TISSUE & CELL 1977 9 (3) 373-394 Published by Longman Group Ltd. Printed in Great Britain

W. J. LARSEN

STRUCTURAL JUNCTIONS.

DIVERSITY A REVIEW

OF GAP

ABSTRACT. Gap junctions are plasma membrane specializations characterized as aggregates of intramembranous particles in two apposed membranes meeting particleto-particle in the 2-4 nm intermembrane ‘gap’. Recent thin-section and freeze-fracture evidence has revealed significant structural variations of gap junctional structure at various stages of development and from different organisms and tissues. It is suggested that a comparative analysis of these differences may provide clues to the specificbiological function(s) of these ubiquitous organelles.

and Karnovsky, 1967) has been implicated in the cell-to-cell conduction of action potentials in some excitable tissues (Pappas et al., 1971; Payton et al., 1969; Dewey and Barr, 1964; Bennett, 1973) and the cell-tocell transfer of small metabolites (Gilula et al., 1972; Gilula, 1974a; Socolar, 1973), nucleotides (Pitts and Simms, 1977) or small regulatory molecules (Loewenstein, 1974; Sheridan, 1973, 1977). In addition recent evidence suggests that the gap junction may be involved in the action of several hormones (Merk et al., 1972; Albertini et al., 1975; Decker, 1976a, b; Azarnia and Larsen, 1977; Larsen, 1977) and vitamin A (Prutkin, 1975; Elias and Friend, 1976). In spite of this concerted effort, however, little is known with respect to the involvement of gap junctions in any specific biological phenomenon. Recent electron microscopic studies, however, primarily those utilizing the freeze fracture technique, are beginning to provide evidence that structural differences in so called ‘gap junctions’ may reflect differences in developmental or functional activities of this structure. It is, therefore, the purpose of this essay to review many of these recent investigations and to illustrate, with a few examples, some of this structural diversity. Developmental and functional questions arising from an analysis of these differences will then be explored.

Introduction A SUBJECT of continuing interest in developmental and cell biology is the question of the relationship of cell contact interactions to the control of cellular differentiation, growth, and physiological function. Numerous studies have been undertaken to investigate the influence of cell contact on growth and cell locomotion, and the role of membraneassociated molecules in cell adhesion, cell sorting, morphogenesis, and hormone action. Other studies, carried out primarily in the last three decades, have been concerned with the apparent presence of permeable intercellular pathways in both excitable and inexcitable cells and the character and significance of junctional specializations observed with the electron microscope. The presence of specialized junctions in almost all tissues has stimulated an increasing number of studies directed to the problem of their specific functional significance, and it is not surprising that many of the hypothetical functions proposed are related to the control of cell differentiation, growth and physiological function. One of these membrane junctions commonly referred to as the ‘gap junction’ (Revel Department of Anatomy, University of Iowa College of Medicine, Iowa City, Iowa 52242. Received 26 May 1977. 25

373

314

LARSEN

Historical Background

Gap junctional profiles in thin section In stained thin sections, gap junctional

spite of the structural variability just alluded to, all of the ‘gap junctions’ discussed in this essay possess several common features distinguishing this membrane specialization from several others. Initial descriptions of the gap junction by Karrer (1960), Dewey and Barr (1962), Robertson (1963), Revel and Karnovsky (1967) and Kreutziger (1968) established morphological criteria essential in alleviating confusion about the identity of the ‘gap junction’, although, as we shall discuss below, some difficulties still exist. The methods used to reveal these relevant structural details involve the electron microscopic analysis of (1) stained thin sections; (2) unstained thin sections of lanthanum treated tissue; (3) negatively stained preparations of isolated gap junctional membrane; and (4) platinum-carbon replicas of freezefractured membranes. More recently the technique of X-ray diffraction has been utilized in investigations of gap junctional structure. We shall first briefly discuss generally accepted interpretations of the images obtained with each of these techniques and then summarize this discussion with a simple diagram of a generalized gap junction.

profiles are usually characterized by the close apposition of membranes from adjacent cells. In very thin sections of tissues stained in block with uranyl acetate prior to staining of the section with uranium and lead salts, the junctional profile may appear to be septilaminar resulting, presumably, from the close apposition of two unit membranes (Fig. la). The entire width of the junction is usually described as 15-18 nm and the space between the membranes as approximately 2-4 nm. Although the presence of seven layers is often considered essential to distinguish tight from gap junctions, it is interesting to note that many original investigators described gap junctions in several tissues as five layered structures (Fig. 1b). The ‘quintuple-layered structures’ described in vascular muscle by Karrer (1960) were probably ‘gap junctions’ since it has not been possible to discern tight junctions in vascular muscle with freeze fracture techniques. Other investigators also described pentalaminar junctions (Dewey and Barr, 1964) which it now seems in retrospect were gap junctions (see review by McNutt and Weinstein, 1973). Probable explanations for such descriptions include the overlapping of images in thick sections, insuffi-

In

a

b

Fig. I. Diagrams of thin section profiles of apparent gap junctions. (a) Classical profile with 2-4 nm ‘gap’. (b) Profile of junction fixed with permanganate (Dewey and Barr, 1964).(c) Profile with intercellular densities with periodicity of 8 nm. (d) Beaded profile produced

by adding

Hz02 to the fixative (Peracchia,

1973a).

DIVERSITY

OF GAP

JUNCTIONS

375

cient resolution of earlier instruments and fixation effects as first suggested by Dewey and Barr (1964). More recent studies describe apparent gap junctional profiles characterized by intercellular densities with an 8 nm periodicity (Fig. lc) and these images are more difficult to explain. Although this profile differs significantly from the profiles just described, it seems likely that they represent gap junctions since details observed with other ultrastructural techniques (discussed below) are similar to those observed in tissue characterized by gap junctions with typical septilaminar profiles. Although the gap junctions of crayfish septate axon appear to be basically septilaminar, these membranes have a beaded appearance when fixed in glutaraldehyde containing hydrogen peroxide (Fig. Id) (Peracchia, 1973a). Profiles C$ gap lanthanum

junctions

infiltrated

with

An investigation by Revel and Karnovsky (1967) provided the first firm criteria for distinguishing gap and tight junctions from one another. Their method of lanthanum staining revealed areas of close cell-to-cell apposition characterized by 24 nm ‘gaps’ infiltrated with lanthanum. In apparent en face sections of this structure, unstained intercellular bridges were observed interrupting the intercellular space. This technique has proven to be one of the most useful methods for unequivocally demonstrating gap junctions in a wide variety of tissues and in lieu of freeze fracture data, this method has generally been considered to be sufficient to establish the presence of gap junctions in a variety of tissues. Negative staining of isolated gap junctions

Gap junctions were first isolated and negatively stained by Benedetti and Emmelot (1968a) and later by Evans and Gurd (1972), Goodenough and Stoekenius (1972), Gilula (1974b) and Duguid and Revel (1976). This procedure reveals particles in gap junctional membranes, 68 nm in diameter with a central electron-dense pit or depression. The center-to-center particle spacing is usually on the order of 85-9 nm (Goodenough, 1975) but may depend in part on the method of isolation and further treatment by trypsin

Benedetti and Emmelot, 1968b; Goodenough, 1976). Photographic rotation techniques suggest that the particles in gap junctions of crayfish septate axons are composed of six subunits (Peracchia, 1973b), a result also obtained with X-ray diffraction techniques (see this section below) for mouse liver gap junctions (Goodenough, 1975). Freeze fracture of gap jmctions

One of the most useful techniques for the study of gap junctions is freeze fracture. The basis of this procedure involves the fracturing of membrane through the hydrophobic region of the lipid bilayer at low temperature, in a vacuum, and the replication of exposed fracture faces by sputtering a thin layer (approximately 2 nm) of platinum onto it at a 45” angle.The details of this procedure and interpretations of images produced with this technique have been described in several recent reviews (McNutt and Weinstein, 1973; Branton et al., 1975; Weinstein et al., 1976). On the basis of the freeze fracture and freeze etch studies of Chalcroft and Bullivant (1970), Goodenough and Gilula (1974) and Peracchia (1973b), it now seems clear that gap junctions are composed of aggregates of intramembranous particles in apposed membranes, paired particle to particle across the intercellular space. Many original studies suggested that gap junctional particles were homogeneous with respect to size but exceptions have been noted and some will be discussed below. The initial freeze fracture description of gap junctions by Kreutziger (1968) has been followed with hundreds of studies reporting the occurrence of similar structures in a variety of tissues. X-ray diffraction junctions

studies

of

isolated

gap

Recent X-ray diffraction studies of isolated mouse liver gap junctions also suggest that the gap junctional particles span the membrane (Goodenough, 1975). The approximate center-to-center spacing of particles in these preparations is about 8.6 nm and particles are hexagonally packed with a lattice constant of 8.2-8.8 nm. These studies also suggest that gap junctional particles are composed of six subunits with a central aqueous pore.

316 The stereotypical

LARSEN

gap junction

The generalized image of the gap junction synthesized from these original studies is based upon the following generalities: (I) The gap junction is a bi-membranous organelle; (2) particle aggregates in each membrane generally occur as irregular plaques; (3) the junctional particles are hexagonally or (more generally) polygonally packed; and (4) meet particle-toparticle in the intercellular space. (5) These particles are generally homogeneous in size. A simplified three-dimensional representation of this structure is illustrated in Fig. 2. Recent studies have suggested, however, that ‘gap junctions’ from a variety of tissues vary significantly in many structural details. Gap junctions, for example, may not always be formed by membrane from two cells of the same type, but may arise through the interaction of membranes from two different kinds of cells. Alternatively, gap junctions, may not necessarily depend on their formation through the interaction of membranes from two separate cells, but may form at points of contact between processes of the same cell. Apparent gap junctional vesicles may also be found isolated within the cytoplasm of some cells, representing yet another topological variation. Significant variations in the shapes of gap junctions from different cell types have now been described. Particles may not always be packed in a simple hexagonal or polygonal lattice and the centerto-center spacing of particles within the same junction may vary widely. In addition, intramembranous particles within or surrounding some gap junctions may differ in size. Gap junctional particles may not always occur on the same fracture face in freeze fracture replicas. The size of gap junctions may vary considerably from one cell to another and gap junctions from cells of different types may be intimately associated with different cytoplasmic and plasma membrane organelles. Since we are now limited by semantic considerations and by want of a more flexible term, we will apply the notation ‘gap junction’ to all of the structures we shall consider in this essay. The need for qualifying adjectives peculiar to specific variants will be obvious as our discussion continues.

ir

lb’ Fig. 2 A three-dimensional diagram of part of a typical gap junction. The ‘gap’ is continuous with extracellular snace and is usually 24 nm wide. The total thickness of the junction is generally considered to be 15-18 nm but it is possible that part of this may be contributed by cytoplasmic dense material (cdm) (Larsen, 1975). In this diagram the intramembranous particles (imp) are shown in section but an en face view would reveal hexagonal or polygonal packing, lb, lipid bilayer; its, intercellular space.

Variants of ‘Typical Gap Junctional Structure’ The source of membranes contributing to the formation of ‘gap junctions’ and their topology

It is probably safe to say that the majority of gap junctions reported in the literature are junctions resulting from the interaction of the plasma membrane from two different cells of the same cell type. Such structures have been reported to occur throughout the animal kingdom in organisms as primitive

DIVERSITY

OF GAP

JUNCTIONS

as the Coelenterates and in almost all tissues except adult striated muscle. In general, it seems likely that all cells within any given tissue mass or organ are connected to each other by gap junctions. Gap junctions may also form, however, through the interaction of the plasma membranes of cells of two ‘different types’. Johnson et al. (1973) have demonstrated gap junctions in Limulus in stained thin sections between epithelial and reserve cells of the midgut. They have referred to these structures as heterocellular gap junctions in contrast to the homocellular gap junctions formed between cells of the same type. Heterocellular gap junctions have also been reported between basal and principal cells of an insect rectum (Noirot and NoirotTimothee, 1976), non-pigmented and pigmented cells of the rabbit ciliary epithelium (Kogon and Pappas, 1975), embryonic pigmented and neural retina of the human (Fisher and Linberg, 1975), and between granulosa cells and ovum in developing rabbit follicles (Anderson and Albertini, 1976). In addition, they have been reported to occur between supporting and hair cells of the organ of Corti in a lizard ear (Nadol et al., 1976). Gap junctions may also occur between fibroblasts and cardiac muscle cells, but clear evidence has not yet been presented (Goshima, 1970; Hyde et al., 1969). Although it has been reported that gap junctions may form between vascular muscle and accompanying fibroblasts, no unequivocal evidence has been published. Finally it has been suggested that gap junctions may form between cells of different types in tissue culture, but it should be mentioned that no ultrastructural evidence was presented in the original study by Michalke and Loewenstein (1971). In the last few years a curious structure now referred to as the ‘reflexive gap junction’ (Herr, 1976) has been reported to occur in a number of tissues. This gap junction is formed through the interaction of membrane arising from the same cell and it now seems clear that several cells are capable of making gap junctions with themselves. These structures have been reported in at least seven different tissues including arterial smooth muscle (Iwayama, 1971), mesangial and lacis cells of the kidney (Pricam et al., 1974), human ovarian decidual cells (Herr, 1976),

377

luteal cells (Albertini, 1976), Leydig cells (Fig. 3) (Connell and Connell, 1977), and salivary gland cells of the adult blowfly (Oschman, 1976). Freeze fracture of reflexive gap junctions in human ovarian decidual cells reveal features common to intercellular gap junctions (Figs 4, 5). One additional topological variant of the gap junction has now been reported to occur in a large number of tissues and has been referred to as an ‘annular gap junction’ (Merk et al., 1972) or as spherae occlusae (Espey and Stutts, 1972). These structures have been termed ‘annular’ because of their appearance in thin section (Figs. 6, 7). Evidence from serial sectioning studies (Espey and Stutts, 1972; Merk et al., 1972) and recent freeze fracture evidence (Figs. 8, 9) (Wille and Larsen, 1977) suggest that some of these annular profiles represent transections of gap junctional spheres completely isolated from the surface plasma membrane. This interpretation is also supported by lanthanum tracer studies providing indirect evidence that not all such profiles are continuous with the extracellular space (Garant, 1972; Merk et al., 1972). More complex interiorized gap junctional profiles have been reported to occur in several tissues involving the apparent enclosure of gap junctional spheres within others (Fig. 10). Profiles of apparent cytoplasmic gap junctional vesicles have been observed in numerous tissues listed in Table 1. The size of individual gap junctional particle aggregates

In freeze fracture preparations, gap junctions usually appear as irregular aggregates of particles on the P-fracture face and pits on the E-fracture face. These aggregates may vary from 20-30 nm to several micra in diameter (Figs. 1l-l 3). Typical particle aggregates tentatively identified as gap junctions may contain only a few particles (Figs. 11, 12) or many thousand particles (Fig. 13). Some junctions may cover areas of 20-30 pm2 (Wille and Larsen, unpublished observation) but such large junctions appear to be exceptional. Gap junctions of several epithelia, however, are considered to be extensive and a few of the best documented examples are listed in Table 2. In contrast to this, gap junctions appear to

378

LARSEN

Fig. 3. Reflexive gap junction from canine testicular interstitial cell. Note cytoplasmic continuity between both sides of the junction (open arrows). This reflexive junction also has a circular junctional profile associated with it. Lanthanum has infiltrated the intercellular ‘gap’. x 124,000 (from Connell and Connell, 1977). Fig. 4. Cross-fracture of a human ovarian decidual cell. Open arrow denotes peduncular processes joined by reflexive gap junction. x 8600. Fig. 5. High magnification of freeze-fractured reflexive gap junction designated in Fig. 13. Note typical E-fracture face pits, and P-fracture face particles x 121,ooO.

DIVERSITY

OF GAP

JUNCTIONS

379

Fig. 6. Profiles of typical intercellular gap junction (closed arrow) and ‘annular’ gap junction (open arrow) in rabbit granulosa cell. x 74,000. Fig. 7. ‘Annular’ gap junction ribosomes. x 100,000.

from rabbit

granulosa

cell containing

swollen

Fig. 8. Freeze-fracture replica of cytoplasmic gap junctional vesicle. Pits occur on the E-fracture face and particles arc found on the P-fracture face. x 32,000. Fig. 9. Two-gap junctional arrows). x 61,000.

spheres in cytoplasm of a rabbit granulosa cell (open

LARSEN

380

Table 1. Tissues possessing annular gap junctions Reference

Cell type Granulosa

Luteal Leydig Embryonic pigment and neural retina h#anocytes in skin Wool follicles Estrogen dependent adenocarcinoma of proximal convoluted tubule Ameloblast Vitamin A-treated skin tumor Liver Cardiac muscle Ependyma Osteoblasts, preosteoblasts Adenocarcinoma of adrenal cortex

Espey and Stutts (1972), Merk et al. (1972, 1973) Enders (1973), Albertini and Andersen (1974a), Zamboni (1974), Albertini et al. (1975), Wille and Larsen (1977) Albertini and Andersen (1974b) Connell and Christensen (1975) Fisher and Linberg (1975) Larsen, unpublished observation Orwin et al. (1973) Letourneau et al. (1975) Garant (1972) Prutkin (1975) Perissel et al. (1976) Dewey and Barr (1974) Brightman (1965) Marquart (1977) Leibovitz et al. (1973)

be completely absent from adult striated muscle although they have been reported to occur in myoblasts during muscle development (Rash and Staehelin, 1974; Keeter etal., 1975). An exhaustive quantitative study failed to reveal the presence of gap junctions in a tumorgenic derivative of a mouse L cell (Azarnia et al., 1974; Larsen et al., 1977). Other cancer cells have been reported to lack gap junctions or to possess them in relatively low frequency (Weinstein et al., 1976; Azarnia and Larsen, 1977).

Gap junctions of diverse shape

Variation in gap junctional shape may also be extreme. Although many gap junctional particle aggregates are basically circular or elliptical (Fig. 14), others may be linear (Fig. 15), branched (Fig. 16) or shaped as irregular plaques with particle-free spaces (Fig. 17) or as irregular solid plaques (Fig. 18). Some gap junctions may occur as plaques with linear tails (Fig. 19) or rarely as regular hexagons (Figs. 20, 21). Gap junctions in human ovarian decidual cells may be calyculate (Herr, 1976) and in some special cases as discussed above, may occur as nearly perfect spheres within the cytoplasm (Fig. 8, 9). Gap junctional particle size

Fig. 10. Diagram of complex annular gap junctional profiles observed in a variety of cells (a) One ann~ar profile is surrounded by another (b) Three con&ntric annular protiles (c) Two annular profiles may be enclosed side-by-side within a third.

Although a distinguishing characteristic of most gap junctions is the apparent uniformity of particle shape and size, significant variations of size may occur. Goodenough and Gilula (1974) in a freeze fracture study,

Table 2 Occurrence of ‘extensive’ gap ,junctions Cell type _ Granulosa, luteal, Leydig Adrenal cortex, liver, pancreas Ameloblasts Pigmented ciliary epithelium Embryonic pigmented and neural retina -~~~ -__

References As in Table 1 Friend and Gilula (I 972) Garant (1972) Kogon and Pappas ( 1975) Fisher and Linberg (1975)

FIN_ I I. Free/e fracture replica of prohahle gap Junctional particle chlcken cardiac muccle cell in tww culture (open arrow). Close packing cught particlcc m an area of membrane contnct supports the possibihty gap,LlnclIoIl x 125,000. Fig. 12. The tional aggregates the extracellular

13.

aggregate in of seven 10 that this is a

area of rabbit granulosa cell membrane contains m~merwb gap junccontaining approximately S-6 to 100-200 part&a. A small piece of leatlet may remazn adherent to some particle aggregates. x 50,000.

Fig. Freeze fracture replica of relatively large gap junction of a granulosa from a mature follicle of a rabbit. Both E- and P-fracture faces are evident. juwtlon is approxlmatrly 3 ,~rn in d~amrtrr. x 36,000.

ccl1 This

LARSEN

tions. A review of recently published micrographs indicates that the primary relationship of gap junctional particles may frequently be hexagonal. Secondary patterns involving diverse arrangements of small units of particle clusters, however, often impose an additional degree of complexity. In addition, even if no secondary pattern exists, centerto-center distances and lattice constants may differ significantly from one junction to the next (Figs. 22,23) or may even vary dramatically within the same junction (Figs. 24, 25). Particle density may also be affected by various exogenous agents. Particle packing density is affected by the infiltration of tissue by solutions containing toxic agents, hypertonic sucrose, or Ca2 -, Mg’ ’ free EDTA (Peracchia and Dulhunty, 1976; Peracchia, 1977). Similar effects on particle packing density are observed when isolated junctions are treated with trypsin (Benedetti and Emmelot, 1968b; Goodenough, 1976). In some junctions small clusters of tightly packed particles appear to be secondarily organized into relatively closed (Figs. 26, 27) or open patterns (Figs. 28-30). One of the most distinctive variations is observed in the ovarian granulosa (Fig. 30) and luteal cell (Albertini and Andersen, 1974a, b; Wille and Larsen, 1977). In these junctions,

described liver gap junctions composed mainly of 7.7 nm particles but also demonstrated a peripheral string of particles 9% 12 nm in diameter. Johnson et al. (1974), Decker and Friend (1974), Decker (1976a) and Albertini and Andersen (1974b) observed larger particles loosely clustered in the vicinity of developing gap junctional particle aggregates (note Fig. 19). The shape of gap junctional particles is difficult to accurately determine with the freeze fracture technique but has been interpreted as ovoid or cylindrical on the basis of negative staining (Peracchia, 1973b) and X-ray diffraction data (Goodenough, 1975). It is conceivable that rotary shadowing procedures (Margaritis et al., 1977) would improve freeze fracture images sufficiently to ascertain additional details of particle substructure. Particle packing patterns

The gap junctional particle packing patterns most often reported are hexagonal or polygonal (Robertson, 1963 ; Revel and Karnovsky, 1967; Pinto da Silva and Gilula, 1972; Hand and Gobel, 1972; McNutt and Weinstein, 1973 ; Gilula, 1974) but it is now clear that these descriptions are often inadequate when applied to numerous gap junc-

Fig. 14. Nearly circular aggregate of gap junctional of third ventricle of the rat brain. x 70,000.

particles

Fig. cardiac

in cultured

15. Linear strings of gap junctional muscle cells. x 124,000.

Fig. 16. Branched cells. x 112,000.

aggregate

particles

of gap junctional

particles

in ciliated ependyma embryonic

in chick

cardiac

chick muscle

Fig. 17. Plaque of gap junctional particles with large particle free areas (open arrow) from cardiac muscle of neonatal rat. x 112,000. Fig. 18. Irregular gap junctional decidual cells. x 68,000.

particle aggregates

on membrane

of human ovarian

Fig. 19. Large gap junctional plaque with linear tail in rabbit ovarian granulosa cell. Note large particles in proximity of aggregate (open arrow). x 60,000. Fig. 20. Hexagonal x 175,000.

gap junction

from tumor of Murine Cloudman

S-91 melanoma.

Fig. 21. Nearly hexagonal gap junction from rabbit ovarian granulosa cell completely surrounded by tight junctional groove on E-fracture face (solid arrow). x 89,000.

LAKSEN

groups of approximately 20-60 tightly packed particles may be separated from each other by an interconnecting system of particlefree aisles such that no particle within the junction is more than two or three particle diameters distant from particle-free membrane. Other variations in particle packing patterns include the linear arrangements already discussed (Figs. 15, 16) and the packing of particles into columns observed in ciliary epithelium (Kogon and Pappas, 1975) and in human ovarian decidual cells (Herr, 1976) after lanthanum staining. Apparent gap junctional particles may be organized in similar arrangements in human Lesch-Nyhan cells in culture (Larsen et al., 1977). Polurity 0J’ gap junctions in fLeeze ,fi-acture replicas

As noted above, freeze fracturing generally splits gap junctions in such a way that particles remain adherent to the outer fracture face of the inner leaflet (P-fracture face) and pits occur on the inner aspect of the

Fig. 22. Hexagonally melanoma. x 385,000.

outer membrane leaflet (E-fracture face). Such polarity appears universal among all vertebrate gap junctions (Fig. 31). Freeze fracture replicas of gap junctions of the Arthropods, however, appear reversed with respect to the distribution of particles and pits on the P and E fracture face (Flower, 1972; Gilula, I974a). A recent freeze fracture study of the annelid intestine (Larsen, unpublished observations) has also revealed junctions with reversed polarity (Fig. 32) in freeze fracture

replicas.

The association qf’ ‘gap ,jtrnctions’ with cytoplasmic structures and intramenhrunous specializations Cytoplasmic organelles. Gap junctions may be intimately associated with cytoplasmic membranes including those of small vesicles (Peracchia, l973a), rough endoplasmic reticulum (RER) (Nunez, 1971; May, 1977) and smooth endoplasmic reticulum (SER) (Albertini and Andersen, 1974b; Connell and Connell, 1977; Fawcett, 1977). These relationships have been documented with thin

packed gap junctional particles in gap junction from Cloudman

Fig. 23. Loosely packed particles granulosa cells. x I2 1,000.

occur in some very large gap junctions

of rabbit

Fig. 24. Some areas of this granulosa cell gap junction are tightly packed in hexagonal array (solid arrow) while other areas are more loosely organized (open arrow). x 170,000. Fig. 25. Both open (open arrow) and tight (solid arrows) terize this embryonic chick cardiac muscle cell. x 135,000.

particle

packing

charac-

Fig. 26. Particles in this granulosa cell gap junction are packed tightly units of a few particles. These units are in turn closely packed. x 184,000.

into small

Fig. 27. Junctions particles. x 91,000.

groups of

from mouse adrenal

cortex are composed

of indistinct

Fig. 28. This rabbit adrenal cortical gap junction is characterized by small groups of particles separated by small particle-free areas (open arrow). x 148,000. Fig. 29. The E-fracture face of a gap junction from an astrocyte reveals small patches of pits (solid arrow) interspersed with smooth aisles (open arrow). x 113,000. Fig. 30. Many large granulosa cell gap junctions are characterized by small particle islands or columns (solid arrows) separated by particle-free aisles (open arrow). x I25.000.

._

LARSEN

Fig. 31. This micrograph reveals polarity of typical freeze fractured vertebrate gap junction. Note relationship of P and E face. x 72,000. Fig. 32. The polarity of a gap junction from the intestine of Lumbricus terrestris is the same as that seen in some Arthropod gap junctions and inverted with respect to the vertebrate gap junction pictured in Fig. 3 I. x 97.000.

Fig. 33. Apparent gap junctional aggregate (open arrow) and tight junctional (solid arrow) surrounded by particles in rabbit granulosa cell. x 105,00(1.

fibril

Fig. 34. Endothelial cell tight junctional fibrils (solid arrow) interspersed with small particle aggregates (open arrow). x 92,000. Fig. 35. Particle aggregate and tight junctional chick endothelial cell. x 127,000.

fibrils in close association in cultured

Fig. 36. Particle aggregate partially surrounded by tight junctional chick endothelial cell. x 95,000.

fibrils in cultured

Fig. 37. Particle aggregates in continuity with lmear particle assemblies and apparent tight junctional fibrils in cultured chick endothelial cells (solid arrows). A line of particles at upper right merges into short lengths of apparent tight junctional fibrils (open arrow). x 86,000. Fig. 38. A gap junction in close proximity to a desmosome in the membrane of a chick cardiac muscle cell. GJ, gap junctional particle; DES, desmosome. x 133,000. Fig. 39. Small orthogonal assemblies may occur in close proximity to gap junctions (solid arrow) or separated from them (open arrow) in membrane of ependyma of the third ventricle of the rat brain. x 74,000.

LARSEN

388

section and freeze-fracture electron microscopy. It has been suggested that the interaction of gap junctions in crayfish septate axon with small vesicles may involve actual contact through elements associated with both membranes (Peracchia, 1973a). The association of RER with the gap junctions of bat thyroid (Nunez, 1971) and of neuroblasts (May, 1977) is so intimate that ribosomes are absent from that face of the RER membrane interacting with the gap junction cytoplasmic face. The interaction of SER with the cytoplasmic aspect of gap junctions in granulosa cells (Albertini and Andersen, 1974a) and smooth muscle (Fry et al., 1977) is sufficiently close so that during freeze fracturing, the fracture plane may skip back and forth between SER and gap junctional membrane. In some cases, mitochondria have also been found preferentially localized in the vicinity of gap junctions although the intimacy of this association does not appear to approach that of the examples discussed above (Nunez, 1971). Ribosomes. Ribosomes are attracted to the gap junction of mouse liver in tissue removed from the animal several hours before fixation (David-Ferreira and David-Ferreira, 1973). This response has been attributed to the autolytic process following such treatment, but preferential association of ribosomes with gap junctions may also be observed in normal granulosa cells (Merk ef al., 1972, 1973) and normal ameloblasts (Garant, 1972). Ribosomes also seem to be concentrated within apparent gap junctional vesicles in these cells (Fig. 7). It should be mentioned, however, that cytoplasmic gap junctional vesicles may contain other cytoplasmic organelles as well (Merk et a/., 1973 ; Garant, 1972). Opaque deposits. Electron-dense deposits are found closely adherent to the cytoplasmic faces of gap junction membrane when A/B,,-5 cells (Larsen, 1975). Leydig cells (Connell and Connell, 1977) granulosa cells and Lesch-Nyhan cells (Larsen, unpublished results) are fixed in calcium-containing glutaraldehyde at room temperature. Intramembranous structures. Freeze fracture

studies have demonstrated

the close associa-

tion of gap junctional particles with elements of qualitatively different intramembranous structures. In freeze fractured membranes of a wide variety of vertebrate epithelial cells, apparent gap junctional particles often appear in association with tight junctional fibrils (Figs. 33-36). Occasionally, apparent gap junctional particles are found in close association with isolated fibrils (Figs. 33-35). In other cases, particles may appear to be entrapped with an anastomosing network of fibrils (Figs. 36, 21). Since it appears, however, that tight junctional fibrils may be assembled from particulate precursors (Montesano et al., 1975) similar in shape and size to gap junctional particles, it would seem prudent to take some care in the identification of particles associated with tight junctional fibrils. One such example of particles of ambiguous identity is illustrated in Fig. 37. Gap junctional particles may also occur in close association with desmosomal elements in a number of tissues including cardiac muscle (Fig. 38). They may also be found in close association with small rectilinear aggregates of particles in ciliated ependyma of the third ventricle of the rat brain (Fig. 39). Similar rectilinear aggregates have been described in astrocyte membranes (Landis and Reese, 1974) and in mouse ependymal cells (Brightman et al.. 1974).

Implications of Structural Diversity of ‘Gap Junctions’ Terminology

It seems apparent that many membranous specializations assumed to be ‘gap junctions’ vary significantly with respect to several structural details. Although the application of the term ‘gap junction’ to structures resembling more stereotypical forms seems to be generally accepted, relative uncertainty surrounds many other structures. How are so-called ‘reflexive’ gap junctions related to more common forms? Why do gap junctions of embryonic chick heart occur as linear strings of particles? What is the significance of apparent ‘spherical gap junctions’ found deep within the cytoplasm of some cells? Features common to all of these junctions include the interaction of paired particles across a 2-4 nm inter-

DIVERSITY

OF GAP

JUNCTIONS

membrane gap which is, or was, continuous with extracellular space. With respect to almost all other structural details, however, it seems clear that the class of organelles generally termed gap junctions may vary in nearly every way possible. A review of the recent literature suggests that some of this variability may be related to developmental processes involving the formation, growth, and degradation of gap junctions. It also seems possible that certain structural differences may be understood on the basis of phylogenetic considerations and that others may be physiologically significant. Development and turnover of gap junctions

It has been argued by several investigators (Johnson et al., 1974; Decker and Friend, 1975; Decker, 1976; Griepp and Revel, 1977) that the formation of gap junctions first involves the close apposition of adjacent membranes, and that these areas are characterized in freeze fracture replicas as particlefree ‘formation plaques’ which later contain small aggregates of gap junctional particles. These studies have carefully documented many stages of this process. It seems reasonable to suggest that the intial step in the formation of gap junctions involves an interaction of cell surface components which bring the apposed membranes into close range prior to the insertion of gap junctional particles into the membrane. The initial form taken by insipient gap junctional aggregates are small compact particle groups, strings of particles, or hybrids of these two forms (Johnson et al., 1974; Decker and Friend, 1975). In some cases it has been suggested that tight junctional fibrils may act as foci for the early development of gap junctions (Fletcher and Robertson, 1975; Decker and Friend, 1976) and there is no doubt that the association of short lengths of tight junctional fibrils with particles similar in size and shape to those in gap junctions does occur. In many cells, however, where tight junctional fibrils have not been documented, it seems likely that gap junctional formation may be initiated in the absence of such accessory structures. The growth of gap junctions may be characterized by several structural modifications in addition to a progressive increase in 26

389

size. In many tissues, the shape of the junction may become more regular as it enlarges, and this phenomenon has been described in Novikoff hepatoma cells in culture (Johnson et al., 1974) granulosa cells (Albertini and Andersen, 1974a; Wille and Larsen, 1977) in cardiac muscle cells of the embryonic mouse (Gros and Challice, 1976) and in a fibroblast in tissue culture (Larsen, 1977). Although it has been shown that hypertonic sucrose, detergents, trypsin and certain toxic agents may have dramatic effects on the particle packing density and particle packing patterns of some junctions (Peracchia and Dulhunty, 1976; Peracchia, 1977; Benedetti and Emmelot, 1968b; Goodenough and Gilula, 1974; Goodenough, 1976), it seems that some changes may be related to the development of the junction itself. The junctions of a fibroblast cell line in tissue culture exhibit alterations of particle packing configurations related to size and the number of days after seeding (Larsen, 1977). Granulosa cell gap junctions undergo striking changes in particle packing patterns during gap junctional growth and maturation (Wille and Larsen, 1977). Although the mechanism of deposit formation on gap junctional membranes is not yet clear, there appear to be differences in the size and distribution of deposits on the gap junctions of a fibroblast cell line in tissue culture related to length of the gap junctional profile. The deposits on small junctions are large and distributed asymmetrically while those on larger junctions are relatively small and usually precisely paired across the two junctional membranes (Larsen, 1975). Is it possible that these deposit distributions are related to the differences in particle packing patterns observed in immature and mature junctions? Variability related to phylogeny

Reasons for the difference of polarity of freeze fractured gap junctions of Arthropods and those of some other phyla are not understood. It is not known whether the association of particles with the E rather than the P face is related to properties intrinsic to the junction itself or to the plasma membrane in general. Since it seems likely that the Annelids are derived evolutionarily from an ancestor common to the Arthropods (Meg-

LARSEN

litsch, 1967) it is interesting that the polarity of junctions in Lumbricus is the same as in several Arthropod species. Molluscan freezefractured gap junctions, however, are of the same polarity as those described in the vertebrates (Gilula and Satir, 1971). These considerations bring us to the general question of the evolutionary relationships of gap junctions from diverse phyla and tissues. It may be worthwhile to ask whether or not ‘gap junctions’ spanning the phylogenetic spectrum are homologous or simply analogous structures. Has nature revealed its usual conservativism by remodeling and altering a basic and simple design or do the documented similarities in structure we have described reflect a parallel or convergent evolution of organelles arising from uncommon ancestors? Function and structure

Although there is no direct evidence implicating any ‘gap junction’ in any specific cellular function, it seems possible that recently observed structural and behavioral characteristics of several gap junctions may specify further our investigations of this question. It was first noted by Friend and Gilula (1972) who surveyed a number of epithelia that certain glandular tissues possessed relatively extensive gap junctions. These tissues were liver, adrenal cortex, and pancreas. As noted in Table 2, subsequent reports document the presence of extensive gap junctions in a variety of other secretory cells. Furthermore, interiorized gap junctional vesicles also seem to occur predominantly in secretory epithelial cells (Larsen, 1977) and are particularly frequent in these cells during that developmental stage characterized by the most active secretion (Garant, 1972; Larsen, 1977). In addition, the induction of mucous synthesis and secretion in a keratoacanthoma of the rabbit ear (Prutkin, 1975) and in normal chick shank epithelium in organ culture (Elias and Friend, 1976) is accompanied by the formation of interiorized gap junctional vesicles or extensive intercellular gap junctions. Finally, the reflexive gap junctions discussed previously have most often been observed in active secretory cells. Thus it appears that the most

atypical gap junctional structures seem to occur predominantly in secretory epithelia. Exceptions to these generalizations, however, should also be mentioned. Secretory cells are not the only cells possessing reflexive gap junctions. These structures also occur in at least three smooth muscle cell types. Apparent annular gap junctions have also been reported to occur in cardiac muscle cells (Dewey and Barr, 1964). One additional characteristic shared by some secretory and one smooth muscle cell gap junction is their close association with elements of smooth endoplasmic reticulum as noted previously. Thus it appears that gap junctions in certain inexcitable cells may share several uncommon features with gap junctions in some excitable tissues. Since many similarities in the details of control of secretory and contractile processes are known to exist (Douglas, 1968), it is tempting to speculate that these gap junctions may serve in some regulatory function commonly required by these two tissue types. No direct evidence is available to support this hypothesis but these observations provide a basis for further pursuit of this idea. Hormone action

It has also been suggested that the gap junctions in some cells may be involved either directly or indirectly in hormone action (Albertini et al., 1975; Larsen, 1977) and the influence of peptide hormones (Bjersing and Cajander, 1974; Decker, 1976b; Larsen, 1977), estrogen (Merk et al., 1972; Fletcher and Robertson, 1975), vitamin A (Prutkin, 1975; Elias and Friend, 1976) and thyroxin (Decker, 1976a) on the growth and turnover of gap junctions have been recently documented. Most of these studies have been discussed in a recent review (Larsen, 1977). Growth control

The possible role of gap junctions in growth control has also been discussed in several recent reviews (Loewenstein, 1974; Weinstein et al., 1976; Sheridan and Johnson, 1975; Gilula, 1975; Azarnia and Larsen, 1976). Briefly, it has been suggested by some investigators that division rates of some tumor cells may be high because of some defect in the ability of these cells to form appropriate intercellular junctions. Models involving gap junctions in growth control

DIVERSITY

OF GAP

JUNCTIONS

require them to serve as cell-to-cell conduits for regulatory molecules (Loewenstein, 1974; Sheridan, 1976) or as structures involved directly or indirectly in the regulation of hormone action (Albertini et al., 1975; Larsen, 1977).

fall far short of clarifying adequately the functional significance of these cell junctions. It is hoped, however, that these clues may lead us to the consideration of new questions and predictions of increasing specificity. Acknowledgements

Summary and Conclusions

It has been suggested in this essay, that plasma membrane organelles generally referred to as gap junctions may vary significantly in many structural details. Even though such diversity exists, it still seems appropriate, however, to refer to these membrane specializations as ‘gap junctions’ since this term denotes one feature clearly shared by all of these structures. It seems possible that many of the structural differences reported here are related to the life history of particular gap junctions, while other variable characteristics may have phylogenetic or physiological relevance. Nevertheless, these observations

I would like to thank Dr Kent Hermsmeyer and John Herr for providing cardiac muscle and ovarian decidual cells respectively. I am also grateful to Dr Pushpa Deshmukh and Dr Ian Philips for ependymal cells; Clare Wille for assistance in studies on granulosa cells and for Fig. 30; and Dr Carolyn Connell and Academic Press for the use of Fig. 3. I would also like to thank Susan O’Donnell for her expert technical assistance and Thomascyne Buckley and Paul Reimann for photographic assistance. This study was supported by grants from the American Cancer Society and the University of Iowa College of Medicine.

References ALBERTINI,D. F. 1976. Personal communication. ALBERTINI, D. F. and ANDERSEN, E. 1974a. Structural modifications of lutein cell gap junctions during pregnancy in the rat and mouse. Anat. Rec. 181, 171. ALBERTINI,D. F. and ANDERSEN,E. 1974b. The appearance and structure of intercellular connections during the ontogeny of the rabbit ovarian follicle with particular reference to gap junctions. J. Cell. Biol., 63, 234. ALBERTINI, D. F., FAWCETT, D. W. and OLDS, P. J. 1975. Morphological variations in gap junctions of ovarian granulosa cells. Tissue & Cell, 7, 389. ANDERSON,E. and ALBERTINI,D. F. 1976. Gap junctions between the oocyte and companion follicle cells in the mammalian ovary. J. CeN Biol., 71, 680. AZARNIA, R. and LARSEN,W. J. 1977. Intercellular communication and cancer. In Intercellular Communication (ed. W. C. DeMello). Plenum, New York. AZARNIA, R., LARSEN,W. J. and LOEWENSTEIN, W. R. 1974. The membrane junctions in communicating and non-communicating cells, their hybrids, and segregants. Proc. natn. Acad. Sci. U.S.A., 71, 880. BENEDETTI,E. L. and EMMELOT,P. 1968a. Hexagonal array of subunits in tight junctions separated from isolated rat liver plasma membranes, J. Cell Biol., 38, 15.

392

LARSEN

BENEDETTI,E. L. and EMMELOT,P. 1968b. Structure and function of plasma membranes isolated from liver. In The Membranes (eds. A. J. Dalton and F. Haguenau), p. 33. Academic Press, New York. BENNETT,M. V. L. 1973. Function of electrotonic junctions in embryonic and adult tissues. Fedn froc., 32,65. BIERSING, L. and CAJANDER,S. 1974. Ovulation and the mechanism of follicle rupture. IV. Ultrastructure of membrana granulosa of rabbit Graafian follicles prior to induced ovulation. Cc/I Tiss. Res., 153, 1. BRANTON,D., BULLIVANT,S., GILULA, N. B., KARNOVSKY,M. J., MOOR, H., M~HLETHALER,K., NORTHCOTE, D. H., PACKER, L., SATIR, B., SATIR, P., SPETH, V., STAEHELIN,L. A., STEERE, R.. and WEINSTEIN. R. 1975. Freeze-etching nomenclature. Science, 190, 54. BRIGHTMAN, M. W. 1965. The distribution within the brain of ferritin injected into cerebrospinal fluid compartments. J. Cell Biol., 26, 99. BRIGHTMAN,M. W., PRESCOTT,L. and REESE,T. 1974. Intercellular junctions of special ependyma. In Brainendocrine Interaction. II. The Ventricular System. 2nd Int. Symp., Shizuoka, p. 146. Karger, Base]. 1975. CHALCROFT, J. P. and BULLIVANT, S. 1970. An interpretation of liver cell membrane and junction structure based on observation of freeze-fracture replicas of both sides of the structure. J. Cell Biol., 47, 49. CONNELL, C. J. and CHRISTENSEN,A. K. 1975. The ultrastructure of the canine testicular interstitial tissue. Biol. Reprod., 12, 368. CONNPLL, C. J. and CONNELL, G. M. 1977. The interstitial tissue of the testis. In The Testis, IV (eds. A. D. Johnson and W. R. Games), p. 333. Academic Press, New York. DAVID-FERREIRA,J. F. and DAVID-FERREIRA,K. L. 1973. Gap junction: ribosome association after autolysis. J. Cell Biol., 58, 226. DECKER, R. S. 1976a. Hormonal regulation of gap junction differentiation. J. CeN Biol., 69, 669. DECKER, R. S. 1976b. Adrenocorticotropic hormone (ACTH)-induced formation of gap junctions between differentiating Y-l tumor cells in vitro. J. Cell Biol., 70, 412a (Abstr.). DECKER, R. S. and FRIEND, D. S. 1974. Assembly of gap junctions during amphibian neurulation. J. Cell Biol., 62, 32. DEWEY, M. M. and BARR, L. 1962. Intracellular connections between smooth muscle cells: the nexus. .S&ncp 137, 670. DEWEY, M. M. and BARR, L. 1964. A study of the structure and distribution of the nexus, J. Cell Biol., 23. 553. DOUGLAS, W. W. 1968. Stimulus-secretion coupling: the concept and clues from chromaffin and other cells. Br. J. Pharmar., 34, 451. DUGUID, J. and REVEL, J. P. 1976. The protein component of the gap junction. Cold Spring Harb. .Symp. quant. Biol., 40, 45. ELIAS, P. and FRIEND, D. S. 1976. Vitamin A-induced mucous metaplasia: an in virro system for modulating tight and gap junction differentiation. J. Cell Biol., 68, 173. ESPEY, L. L. and STUTTS, R. H. 1972. Exchange of cytoplasm between cells of the membrana granulosa in rabbit ovarian follicles. Biol. Reprod., 6, 168. EVANS, W. H. and GURD, J. W. 1972. Preparation and properties of nexuses and lipid enriched vesicles from mouse liver plasma membrane. Biochem. J. 128, 691. FAWCETT,D. W. 1977. See CONNELL, C. J. and CONNELL,G. M. 1977. FISHER, S. K. and LINBERG, K. A. 1975. Intercellular junctions in the early human embryonic retina. J. Ulrrastruct. Res., 51, 69. FLETCHER, W. H. and ROBERTSON,J. D. 1975. Assembly of an ‘enclosed gap junction‘ by granulosa cells in the developing ovarian follicles of sexually immature rats. J. CeN Biol., 67, I 16a (Abstr.). FLOWER, N. E. 1972. A new junctional structure in the epithelia of insects of the order Dictyoptera. J. Cc/l Sci., 10, 683. FRIEND, D. S. and GILULA, N. B. 1972. Variations in tight and gap junctions in mammalian tissues. 1. Cell Biol., 53, 758. FRY, G. N., DEVINE, C. E. and BURSTOCK,G. 1977. Freeze fracture studies of nexuses between smooth muscle cells. Close relationship to sarcoplasmic reticulum. J. Ce// Bin/., 72, 26. GARANT, P. R. 1972. The demonstration of complex gap junctions between the cells of the enamel organ with lanthanum nitrate. J. Ultrastruct. Res., 40, 333. GILULA, N. B. 1974b. Isolation of rat liver gap junctions and characterization of polypeptides. J. Cell Bio!., GILULA, N. B. 1974a. Junctions between cells. In Cell Communication (ed. R. P. Cox), p .l.l. Wiley, New York. 63, 1I la (Abstr.). GILULA, N. B. 1975. Junctional membranes in normal and neoplastic tissues. In C~~l/u/:rrMembranes and Tumor Cell Behavior, p. 219. Williams and Wilkins Co., Baltimore. GILULA, N. B. and SATIR, P. 1971. Septate and gap junctions in molluscan gill epithelium. J. Cell Biol., 51, 869. GILULA, N. B., REEVES,0. R. and STEINBACH,A. 1972. Metabolic coupling, ionic coupling, and cell contacts. Nature, Lond., 235, 262.

DIVERSITY

OF

GAP

JUNCTIONS

GILULA, N. B., FAWCETT, D. N. and AOKI, A. 1976. The Sertoli cell occluding

393

junctions and gap junctions in and developing mammalian testis. Devl Biol., 50, 142. GOODENOUGH, D. A. 1975. The structure and permeability of isolated hepatocyte gap junctions. Cold Spring Hub. Symp. quant. Biol., 39, 37. GOODENOUGH, D. A. 1976. In vitro formation of gap junction vesicles. J. Cell Biol., 68, 220. GOODENOUGH, D. A. and STOECKENIUS,W. 1972. The isolation of mouse hepatocyte gap junctions. Preliminary chemical characterization and X-ray diffraction. J. Cell Biol., 54, 646. GOODENOUGH, D. A. and GILULA, N. B. 1974. The splitting of hepatocyte gap junctions and zonula occludentes with hypertonic disaccharides. J. CeN Biol., 61, 575. GOSHIMA, K. 1970. Formation of nexuses and electrotonic transmission between myocardial and F L cells in monolayer culture. Expl CeN Res., 63, 124. GRLEPP, E. V. and REVEL, J. P. 1977. Gap junctions in development. In Intercellular Communication (ed. W. C. DeMcllo), p. I. Plenum, New York. GROS, D. and CHALLICE, C. E. 1976. Early development of gap junctions between the mouse myocardial cells. A freeze-etching study. Experientiu, 32, 996. HAND, A. R. and GOBEL, S. 1972. The structural organization of the septate and gap junctions of Hydra. J. Cell Biol., 52, 397. HERR, J. C. 1976. Reflexive gap junctions: gap junctions arising from the same ovarian decidual cell. J. Cell Biol., 69, 495. HYDE, A., BLONDEL, B., MATTER, A., CHENEVAL, J. P., FILLOUX, B. and GIRARIER, L. 1969. Homo- and heterocellular junctions in cell cultures: an electrophysiological and morphological study. Prog. Bruin Res., 31, 283. IWAYAMA, T. 1971. Nexuses between areas of the surface membrane of the same arterial smooth muscle cell. J. Cell Biol., 49, 52 I. JOHNSON,R. G., HERMAN, W. S. and PREUS, D. M. 1973. Homocellular and heterocellular gap junctions in Limulus: a thin-section and freeze-fracture study. J. Ultrustruct. Res., 43, 298. JOHNSON, R. G., HAMMER, M., SHERIDAN, J. D. and REVEL, J. P. 1974. Gap junction formation between reaggregated Novikoff hepatoma cells. Proc. natn. Acad. Sci. U.S.A., 71, 4536. KARRER, H. E. 1960. The striated musculature of blood vessels. II. Cell interconnection and cell surface. J. Cell Biol., 8, 135. KEETER, J. S., PAPPAS, G. D. and MODEL, P. G. 1975. Inter- and intramyotomal gap junctions in the Axolotl embryo. Devl Biol., 45, 21. KELLY, R. 0. and FALLON, J. F. 1976. Ultrastructural analysis of the apical ectodermal ridge during vertebrate limb morphogenesis. Devl Biol., 51, 241. KOGON, M. and PAPPAS, G. D. 1975. Atypical gap junctions of the ciliary epithelium of the albino rabbit eye. J. Cell Biol., 66, 67 I. KREUTZIGER, G. 0. 1968. Freeze-etching of intercellular junctions of mouse liver. 26th Proceedings of the Electron Microscopy Society of America, p. 138. Claitors Publishing Div., Baton Rouge, Louisiana. LANDIS, M. D. and REESE, T. S. 1974. Arrays of particles in freeze-fractured membranes. J. Cell Biol., 60, 316. LARSEN, W. J. 1975. Opaque deposits on gap junction membranes after glutaraldehyde-calcium fixation. J. Cell Biol., 67, 801. LARSEN, W. J. 1977. Gap junctions and hormone action. In Transport of Ions and Water in Epithelia (eds. B. J. Wall, J. L. Oschman, Moreton, B. Gupta). Academic Press, London (in press). LARSEN, W. J., AZARNIA, R. and LOEWENSTEIN,W. R. 1977. Junctional membrane structure of hybrids between communication-competent and communication-incompetent cells. J. Membrane Biol. (in press). LEIBOVITZ, R., MCCOMBS, W. B., JOHNSTON, D., MCCOY, C. and STINSON, J. C. 1973. New human cancer cell culture lines. I. SW-13 small cell carcinoma of the adrenal cortex. J. turn. Cancer Inst., 51, 691. LETOURNEAU,R. J., LI, J. J., ROSEN, S. and VILLE, C. 1975. Junctional specialization in estrogen-induced renal adenocarcainoma of the golden hamster. Cancer Res., 35, 6. LOCKE, M. 1969. The ultrastructure of the oenocytes in the molt/intermolt cycle of an insect. Tissue & Cell, 1, 103. LOEWENSTEIN,W R 1974. Permeable junctions between cells. In Cell Membranes (eds. G. Weissman and R. Claiborne), p. 113. H.P. Publ. Co., New York. MARGARITIS, L. H., ELGSAETER,A. and BRANTON, D. 1977. Rotary replication for freeze-etching. J. Cell Biol., 72, 47. MARQUART, K. H. 1977. So-called annular gap junctions in bone cells of normal mice. Experientia, 15, 270-272. MAY, M. K. 1977. Personal communication. MCNUTT, N. S. and WEINSTEIN,R. S. 1973. Membrane ultrastructure at mammalian intercellular junctions. Prog. Mol. Biol., 26, 47. mature

394

LARSEN

MEGLITSCH, P. 1967. Invertebrate Zoology. Oxford University Press, New York. MERK, F. B., BOTTICELLI,C. R. and ALBRIGHT, .I. T. 1972. An intercellular response to estrogen by granulosa cells in the rat ovary: an electron microscopic study. Endocrinology, 90, 992. MERK, F. B., ALBRIGHT, J. T. and BOTTICELLI,C. R. 1973. The fine structure of granulosa cell nexuses in rat ovarian follicles. hat. Rec., 175, 107. MICHALKE, W. and LOEWENSTEIN,W. R. 1971. Communication between cells of different types. Nature, Lond., 232, 121. MONTESANO,R., FRIEND, D. S., PERRELET,A. and ORCI, L. 1975. In viva assembly of tight junctions in fetal rat liver. J. Cell Biol., 67, 310. NADOL, J. B., MULROY, M. J., GOODENOUGH, D. A. and WEISS, T. F. 1976. Tight and gap junctions in a vertebrate inner ear. Am. J. Anat., 147, 281. NOIROT, C. and NOIROT-TIMOTHBE,C. 1976. Fine structure of the rectum in cockroaches (Dictyoptera): general organization and intercellular junctions. Tissue & CeN, 8, 345. NUNEZ, E. A. 1971. Secretory processes in follicular cells of the bat thyroid. II. The occurrence of organelleassociated intercellular junctions during later hibernation. Am. J. Anat., 131, 227. ORWIN, D. F. G., THOMSON,R. W. and FLOWER, N. E. 1973. Plasma membrane differentiations of keratinizing cells of the wool follicle. I. Gap junctions. J. Ultrastruct. Res., 45, 1. OSCHMAN, J. L. 1976. Personal communication. PAPPAS, G. D., ASADA, Y. and BENNETT, M. V. L. 1971. Morphological correlates of increased coupling resistance at an electrotonic synapse. J. Cell Biol., 49, 173. PAYTON, B. W., BENNE~, M. V. L. and PAPPAS, G. D. 1969. Permeability and structure of junctional membranes at an electrotonic synapse. Science, 166, 1641. PEKACCHIA,C. 1973a. Low resistance junctions in crayfish. 1. Two arrays of globules in junctional membranes. J. Cell Biol., 57, 54. PERACCHIA, C. 1973b. Low resistance junctions in crayfish. II. Structural details and further evidence for intercellular channels by freeze-fracture and negative staining. J. Cell Biol., 57, 66. PERACCHIA, C. 1977. Gap junctions; structural changes after uncoupling procedures. J. Cell Biol., 72, 628. PERACCHIA, C. and DULHUNTY, A. F. 1976. Low resistance junctions in crayfish: structural changes with functional uncoupling. J. Cell Biol., 70, 4 17. PERISSEL,B., CHARBONNE,F., CHESSEBEUF,M. and MALET, P. 1976. Differentiation of the plasma membrane of hepatic cells in monolayer cultures. Cell Tiss. Res., 171, 157. PINTO DA SILVA, P. and GILULA, N. B. 1972. Gap junctions in normal and transformed fibroblasts in culture. Expl Cell Res., 71, 393. PITTS, J. D. and SIMMS,J. W. 1977. Permeability of junctions between animal cells: intercellular transfer of nucleotides but not macromolecules. Expl Cell Res., 104, 153. PRICAM, C., HUMBERT, F., PERRELET,A. and ORCI, L. 1974. Gap junctions in mesangial and lacis cells. J. Cell Biol., 63, 349. PRUTKIN, L. 1975. Mucous metaplasia and gap junctions in the vitamin A acid-treated skin tumor, keratoacanthoma. Cancer Res., 35, 364. RASH, J. F. and STAEHELIN, L. A. 1974. Freeze-cleave demonstration of gap junctions between skeletal myogenic cells in viva. Devl Biol., 36, 455. REVEL, J. P. and KARNOVSKY,M. J. 1967. Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. .I. Cell Biol., 33, C7. ROBERTSON,J. D. 1963. Occurrence of a subunit pattern in the unit membrane of club endings in the Mauthner cell synapses in goldfish brains. J. Cell Biol., 19, 201. SHERIDAN, J. D. 1971. Electrical coupling between fat cells in newt fat body and mouse brown fat. J. Cell Biol., 59, 795. SHERIDAN, J. D. 1976. The cell surface in development. In E.B.A. (mmbranes) (eds. Nicolson and Post). Elsevier, Amsterdam. SHERIDAN, J. D. and JOHNSON, R. G. 1975. Cell junctions and neoplasia. In Molecular Pathology (eds. R. Good, S. Day and J. Yunis). C. C. Thomas, Springfield. SOCOLAR, S. J. 1973. Cell coupling in epithelia. Expl Eye Res., 15, 693. WEINSTEIN,R. S., MERK, F. B. and ALROY, J. 1976. The structure and function of intercellular junctions in cancer. In Advances in Cancer Research (eds. G. Klein and S. Weinhouse), Vol. 23, 23. Academic Press, New York. WILLE, C. L. and LARSEN, W. J. 1977. Particle packing patterns in developing gap junctions of rabbit granulosa cells. Anat. Rec., 187, 749 (Abstr.). ZAMBONI, L. 1974. Fine morphology of the follicle wall and follicle cell-oocyte assxiation. Rio!. Reprod., 10, 125-149.