seminars in CELL BIOLOGY Vol 3, 1992: pp 21-28
Aspects of gap junction structure and assembly f.-P. Revel, ].H. Hoh, S.A. ]ohn*, D. W Laird, K. Puranamt and S.B. Yancty The development of ideas about gap junction structure is summarized, including some recent results obtained by use of atomic force microscopy. Particular attention is paid to novel aspects of the biosynthesis and assembly of connexons and to theformation of new junctions.
filled the space between structural elements of the junction, delineating the part of the junctional elements which bridge the extracellular space. At about the same time, Loewenstein showed electrical and dye coupling between the giant cells of Drosophila salivary glands and other specimens.? In short order Potter et al published their findings on electrical coupling between cells of squid embryos and speculated, prophetically as it turns out, about the roles of. gap junctions'' while Subak-Sharpe et al9 ,10 showed that cellular metabolites, not just ions or exogenously introduced dyes, could also be shared by neighboring cells. Gilula et alll later demonstrated that all of these exchanges correlated well with the presence of gap junctions. The physiological properties of gap junctions soon led to the idea that a special region of the cell membrane was involved in coupling (see ref 12, for a review). At first, however, the nature of this structure was not at all obvious, with some workers arguing for the involvement of septate junctions,7,13 and others holding that tight junctions played a central role in cell-cell coupling. 4,8,14 Another structure, the 'close junction', which might act as pathway for intercellular exchanges was described by Trelstad et al in their ambiguously titled report on cell contacts in chick embryos.P A detailed analysis of this novel junction, as found between cells of both excitable and non excitable tissues, was published by Revel and Karnovsky.l'' Their study showed distinct areas where plasma membranes of adjoining cells were separated by a narrow gap, which was bridged by structures set in a hexagonal pattern. The 'gap junctions' as they came to be known, were soon confirmed to be widely distributed, 17 found between cells and in tissues where physiological coupling was evident. ll,18,19 Today we recognize that gap junctions are essentially ubiquitous. They have been described between the cells of early embryos in many species (for reviews see refs 20, 21; also ref 22) and in primitive organisms. Gap junctions are found in Coelenterates23,24 and the transfer of morphogenetic factors in Hydra25 is, at least partially, inhibited by anti gap junction antibodies. Gap junctions-v and electrical coupling (Lal and Revel, unpublished) are
Key words: gap junction / formation / Cx43 / monensin / A-CAM / atomic force microscopy / phylogeny
The discovery of 'gap junctions' According to Socolar;! exchanges of small molecules between cells were recognized as early as 1925 when Schmidtman observed that dyes introduced into one cell spread to their neighbors. While her observations were puzzling at the time, today we would casually ascribe them to the existence of gap junctions, clusters of transmembrane channels specialized for cell to cell exchanges. The structures responsible for such exchanges were originally seen in heart muscle by Sjostrand et al 2 but first dearly illustrated by Karrer-' who even postulated that the 'pentalaminar' junctions he saw served a role in the propagation of electrical signals between heart cells. Shortly thereafter, Dewey and Barr! argued that the 'nexus', a specialization of smooth muscle cell membranes resulting from the 'direct flision' of their outer layer, was the site of stimulus propagation. The structural complexity of such electrical synapses was brought out first by Robertson, in his work on the Mauthner cells of the goldfish brain. Using permanganate as a fixative, he demonstrated a honeycomb like specialization of the membranes involved in the contact. 5 The copious precipitates which form on reduction of KMn04 presumably
From the Division ofBiology, California Institute ofTechnology, Pasadena, CA 91125, USA "Present address: Department of Physiology, UCLA, Los Angeles, CA, USA t Present address: Hughes Medical Institute, Duke University, Durham, NC, USA ©1992 Academic Press Ltd 1013-1682/92/010021 + 08$5. 00/0
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22
found between cells of extremely primitive organisms such as Mesozoans. There are even reports of cell coupling in reaggregating' Sponges.V While junctionlike clusters of membrane particles have been found in freeze cleave preparations of warm water Sponges (Mann and Revel, unpublished) the morphological demonstration .of gap junctions in Porifera is open to debate. 28
Structure of connexons As just indicated, it was recognized early on that the cell junctions associated with physiological coupling consisted of discrete structures, named 'connexons' by Goodenough.f which are anchored in the cell membrane. Connexons extend far enough into the extracellular space to contact a similar structure extending out of the membrane of the neighboring cell. 16,29-33 Each connexon is one cell's contribution to the bipartite, cytoplasm to cytoplasm , structure which serves for cytosolic exchanges. Gap junctions are recognizable morphologically because thousands of connexons are usually clustered in large arrays, or ~ pl aqu e s ' . The functional interpretation of gap junctions as devices for intercellular communication requires that each connexon pair form an aqueous channel through which ions and small molecules can pass, without leaking into the extracellular space. Connexons are comprised of proteins of distinctive structure, called 'connexins' as a class (connexin is abbreviated Cx, followed by the molecular mass determined from cDNA, e.g. Cx43). In the following sections we present some of the data on morphological aspects of the connexons.
Visualization of the aqueous channel Visualization in fixed samples
A dimple seen on connexons in freeze cleave preparations has often been interpreted as representing the channel pore. The dimple is especially well seen in deep etched preparations.I" A central dot of a size compatible with the 1.5-2.0 nm diameter expected from permeation by tracer molecules'P and which could thus correspond to the channel pore, is also found in samples negatively stained with phosphotungstic acid or uranyl salts36 as well as with alkali metal ions .37 A priori this fulfils the expectation that materials should have access to the channel at the cytoplasmic face of the junctions.
A central structure is also seen in lanthanum permeated, sectioned material. This finding is unexpected since lanthanum salts fill the extracellular space and thus should not be able to enter a channel connected only to cytoplasm, if the channel wall is, as required by the physiological data, impermeable to aqueous solutes. An obvious way out is to argue that the permeability of the connexon has been changed by tissue preparation for microscopy. 38 The dimple associated with connexons in freeze cleaved samples is seen only on the P face of the cleaved membrane, that is on the aspect of the connexon which, in life, faced another connexon in the extracellular space. Deep etch preparations reveal not only the cleaved faces, but also the natural cytoplasmic face of membranes and junctions and should therefore also reveal the other end of the channel. In regions where the cytoplasmic face is exposed however, there is no evidence of connexons, or pores.P" Perhaps the channel is closed, or the cyto-plasmic domains of connexins are so tightly packed that the pore opening cannot be detected (see further discussion, below). Alternately there are other structures like the 'fuzzy layer' detected by Manjunath and Page39 in association with heart connexons, which might obscure the connexons and the channel mouth opening as revealed by deep etching The fuzzy layer, however, is not particularly prominent in liver junctions, which were used in the deep etch work. Visualization in unfixed samples
One approach to examination of 'native' structures is to use X-ray diffraction of packed gap junction plaques. Even though such analyses do not usually require fixation, a light treatment with glutaraldehyde has been used, without any apparent effect on the structures.t? The pioneering work of Caspar et al41 ,42 led to a gap junction model where each connexon consists of six subunits, which form a hollow cylinder. This model has been modified and elaborated on,43 notably by Unwin and collaborators. vvff The X-ray data provides experimental support for models in which the wall of the connexon is formed of protein subunits which surround a solvent filled structure, presumably the channel through which solutes can flow. An important feature of many of the models proposed is that the solvent containing channel is broad in the middle and often seems closed at the cytoplasmic surface. 43 ,44
23
Gap junction structure and assembly
Much of the X-ray data is based on an analysis ofliver derived junctions, containing principally the connexin protein Cx32 as well as some Cx26. 47,48 Comparison of the sequence data for all known connexins suggests that all should have the same general configuration (see ref 49 for discussion) with the possible exception of Cx38 in Xenopus. 50 It is therefore likely that models based on X-ray analysis of one type of junction will be applicable to all others at least in general terms.
Atomic force microscopy The atomic force microscope (AFM) provides a novel way to image biological structures, theoretically permitting one to trace out the surface contour of even unfixed biological structures at high resolution. 51-53 When imaging biological objects, however, it is as yet not possible to reach the atomic resolution which is easily achievable with non-biological materials.54 Isolated gap junctions deposited on a glass substrate reveal their cytoplasmic faces, which are found to be smooth, devoid of detectable structures'
Aspects of gap junction structure and assembly f.-P. Revel, ].H. Hoh, S.A. ]ohn*, D. W Laird, K. Puranamt and S.B. Yancty The development of ideas about gap junction structure is summarized, including some recent results obtained by use of atomic force microscopy. Particular attention is paid to novel aspects of the biosynthesis and assembly of connexons and to theformation of new junctions.
filled the space between structural elements of the junction, delineating the part of the junctional elements which bridge the extracellular space. At about the same time, Loewenstein showed electrical and dye coupling between the giant cells of Drosophila salivary glands and other specimens.? In short order Potter et al published their findings on electrical coupling between cells of squid embryos and speculated, prophetically as it turns out, about the roles of. gap junctions'' while Subak-Sharpe et al9 ,10 showed that cellular metabolites, not just ions or exogenously introduced dyes, could also be shared by neighboring cells. Gilula et alll later demonstrated that all of these exchanges correlated well with the presence of gap junctions. The physiological properties of gap junctions soon led to the idea that a special region of the cell membrane was involved in coupling (see ref 12, for a review). At first, however, the nature of this structure was not at all obvious, with some workers arguing for the involvement of septate junctions,7,13 and others holding that tight junctions played a central role in cell-cell coupling. 4,8,14 Another structure, the 'close junction', which might act as pathway for intercellular exchanges was described by Trelstad et al in their ambiguously titled report on cell contacts in chick embryos.P A detailed analysis of this novel junction, as found between cells of both excitable and non excitable tissues, was published by Revel and Karnovsky.l'' Their study showed distinct areas where plasma membranes of adjoining cells were separated by a narrow gap, which was bridged by structures set in a hexagonal pattern. The 'gap junctions' as they came to be known, were soon confirmed to be widely distributed, 17 found between cells and in tissues where physiological coupling was evident. ll,18,19 Today we recognize that gap junctions are essentially ubiquitous. They have been described between the cells of early embryos in many species (for reviews see refs 20, 21; also ref 22) and in primitive organisms. Gap junctions are found in Coelenterates23,24 and the transfer of morphogenetic factors in Hydra25 is, at least partially, inhibited by anti gap junction antibodies. Gap junctions-v and electrical coupling (Lal and Revel, unpublished) are
Key words: gap junction / formation / Cx43 / monensin / A-CAM / atomic force microscopy / phylogeny
The discovery of 'gap junctions' According to Socolar;! exchanges of small molecules between cells were recognized as early as 1925 when Schmidtman observed that dyes introduced into one cell spread to their neighbors. While her observations were puzzling at the time, today we would casually ascribe them to the existence of gap junctions, clusters of transmembrane channels specialized for cell to cell exchanges. The structures responsible for such exchanges were originally seen in heart muscle by Sjostrand et al 2 but first dearly illustrated by Karrer-' who even postulated that the 'pentalaminar' junctions he saw served a role in the propagation of electrical signals between heart cells. Shortly thereafter, Dewey and Barr! argued that the 'nexus', a specialization of smooth muscle cell membranes resulting from the 'direct flision' of their outer layer, was the site of stimulus propagation. The structural complexity of such electrical synapses was brought out first by Robertson, in his work on the Mauthner cells of the goldfish brain. Using permanganate as a fixative, he demonstrated a honeycomb like specialization of the membranes involved in the contact. 5 The copious precipitates which form on reduction of KMn04 presumably
From the Division ofBiology, California Institute ofTechnology, Pasadena, CA 91125, USA "Present address: Department of Physiology, UCLA, Los Angeles, CA, USA t Present address: Hughes Medical Institute, Duke University, Durham, NC, USA ©1992 Academic Press Ltd 1013-1682/92/010021 + 08$5. 00/0
21
j. -r. Revel el at
22
found between cells of extremely primitive organisms such as Mesozoans. There are even reports of cell coupling in reaggregating' Sponges.V While junctionlike clusters of membrane particles have been found in freeze cleave preparations of warm water Sponges (Mann and Revel, unpublished) the morphological demonstration .of gap junctions in Porifera is open to debate. 28
Structure of connexons As just indicated, it was recognized early on that the cell junctions associated with physiological coupling consisted of discrete structures, named 'connexons' by Goodenough.f which are anchored in the cell membrane. Connexons extend far enough into the extracellular space to contact a similar structure extending out of the membrane of the neighboring cell. 16,29-33 Each connexon is one cell's contribution to the bipartite, cytoplasm to cytoplasm , structure which serves for cytosolic exchanges. Gap junctions are recognizable morphologically because thousands of connexons are usually clustered in large arrays, or ~ pl aqu e s ' . The functional interpretation of gap junctions as devices for intercellular communication requires that each connexon pair form an aqueous channel through which ions and small molecules can pass, without leaking into the extracellular space. Connexons are comprised of proteins of distinctive structure, called 'connexins' as a class (connexin is abbreviated Cx, followed by the molecular mass determined from cDNA, e.g. Cx43). In the following sections we present some of the data on morphological aspects of the connexons.
Visualization of the aqueous channel Visualization in fixed samples
A dimple seen on connexons in freeze cleave preparations has often been interpreted as representing the channel pore. The dimple is especially well seen in deep etched preparations.I" A central dot of a size compatible with the 1.5-2.0 nm diameter expected from permeation by tracer molecules'P and which could thus correspond to the channel pore, is also found in samples negatively stained with phosphotungstic acid or uranyl salts36 as well as with alkali metal ions .37 A priori this fulfils the expectation that materials should have access to the channel at the cytoplasmic face of the junctions.
A central structure is also seen in lanthanum permeated, sectioned material. This finding is unexpected since lanthanum salts fill the extracellular space and thus should not be able to enter a channel connected only to cytoplasm, if the channel wall is, as required by the physiological data, impermeable to aqueous solutes. An obvious way out is to argue that the permeability of the connexon has been changed by tissue preparation for microscopy. 38 The dimple associated with connexons in freeze cleaved samples is seen only on the P face of the cleaved membrane, that is on the aspect of the connexon which, in life, faced another connexon in the extracellular space. Deep etch preparations reveal not only the cleaved faces, but also the natural cytoplasmic face of membranes and junctions and should therefore also reveal the other end of the channel. In regions where the cytoplasmic face is exposed however, there is no evidence of connexons, or pores.P" Perhaps the channel is closed, or the cyto-plasmic domains of connexins are so tightly packed that the pore opening cannot be detected (see further discussion, below). Alternately there are other structures like the 'fuzzy layer' detected by Manjunath and Page39 in association with heart connexons, which might obscure the connexons and the channel mouth opening as revealed by deep etching The fuzzy layer, however, is not particularly prominent in liver junctions, which were used in the deep etch work. Visualization in unfixed samples
One approach to examination of 'native' structures is to use X-ray diffraction of packed gap junction plaques. Even though such analyses do not usually require fixation, a light treatment with glutaraldehyde has been used, without any apparent effect on the structures.t? The pioneering work of Caspar et al41 ,42 led to a gap junction model where each connexon consists of six subunits, which form a hollow cylinder. This model has been modified and elaborated on,43 notably by Unwin and collaborators. vvff The X-ray data provides experimental support for models in which the wall of the connexon is formed of protein subunits which surround a solvent filled structure, presumably the channel through which solutes can flow. An important feature of many of the models proposed is that the solvent containing channel is broad in the middle and often seems closed at the cytoplasmic surface. 43 ,44
23
Gap junction structure and assembly
Much of the X-ray data is based on an analysis ofliver derived junctions, containing principally the connexin protein Cx32 as well as some Cx26. 47,48 Comparison of the sequence data for all known connexins suggests that all should have the same general configuration (see ref 49 for discussion) with the possible exception of Cx38 in Xenopus. 50 It is therefore likely that models based on X-ray analysis of one type of junction will be applicable to all others at least in general terms.
Atomic force microscopy The atomic force microscope (AFM) provides a novel way to image biological structures, theoretically permitting one to trace out the surface contour of even unfixed biological structures at high resolution. 51-53 When imaging biological objects, however, it is as yet not possible to reach the atomic resolution which is easily achievable with non-biological materials.54 Isolated gap junctions deposited on a glass substrate reveal their cytoplasmic faces, which are found to be smooth, devoid of detectable structures'
