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.
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.
Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California;
Catherine
C
Cheng
Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California;
Roberta B
RB
Nowak
Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, New York; and.
Junyuan
J
Gao
Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, New York; and.
Xiurong
X
Sun
Department of Neurobiology, Morehouse School of Medicine, Atlanta, Georgia.
Sondip K
SK
Biswas
Department of Neurobiology, Morehouse School of Medicine, Atlanta, Georgia.
Woo-Kuen
WK
Lo
Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, New York; and.
Richard T
RT
Mathias
Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California; velia@scripps.edu.
Velia M
VM
Fowler
D000818:Animals; D002454:Cell Differentiation; D017630:Connexins / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism*; D006706:Homeostasis / Q000502:physiology*; D007381:Intermediate Filament Proteins / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism; D007908:Lens, Crystalline / Q000166:cytology* / Q000378:metabolism*; D018345:Mice, Knockout; D008822:Mice, Transgenic; D000099264:Connexin 50
D017630:Connexins; D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D007473:Ion Channels; D000099264:Connexin 50; C082917:GJA3 protein, human
CP49; beaded intermediate filament; impedance; membrane skeleton; tropomodulin 1