Gap junctions contain different amounts of cholesterol which undergo unique sequestering processes during fiber cell differentiation in the embryonic chicken lens

Abstract

Purpose: To determine the possible changes in the distribution of cholesterol in gap junction plaques during fiber cell differentiation and maturation in the embryonic chicken lens. The possible mechanism by which cholesterol is removed from gap junction plaques is also investigated.

Methods: Filipin cytochemistry in conjunction with freeze-fracture TEM was used to visualize cholesterol, as represented by filipin-cholesterol complexes (FCCs) in gap junction plaques. Quantitative analysis on the heterogeneous distribution of cholesterol in gap junction plaques was conducted from outer and inner cortical regions. A novel technique combining filipin cytochemistry with freeze-fracture replica immunogold labeling (FRIL) was used to label Cx45.6 and Cx56 antibodies in cholesterol-containing gap junctions. Filipin cytochemistry and freeze-fracture TEM and thin-section TEM were used to examine the appearance and nature of the cholesterol-containing vesicular structures associated with gap junction plaques.

Results: Chicken lens fibers contain cholesterol-rich, cholesterol-intermediate and cholesterol-free gap junction populations in both outer and inner cortical regions. Filipin cytochemistry and FRIL studies confirmed that cholesterol-containing junctions were gap junctions. Quantitative analysis showed that approximately 86% of gap junctions in the outer cortical zone were cholesterol-rich gap junctions, whereas approximately 81% of gap junctions in the inner cortical zone were cholesterol-free gap junctions. A number of pleiomorphic cholesterol-rich vesicles of varying sizes were often observed in the gap junction plaques. They appear to be involved in the removal of cholesterol from gap junction plaques through endocytosis.

Conclusions: Gap junctions in the young fibers are enriched with cholesterol because they are assembled in the unique cholesterol-rich cell membranes in the lens. A majority of cholesterol-rich gap junctions in the outer young fibers are transformed into cholesterol-free ones in the inner mature fibers during fiber cell maturation. A distinct endocytotic process appears to be involved in removing cholesterol from the cholesterol-containing gap junctions, and it may play a major role in the transformation of cholesterol-rich gap junctions into cholesterol-free ones during fiber cell maturation.

Figure 1

A large intact replica on the Girder finder grid with index number for systematic examinations of fiber gap junctions in the embryonic chicken lens. A low magnification shows an overview of a large intact replica (outlined by circle line) on the Girder finder grid with index number for systematic examinations of fiber gap junctions from outer to inner cortical regions of the embryonic chicken lens at E15. The peripheral area along the circle line represents the superficial surface, and the center represents the nucleus of the lens. This freeze-fracture replica was prepared from a 300 μm lens slice initially cut with a Vibratome for the longitudinal orientation of cortical fiber cells. In this study, initial identifications of various lens regions were made and recorded using the index number (the distance between two parallel grid bars is 100 μm), followed by thorough examinations of the structures of interest in each region. The scale bar is equal to 100 μm.

Figure 2

Different structural configurations of gap junctions in various cortical regions of the embryonic chicken lens. Representative micrographs show different structural configurations of gap junctions (gj) found in various cortical regions in the embryonic chicken lens. A and B: The assembly of forming gap junction plaques in the superficial fibers. C: A cluster of well-formed gap junction plaques in the outer cortical fibers. D: Several mature gap junction plaques found in the inner cortical fibers. Note the presence of vesicular structures (arrows) associated with gap junctions found in both outer and inner cortical fibers. pf, P-face of the membrane; ef, E-face of the membrane. The scale bars are equal to 200 nm.

Figure 3

Freeze-fracture replica immunogold labeling of Cx56 and Cx45.6 in gap junctions of the embryonic chicken lens. Freeze-fracture replica immunogold labeling (FRIL) demonstrates that both Cx56 and Cx45.6 polyclonal antibodies are specifically localized in gap junction plaques distributed in the outer cortex (A and C) and inner cortex (B and D), respectively. The scale bars are equal to 200 nm.

Figure 4

Heterogeneous distribution of cholesterol in gap junction plaques in the outer cortical fiber cells. Gap junctions in the outer cortex (0-200 μm deep) contain different amounts of cholesterol as determined by filipin cytochemistry and freeze-fracture TEM. Newly formed gap junctions (A and B) and well-formed cholesterol-rich gap junctions (C and D) contain a large number of filipin-cholesterol complexes (FCCs). Some FCCs (25-35 nm particles) in the P-face of the non-junctional membrane are indicated by the arrows in A. At high magnification, the border of such cholesterol-rich gap junction is outlined by arrowheads for a clearer visualization (D). Cholesterol-intermediate gap junctions contain considerably less FCCs (E and F). Cholesterol-poor or -free gap junctions contain only a few or no FCCs in the junctional plaques (G and H). pf, P-face of the membrane; ef, E-face of the membrane. The scale bars are equal to 200 nm.

Figure 5

Heterogeneous distribution of cholesterol in gap junction plaques in the inner cortical fiber cells. Gap junctions in the inner cortex (200-400 μm deep) contain different amounts of cholesterol in the embryonic chicken lens. Three subtypes of cholesterol-rich (A and B), cholesterol-intermediate (C and D) and cholesterol-free gap junctions (E and F) were all found in the inner mature fiber cells of the embryonic lens. The scale bars are equal to 400 nm.

Figure 6

Immunogold labeling of Cx56 in cholesterol-containing gap junctions by innovative technique combining filipin cytochemistry and FRIL. In view of the presence of similar aquaporin junctions in lens fibers, our innovative technique combining filipin cytochemistry and FRIL demonstrates that both cholesterol-intermediate (A) and cholesterol-free (B) junctions are real gap junctions (gj) because they contain connexin Cx56. Arrows denote some filipin-cholesterol complexes in (A). The scale bars are equal to 200 nm.

Figure 7

Specific association of unique vesicular structures with gap junction plaques in the outer cortical fibers. A: A low magnification shows a number of vesicular structures (vs) specifically associated with gap junction plaques (gj) in the outer cortical fibers. Asterisks denote the presence of small concaved connexon particles-less or -free areas often associated with the vesicular structures. B and C show the indication of endocytosis (open arrow) of the P-face (pf) of the connexon-free membrane within the gap junction plaques. D and E exhibit the close association of vesicular structures with the adjacent connexon-less membrane areas (asterisk). F: A cluster of vesicular structures with different shapes and sizes are accumulated in two adjacent gap junction plaques. The distinct separation between the P-face of the junction membrane and the vesicular structures is indicated by the arrows. These vesicular structures are shown to be enriched with cholesterol (see Figure 9). The scale bars are equal to 200 nm.

Figure 8

Specific association of unique tubulovesicular structures with gap junction plaques in the inner cortical fibers. A number of vesicular structures with characteristic tubular configurations are observed in gap junction plaques distributed in both outer and inner cortical fibers. They are more often found in the inner cortex (A, C, and D). Asterisks denote the cytoplasm in which the tubulovesicular structures (vs) are located. The distinct separation between the P-face of the junction membrane and the underlying tubulovesicular structures is indicated by the arrows. E: Thin-section TEM reveals the presence of a cluster of vesicular structures of a single unit membrane in the cytoplasm closely associated with a fiber gap junction (gj). The scale bars are equal to 200 nm.

Figure 9

Filipin-treated experiments reveal the presence of discrete filipin-cholesterol particles specifically associated with individual cholesterol vesicles (vs) in the different subtypes of cholesterol-containing gap junctions (gj). A: A low magnification shows a number of cholesterol vesicles distributed randomly within the gap junction plaques. B through E Higher magnifications illustrate the specific accumulation of filipin-cholesterol particles in the vesicular structures (vs) of various forms. The arrows indicate the distinct separation of individual cholesterol vesicles from the gap junction membranes. The scale bars in A through C are equal to 200 nm and in D and E are equal to 100 nm.