Gap junctions are selectively associated with interlocking ball-and-sockets but not protrusions in the lens

[摘要] Purpose: Ball-and-sockets andprotrusions are specialized interlocking membrane domains between lensfibers of all species studied. Ball-and-sockets and protrusions aresimilar in their shape, size, and surface morphology, and aretraditionally believed to play a key role in maintaining fiber-to-fiberstability. Here, we evaluate the hypothesis that ball-and-sockets andprotrusions possess important structural and functional differencesduring fiber cell differentiation and maturation. Methods: Intact lenses of leghornchickens (E7 days to P62 weeks old) and rhesus monkeys (1.5–20 yearsold) were studied with SEM, freeze-fracture TEM, freeze-fractureimmunogold labeling (FRIL), and filipin cytochemistry for membranecholesterol detection. Results: SEM showed thatball-and-sockets were distributed along the long and short sides ofhexagonal fiber cells, whereas protrusions were located along the cellcorners, from superficial to deep cortical regions in both chicken andmonkey lenses. Importantly, by freeze-fracture TEM, we discovered theselective association of gap junctions with all ball-and-socketsexamined, but not with protrusions, in both species. In the embryonicchicken lens (E18), the abundant distribution of ball-and-socket gapjunctions was regularly found in an approximate zone extending at least300 μm deep from the equatorial surface of the superficial corticalfibers. Many ball-and-socket gap junctions often protruded deeply intoneighboring cells. However, in the mature fibers of monkey lenses,several ball-and-sockets exhibited only partial occupancy of gapjunctions with disorganized connexons, possibly due to degradation ofgap junctions during fiber maturation and aging. FRIL analysisconfirmed that both connexin46 (Cx46) and connexin50 (Cx50) antibodiesspecifically labeled ball-and-socket gap junctions, but notprotrusions. Furthermore, filipin cytochemistry revealed that theball-and-socket gap junctions contained different amounts ofcholesterol (i.e., cholesterol-rich versus cholesterol-free) as seenwith the filipin-cholesterol-complexes (FCC) in different corticalregions during maturation. In contrast, the protrusions containedconsistently high cholesterol amounts (i.e., 402 FCCs/μm2membrane) which were approximately two times greater than that of thecholesterol-rich gap junctions (i.e., 188 FCCs/μm2 membrane)found in ball-and-sockets. Conclusions: Gap junctions are regularlyassociated with all ball-and-sockets examined in metabolically activeyoung cortical fibers, but not with protrusions, in both chicken andmonkey lenses. Since these unique gap junctions often protrude deeplyinto neighboring cells to increase membrane surface areas, they maysignificantly facilitate cell-to-cell communication between youngcortical fiber cells. In particular, the large number ofball-and-socket gap junctions found near the equatorial region mayeffectively facilitate the flow of outward current toward theequatorial surface for internal circulation of ions in the lens. Incontrast, a consistent distribution of high concentrations ofcholesterol in protrusions would make the protrusion membrane lessdeformable and would be more suitable for maintaining fiber-to-fiberstability during visual accommodation. Thus, the ball-and-sockets andprotrusions are two structurally and functionally distinct membranedomains in the lens.

[学科分类] 生物化学/生物物理