap junction plaque accretion into micrometer-sized domains or alter the stability of large plaques. This is the first work to reveal that normal gap junction plaque localization and size are associated with normal lens coupling conductance.

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

“… 53 , 54 Meanwhile, double KO of intermediate filament protein, CP49 (phakinin), and actin-capping protein, tropomodulin 1, increased coupling resistance while decreasing the size of Cx46-containing gap junctional plaques. 47 This study demonstrates that the reverse case is also true: disrupting Cx50 leads to cytoskeletal changes. The mechanisms for reciprocal regulation between gap junctions and the cytoskeleton remain not well understood.…”

“…Equal quantities of Cx46 distributed in smaller plaques were previously shown to increase coupling resistance. 47 Therefore, the observed changes in Cx46 gap junction assembly may contribute to a portion of the coupling deficits measured in Cx50 KO lenses. 48 …”

Disruption of the lens circulation causes calcium accumulation and precipitates in connexin mutant mice

“…Transgenic mice with lens expression of a mutant ubiquitin (K6W) develop cataracts and have a decrease in MF gap junction coupling conductance and accumulation of calcium ions in the lens core (24); however, these changes are less pronounced than those in Cx46fs380 homozygous lenses. Mice null for both tropomodulin and phakinin have smaller and more dispersed Cx46 gap junction plaques (without changes in total levels of Cx46), a 50% decrease in gap junctional coupling conductance, and increased intracellular hydrostatic pressure and [Na ϩ ] i (11). These mice do not develop overt cataracts (18); the extent of the changes in their parameters is similar to that in 2-mo-old Cx46fs380 heterozygous lenses, which also do not have cataracts at this age but have very low levels of Cx46.…”

The lens actin filament cytoskeleton: Diverse structures for complex functions

“…Loss of either Tmod1 or CP49 causes decreased lens mechanical stiffness, presumably due to disruption of the spectrin-actin or beaded intermediate filament networks, respectively (Gokhin et al, 2012; Nowak et al, 2009). Unexpectedly, the membrane skeleton and beaded intermediate filaments synergize to promote the formation of large micron-size gap junction plaques in differentiating fiber cells that are crucial for normal lens ion and fluid homeostasis (Cheng et al, 2015). Gap junction plaques rest in lacunae within the spectrin-actin network along fiber cell membranes, suggesting that the membrane skeleton plays a role in accretion or stability of large gap junction plaques in the lens (Cheng et al, 2015).…”

“…Unexpectedly, the membrane skeleton and beaded intermediate filaments synergize to promote the formation of large micron-size gap junction plaques in differentiating fiber cells that are crucial for normal lens ion and fluid homeostasis (Cheng et al, 2015). Gap junction plaques rest in lacunae within the spectrin-actin network along fiber cell membranes, suggesting that the membrane skeleton plays a role in accretion or stability of large gap junction plaques in the lens (Cheng et al, 2015). It remains unclear whether the membrane skeleton interacts directly with gap junction plaques, or if gap junction plaque stability/assembly is affected indirectly by membrane skeleton/beaded filament network interactions through a linker protein (Fuchs and Yang, 1999; Wiche et al, 2015; Wiche and Winter, 2011).…”