Gap junction processing and redistribution revealed by quantitative optical measurements of connexin46 epitopes in the lens.
OBJECTIVE
To map changes in the structure and function of fiber cell gap junctions that occur with lens differentiation.


METHODS
Equatorial lens sections were fluorescently labeled with antibodies to the gap junction protein connexin (Cx)46, the membrane marker wheat germ agglutinin, and the nuclear stain propidium iodide. Two-photon microscopy and digital image analysis were used to quantify label and cell morphology as a function of radial distance (r/a) across the lens. Loop- and tail-specific Cx46 antibodies were used to identify regions of posttranslational modification. Local fiber cell coupling was imaged in situ using two-photon flash photolysis of caged fluorescein.


RESULTS
Antibody labeling showed that the cytoplasmic tail of Cx46 was removed in two zones (r/a approximately 0.9 and r/a approximately 0.7). In addition, with increasing depth, the large radially aligned plaques of peripheral fiber cells became fragmented and dispersed around the cell membrane, and cells became more circular in cross section. Fluorescein transfer between peripheral fiber cells was highly anisotropic and occurred predominantly within a column of fiber cells, resulting in radially directed transport. In regions beyond the zone of nuclear loss, transport was more isotropic and occurred across columns of fiber cells.


CONCLUSIONS
The cleavage of Cx46 is associated with a spatial redistribution of gap junction plaques. The distribution of gap junction plaques around the cell membrane can explain the observed directionality of intercellular solute transfer. The findings suggest that the processing and redistribution of gap junction proteins is central to controlling radial and circumferential solute gradients in different regions within the lens.
Jacobs
Marc D
MD
Department of Physiology, School of Medical Sciences, The University of Auckland, Auckland, New Zealand.
Soeller
Christian
C
Sisley
Aran M G
AM
Cannell
Mark B
MB
Donaldson
Paul J
PJ
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D002462:Cell Membrane; D017630:Connexins / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology; D000939:Epitopes / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives* / Q000378:metabolism; D017629:Gap Junctions / Q000502:physiology*; D007091:Image Processing, Computer-Assisted; D007202:Indicators and Reagents / Q000378:metabolism; D007908:Lens, Crystalline / Q000166:cytology* / Q000378:metabolism*; D010782:Photolysis; D011419:Propidium / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D017630:Connexins; D000939:Epitopes; D007202:Indicators and Reagents; D014909:Wheat Germ Agglutinins; C082917:GJA3 protein, human; C047939:fluorescein isothiocyanate-wheat germ agglutinin; D011419:Propidium; D016650:Fluorescein-5-isothiocyanate