Structure of rapidly frozen gap junctions.

Abstract

The structure of gap junctions in the rabbit ciliary epithelium, corneal endothelium, and mouse stomach and liver was studied with the freeze-fracturing technique after rapid freezing to near 4°K from the living state . In the ciliary epithelium, the connexons were randomly distributed, separated by smooth membrane matrix . In the corneal endothelium, both random and crystalline arrangements of the connexons were observed . In the stomach and liver, the connexons were packed but not crystalline . Experimental anoxia or lowered pH caused crystallization of the connexons within 20-30 min . In the ciliary epithelium, the effects of prolonged anoxia or low pH could not be reversed . In addition, invaginated or annular gap junctions increased in number, but their connexons were usually distributed at random . Rapid freezing thus demonstrates that gap junctions of different tissues are highly pleiomorphic in the living state, and this may explain their variations in structure after chemical fixation . The slow time-course and irreversibility of the morphological changes induced by prolonged anoxia or low pH suggest that connexon crystallization may be a long-term consequence rather than the morphological correlate of the switch to high resistance .When gap junctions are chemically fixed and subsequently analyzed with the freeze-fracturing technique, the clustering of their subunits (particles or connexons) in the plane of the membrane varies in different tissues and often in different cells of the same tissue: hexagonal packing and complete randomness are the extreme configurations, but a great variety of intermediate arrangements have been described (4,10,20) . The functional correlate of these variations is not known, but it seems unlikely that they could reflect differences in the resistance state ofthe junctions. In fact, it was shown that aldehyde fixation abolishes coupling, and therefore freeze-fractured gap junctions in fixed tissues are probably visualized in their highresistance state (3) . Nevertheless, differences in the degree of clustering of the connexons were observed in tissues exposed to uncoupling agents before fixation as compared with their untreated controls (12, 21, 22); thus, it was proposed that the switch to high resistance is accompanied by movement of the connexons in the plane of the membrane and their lateral association in a compact, crystalline lattice . There are exceptions, however, to this behavior, because uncoupling agents did not seem to affect the state of connexon aggregation in the gap junctions of the lens (11).These observations raise important questions on the structure of gap junctions in vivo, and on the time-course of crystallization and its precise relationships with uncoupling. Such questions, however, can only be answered if the structure of gap junctions can be analyzed and experimentally manipulated in absence of chemical fixation. For this reason, we used the technique of rapid-freezing to near 4°K (14, 28) on a variety of tissues. With this method, freezing...

Gap junction connexon configuration in rapidly frozen myocardium and isolated intercalated disks.

“…Raviola et al (33) were able to freeze samples of stomach from anaesthetized rats within 20 s ofthe suspension of blood supply, and the junctions from that organ showed random arrays . Their samples of similarly prepared liver had junctions in hexagonal arrays.…”

“…After isolating or incubating disks in the various media, it was necessary to stabilize the gap junctions with glutaraldehyde to prevent structural changes during the subsequent pelleting necessary for electron microscopy. Aldehyde fixation is known to induce uncoupling and alterations in gap junction structure in some tissues (3,33) but apparently has little affect on connexon configuration in the heart (2,22,25). In the homogenized hearts where there are no tissue penetration problems, fixation would be expected to be rapid and had no detectable effect on the structure of disks isolated using the standard technique and frozen with or without pretreatment .…”

“…Others have shown, however, that the relationship between morphology and functional state may be more complex, and have therefore tried either to define their experimental conditions more rigorously (22) and/or to use ultrarapid freezing techniques (13,22,33,39) in an attempt to get closer to the true functional (or nonfunctional) configuration of the junction. Recently there have even been indications that coupling in some cell types may be associated with the presence of THE JOURNAL of CELL BioLocr " VOLUME 99 AUGUST 1984 [453][454][455][456][457][458][459][460][461][462][463] © The Rockefeller University Press -0021-952-5/84/08/0453/11 $1 .00 ordered arrays of connexons (22,39), a complete reversal of the conclusions in the works noted above.…”

Connexin36 vs. connexin32, “miniature” neuronal gap junctions, and limited electrotonic coupling in rodent suprachiasmatic nucleus

“…Within gap junctions, P-face connexon IMPs are of uniform 9-nm diameter, which is noticeably larger than virtually all other IMPs in the nearby plasma membrane P-face. Connexon E-face pits are equally distinct, making even small clusters of connexon P-face particles and E-face pits easily identifiable by their size and 10-nm center-to-center spacing (Gilula et al, 1972;Raviola and Gilula, 1975;Raviola et al, 1980;Rash et al, 1996;Meier et al, 2004). …”

Dye transfer between cells of the embryonic chick lens becomes less sensitive to CO2 treatment with development.

“…The junctions in these different tissues also may be distinguished in freeze-fracture replicas by their connexon (subunit) packing . For example, linear connexon aggregations are observed in fixed and unfixed freeze-fractured junctional plaques in myocardium (2, 7) but not in lens (12) or liver (30) . There are dramatic interspecies differences in myocardial gapjunction structure as well (18) .…”