Tight junction development between cultured hepatoma cells: Possible stages in assembly and enhancement with dexamethasone

Freeze-fracture and thin-section methods were used to study tight junction formation between confluent H4-II-E hepatoma cells that were plated in monolayer culture in media with and without dexamethasone, a synthetic glucocorticoid. Three presumptive stages in the genesis of tight junctions were suggested by these studies: (1) “formation zones” (smooth P-fracture face ridges deficient in intramembranous particles), apparently matched across a partially reduced extracellular space, develop between adjacent cells; (2) linear strands and aggregates of 9–11 nm particles collect along the ridges of the formation zones. The extracellular space was always reduced when these structures were found matched with pits in gentle E-face depressions; (3) the linear arrays of particles on the ridges associate within the membranes to form the fibrils characteristic of mature tight junctions. The formation zones resemble tight junctions in terms of size, complexity and the patterns of membrane ridges. Although some of the beaded particle specialization may actually be gap junctions, it is unlikely that all can be interpreted in this way. No other membrane structures were detected that could represent developmental stages of tight junctions. Dexamethasone (at 2 × 10^?6 M) apparently stimulated formation of tight junctions. Treated cultures had a greater number of formation zones and mature tight junctions, although no differences in qualitative features of the junctions were noted.     

Morphogenesis of tight junctions in the peritoneal mesothelium of the mouse embryo

The peritoneal mesothelium of mouse embryos (12 to 18 day of gestation) was studied by freeze-fracture and in sections in order to reveal the initial formation of the tight junctions. Freeze-fracture observations showed three types of tight junctions. Type I consists of belt-like meshworks of elevations on the P face and of shallow grooves on the E face. No tight junctional particle can be seen either on the elevations or in the grooves. Type II shows rows of discontinuous particles on the elevations on the P face. Type III consists of strands forming ridges on the P face. On the E face, the grooves of Type II and III appear to be narrower and sharper than those of Type I. Quantitatively, Type I junctions are most numerous during the early stages (day 12-13) of embryonic development, while Type III junctions become more common in the later stages, and are the only type seen by day 18. Observations on sections, however, fail to distinguish between the three types. The results suggest that an initial sign of tight junction formation is close apposition of the two cell membranes in the junctional domain, without tight junctional particles. Later, the particles appear to be incorporated in the tight junctions and the strands form by fusion of the particles.     

Formation of tight and gap junctions in the inner enamel epithelium and preameloblasts in human fetal tooth germs

Human fetal primary tooth germs in the cap stage were fixed with a glutaraldehyde-formaldehyde mixture, and formative processes of tight and gap junctions of the inner enamel epithelium and preameloblasts were examined by means of freeze-fracture replication. Chains of small clusters of particles on the plasma membrane P-face of the inner enamel epithelium and preameloblasts were the initial sign of tight junction formation. After arranging themselves in discontinuous, linear arrays in association with preexisting or forming gap junctions, these particles later began revealing smooth, continuous tight junctional strands on the plasma membrane P-face and corresponding shallow grooves of a similar pattern on the E-face. Although they exhibited evident meshwork structures of various extents at both the proximal and distal ends of cell bodies, they formed no zonulae occludentes. Small assemblies of particles resembling gap junctions were noted at points of cross linkage of tight junctional strands; but large, mature gap junctions no longer continued into the tight junction meshwork structure. Gap junctions first appeared as very small particle clusters on the plasma membrane P-face of the inner enamel epithelium. Later two types of gap junctions were recognized: one consisted of quite densely aggregated particles with occasional particle-free areas, and the other consisted of relatively loosely aggregated particles with particle-free areas and aisles. Gap junction maturation seemed to consist in an increase of particle numbers. Fusion of gap junctions in the forming stage too was recognized. The results of this investigation suggest that, from an early stage in their development, human fetal ameloblasts possess highly differentiated cell-to-cell interrelations.     

Changes in the blood-brain barrier of the central nervous system in the blowfly during development, with special reference to the formation and disaggregation of gap and tight junctions

In the central nervous system (CNS) of pupal Calliphora, dramatic alterations occur in the perineurial and glial gap junctions. Having formed macular plaques by late larval stages, in early pupae cell migration causes the EF intramembranous junctional particles to disaggregate and move apart into linear and then disorganised arrays as shown by freeze-fracture. After nerve and glial cell reorganisation into the adult pattern, the gap junctions begin to reform in the late pupae, again seemingly by particle migration into linear arrays and clusters. Ultimately the particles form numerous macular plaques between both perineurial and glial cells. Statistical analyses support the contention that these are performed EF particles which undergo translateral movement from macular larval junctions into the disaggregated particles of early pupae and that the same particles appear to undergo realignment and reclustering in late pupae to form the mature gap junctions of adults. This is the first report to indicate breakdown and reformation of gap junctions in vivo involving reutilisation of the same intramembranous particles. Perineurial “tight” junctions are not to be found in early pupal stages and their absence can be correlated with the free entry of ionic lanthanum into the CNS observed during that period. In late pupae, when the tight junctional moniliform ridges have apparently reformed, the entry of the tracer lanthanum becomes restricted to the level of the perineurium, penetrating no deeper. This is also the case in the adult, where the blood-brain barrier is maintained. PF particles in the form of short linear ridges and clustered particle arrays in nerve cell membranes are present throughout pupal and adult stages; their continued presence throughout the whole of development suggests some role in neuronal function, as yet unclear.     

Changes in the blood-brain barrier of the central nervous system in the blowfly during development, with special reference to the formation and disaggregation of gap and tight junctions. I. Larval development

In the central nervous system (CNS) of full-grown larvae of the blowfly Calliphora erythrocephala, the glial-ensheathed nerve cells are completely surrounded by a layer of perineurial cells which form a “blood-brain barrier” between the circulating haemolymph and the CNS. A variety of intercellular junctions, including gap and tight junctions, are found between adjacent perineurial cells and some also between apposing glial cells; these have been characterized by freeze-fracturing as well as by tracer studies and analysis of thin sections. They are found not to be present between such cells in the undifferentiated CNS in the newly hatched larvae, nor are the nerve cells encompassed by glial cells; ionic lanthanum can penetrate to the axonal surfaces at this stage. However, over the 5 days of larval growth and development the glial cells produce attentuated cytoplasmic processes that ensheath the nerve cells, and the perineurium is formed; junctional complexes are assembled and a larval blood-brain barrier is produced which excludes tracers. Freeze-fracture preparations suggest that the inverted gap junctions which develop have done so by migration of individual intramembranous EF particles to form, at first, linear arrays and small clusters and, ultimately, macular aggregations in the perineurium; these lie between the undulating rows of PF particles forming the septate junctions. These septate junctions are formed by the organization of arrays of PF particles into multiple rows. Extensive PF particles fusing into ridges with EF grooves to form perineurial “tight” junctions are also observed, seemingly in the process of development; entry of exogenous lanthanum followed by its exclusion parallels the completion of ridge formation. These ridges are simple linear arrays of particles which may be discontinuous, lying in parallel with one another and the surface. Clustered particle arrays as well as scattered short ridges on the axonal PF, however, appear to be present unchanged throughout larval life; their role may therefore be associated with neural membrane function although there are suggestions that some may form axo-glial junctions. This is the first report on the lateral migration of intramembranous particles as the mode of formation of gap junctions in the nervous system of an invertebrate.     

Segmental differentiations of cell junctions in the vascular endothelium. The microvasculature

Small vascular units consisting of an arteriole, its capillaries, and the emerging venule (ACV units) were identified in the rat omentum and mesentery. They were fixed in situ and processed for electron microscopy either as whole units or as dissected segments. Systematic examination of the latter (in thin sections, as well as in freeze-cleaved preparations) showed that the intercellular junctions of the vascular endothelium vary characteristically from one segment to another in the microvasculature. In arterioles, the endothelium has continuous and elaborate tight junctions with interpolated large gap junctions. The capillary endothelium is provided with tight junctions formed by either branching or staggered strands; gap junctions are absent at this level. The pericytic venules exhibit loosely organized endothelial junctions with discontinuous low-profile ridges and grooves, usually devoid of particles. No gap junctions were found in these vessels. The endothelium of muscular venules has the same type of junctions (discontinuous ridges and grooves of low profile); in addition, it displays isolated gap junctions of smaller size and lower frequency than in arterioles. The term communicating junction (macula communicans) is proposed as a substitute for gap junctions, since the latter is inappropriate, in general, and confusing in the special case of the vascular endothelium.     

Definitive Evidence for the existence of tight junctions in invertebrates

Extensive and unequivocal tight junctions are here reported between the lateral borders of the cellular layer that circumscribes the arachnid (spider) central nervous system. This account details the features of these structures, which form a beltlike reticulum that is more complex than the simple linear tight junctions hitherto found in invertebrate tissues and which bear many of the characteristics of vertebrate zonulae occludentes. We also provide evidence that these junctions form the basis of a permeability barrier to exogenous compounds. In thin sections, the tight junctions are identifiable as punctate points of membrane apposition; they are seen to exclude the stain and appear as election- lucent moniliform strands along the lines of membrane fusion in en face views of uranyl-calcium-treated tissues. In freeze-fracture replicas, the regions of close membrane apposition exhibit P-face (PF) ridges and complementary E-face (EF) furrows that are coincident across face transitions, although slightly offset with respect to one another. The free inward diffusion of both ionic and colloidal lanthanum is inhibited by these punctate tight junctions so that they appear to form the basis of a circumferential blood-brain barrier. These results support the contention that tight junctions exist in the tissues of the invertebrata in spite of earlier suggestions that (a) they are unique to vertebrates and (b) septate junctions are the equivalent invertebrate occluding structure. The component tight junctional 8- to 10-nm-particulate PF ridges are intimately intercalated with, but clearly distinct from, inverted gap junctions possessing the 13-nm EF particles typical of arthropods. Hence, no confusion can occur as to which particles belong to each of the two junctional types, as commonly happens with vertebrate tissues, especially in the analysis of developing junctions. Indeed, their coexistance in this way supports the idea, over which there has been some controversy, that the intramembrane particles making up these two junctional types must be quite distinct entities rather than products of a common precursor.     

Tight junctions in the choroid plexus epithelium. A freeze-fracture study including complementary replicas

The tight junctions of the choroid plexus epithelium of rats were studied by freeze-fracture. In glutaraldehyde-fixed material, the junctions exhibited rows of aligned particles and short bars on P-faces, the E-faces showing grooves bearing relatively many particles. A particulate nature of the junctional strands could be established by using unfixed material. The mean values of junctional strands from the lateral, third, and fourth ventricles of Lewis rats were 7.5 +/- 2.6, 7.4 +/- 2.2, and 7.5 +/- 2.4; and of Sprague-Dawley rats 7.7 +/- 3.4, 7.4 +/- 2.3, and 7.3 +/- 1.6. Examination of complementary replicas (of fixed tissue) showed that discomtinuities are present in the junctional strands: 42.2 +/- 4.6% of the length of measured P-face ridges were discontinuities, and the total amount of complementary particles in E-face grooves constituted 17.8 +/- 4.4% of the total length of the grooves, thus approximately 25% of the junctional strands can be considered to be discontinuous. The average width of the discontinuities, when corrected for complementary particles in E-face grooves, was 7.7 +/- 4.5 nm. In control experiments with a "tighter" tight junction (small intestine), complementary replicas revealed that the junctional fibrils are rather continuous and that the very few particles in E-face grooves mostly filled out discontinuities in the P-face ridges. Approximately 5% of the strands were found to be discontinuous. These data support the notion that the presence of pores in the junctional strands of the choroid plexus epithelium may explain the high transepithelial conductance in a "leaky" epithelium having a high number of junctional strands. However, loss of junctional material during fracturing is also considered as an alternative explanation of the present results.     

Junctions in the central nervous system of the cat

Interendothelial membrane contacts in different segments of brain blood vessels were investigated by the freeze-etching technique. The study demonstrated that the endothelial cells of the pre- and postcapillary segments were coupled by elaborate zonulae occludentes. These tight junction formations encompassed gap junctions of different sizes and distribution. The globules of the pre- and postcapillary tight junctions revealed a great fragility which led to an atypical distribution of the sealing elements. In the "typical" brain capillaries the endothelial cells were connected by continuous tight junctions. In contrast to the structural continuity of these formations the fenestrated segments of capillaries in the choroid plexus and area postrema demonstrated discontinuous fasciae occludentes. They were composed of rows of individual particles which should be regarded primarily as focal in nature.     

Square arrays and their role in ridge formation in human lens fibers

Square arrays in human lens fibers were studied with freeze-fracture and thin-section TEM. In superficial fibers a number of patches of square array particles in the P face and pits in the E face are found in the smooth membrane. In the deeper cortex and the nucleus, fiber cells have undulating membranes and many ridges. Numerous patches of the particles (P face) are distributed in the concave regions, and the pits (E face) in the convex areas of the bumpy membrane. In most ridges, patches of the particles occur at regular intervals in the "valley" portion, while the pits are on the "crest" portion of ridges. Also, continuous square arrays having the same "valley" location as the regularly arranged patches are found in areas with extensive ridge patterns. The overlapping of the outer portions of two adjacent square arrays is found on the sides between the "crest" and the "valley" of the ridges. Structurally, square arrays are located in a nonjunctional part of the membrane; in an orthogonal crystalline arrangement; and with a particle size of about 6 nm and center-center spacing about 6.4 nm. They are structurally different from gap junctions found in the lens fibers. Thin-section studies reveal two types of cellular contacts: thin pentalamellar structures (about 12-13 nm in overall thickness) associated with the ridge patterns are believed to be square arrays; thick heptalamellar structures (about 16-17 nm in overall thickness) with a narrow gap in between the two central laminae are believed to be gap junctions. This study strongly suggests that square arrays are specifically involved in ridge formation in human lens fibers.     

A model for de novo synthesis and assembly of tight intercellular junctions. Ultrastructural correlates and experimental verification of the model revealed by freeze-fracture

The structure and function of intercellular tight (occluding) junctions, which constitute the anatomical basis for highly regulated interfaces between tissue compartments such as the blood-testis and blood-brain barriers, are well known. Details of the synthesis and assembly of tight junctions, however, have been difficult to determine primarily because no model for study of these processes has been recognized. Primary cultures of brain capillary endothelial cells are proposed as a model in which events of the synthesis and assembly of tight junctions can be examined by monitoring morphological features of each step in freeze-fracture replicas of the endothelial cell plasma membrane. Examination of replicas of non-confluent monolayers of endothelial cells reveals the following intramembrane structures proposed as 'markers' for the sequential events of synthesis and assembly of zonulae occludentes: development of surface contours consisting of elongate terraces and furrows (valleys) orientated parallel to the axis of cytoplasmic extensions of spreading endothelial cells, appearance of small circular PF face depressions (or volcano-like protrusions on the EF face) that represent cytoplasmic vesicle-plasma membrane fusion sites, which are positioned in linear arrays along the contour furrows, appearance of 13-15 nm intramembrane particles at the perimeter of the vesicle fusion sites, and alignment of these intramembrane particles into the long, parallel, anastomosed strands characteristic of mature tight junctions. These structural features of brain endothelial cells in monolayer culture constitute the morphological expression of: reshaping the cell surface to align future junction-containing regions with those of adjacent cells, delivery and insertion of newly synthesized junctional intramembrane particles into regions of the plasma membrane where tight junctions will form, and aggregation and alignment of tight junction intramembrane particles into the complex interconnected strands of mature zonulae occludentes. The distribution of filipin-sterol complex-free regions on the PF intramembrane fracture face of junction-forming endothelial plasmalemmae corresponds precisely to the furrows, aligned vesicle fusion sites and anastomosed strands of tight junctional elements.(ABSTRACT TRUNCATED AT 400 WORDS)     

“Tight” junctions in the sheath of normal and regenerating motor nerves of the crayfish,Orconectes virilis

Summary Tight or occluding intercellular junctions occur between adjacent glial processes in normal and regenerating crayfish motor nerve sheaths. Although infrequent, these junctions possess the ridge and groove configuration characteristic of freeze-cleaved occluding junctions. When present, nerve sheath tight junctions consist of a single, or at most a few, parallel intramembrane ridges situated on the EF membrane face of the glial plasma membrane. Consequently, such contacts are rarely recognized in thin sections of plasticembedded nerve sheaths. Crayfish nerve sheath tight junctions are of the fascia occludens type and, therefore, do not impede solute flow across the nerve sheath. Fasciae occludentes of regenerating nerve sheaths occur in close proximity to discoid plaque-like aggregates of particles assumed to represent maculae adhaerentes. This relationship, which was not observed in normal nerve sheaths, suggests a functional association between the two types of junctions, perhaps developmental transformation of one junction type into the other. Although ridges and grooves of tight junctions occur next to crossfractured trans-glial channels, no functional significance is proposed for this relationship. This study is the first report of tight intercellular junctions in crustacean glial nerve sheaths.Supported by the National Research Council of Canada     

Freeze-fracture of capillary endothelium in rat brain

Summary The rat brain capillary was studied with freeze-fracture technique. The attached plasmalemmal vesicles were quite few in number on the luminal front and sometimes numerous on the contraluminal side. The fracture appearance of some tight junctions showed interconnecting ridges on face A and complementary furrows devoid of particles on face B, comparable to the common tight junction in the normal epithelia. Other tight junctions revealed a preferential disposition of quasicontinuous rows of particles on shallow furrows of face B, resembling the tight junctional strands of capillary endothelium in non-cerebral tissues. Either behavior is probably due to the difference in the fracture plane around the single fibril. In addition, the tight junctional strand could surround the perimeter of the endothelial cell completely although the exposed strand of tight junction was limited in length.     

A vertebrate-like blood--brain barrier, with intraganglionic blood channels and occluding junctions, in the scorpion

India ink and ionic lanthanum injections have revealed that the central nervous system (CNS) of the scorpion possesses a highly vascularized cephalothoracic ganglionic mass. It, together with other abdominal ganglia which form a ventral nerve cord, are all ensheathed by an outer layer of modified glial, or perineurial, cells. These cells resemble those which line the blood channels permeating the CNS, in exhibiting both inverted gap and tight junctions. Although the latter show close or fused membrane appositions, lanthanum appears to penetrate past a number, but not all, of them. Freeze-fracturing reveals that these junctions are composed of E-face particles aligned into a network of rows, or ridges, which are frequently discontinuous, especially near the periphery of the perineurium. This produces a somewhat 'leaky' system but occlusion to tracers occurs ultimately, for in the CNS none can be found beyond the perineurium. The existence of this perineurial blood-brain barrier is also demonstrable electrophysiologically where cations such as Mg2+ are unable to penetrate beyond the perineurial layer although they can, it seems, leak in via the blood vascular system. Relative differences in tightness between the perineurium and the cells lining the blood channels may be attributed to differences in the relative number of discontinuous ridges. This is borne out by the observation that the peripheral nervous system has a highly attenuated perineurium with many fewer junctions, and some of these nerves tend to be leaky with respect to tracer penetration. In fixed material the junctional ridges may fracture on to the E-face or partly on both the EF and PF, while in unfixed tissue they are usually found on the PF. In both cases they exhibit complementary grooves that are coincident with the ridges across membrane transitions; in such cases the cell membranes are fused with concomitant obliteration of the intercellular space. These tight junctions, often closely associated with EF gap junctional particle aggregates which may be very loosely clustered, appear to form the basis of the observed blood-brain barrier in the scorpion CNS.     

Freeze-fracture and tracer studies on the intercellular junctions of insect rectal tissues.

Both rectal pads of the cockroach and rectal papillae of the blowfly possess highly infolded lateral borders; these are associated by desmosomes and septate junctions that maintain the physical integrity of the cell layer at the luminal and basal intercellular regions. Adjacent cells are coupled by gap junctions that allow for cell-to-cell communication and which occur at intervals along the undulating lateral clefts. In rectal pads, occluding basal tight junctions are found as well as extensive scalariform junctions. The latter, like the stacked membrane infoldings of rectal papillae, exhibit intercellular columns and numerous intramembranous P face particles; these are undoubtedly involved in ion transport. In the inter-stack clefts of papillae, reticular septate junctions are encountered which, after freeze-fracture, possess a striking network of PF ridges and EF grooves that are discontinuous and not always complementary. These may serve to regulate the speed and extent of distension of the clefts during solute movement to allow for even and effective fluid flow in this transporting epithelium.     

Vitamin-A-induced mucous metaplasia. An in vitro system for modulating tight and gap junction differentiation

Stratified squamous epithelia from 14-day chick embryo shank skin contain rare tight-junctional strands and only small gap junctions. Exposure of this tissue to retinoic acid (vitamin-A) (20 U/ml) in organ culture, however, induces mucous metaplasia, accompanied by tight-junction formation and gap-junction growth; untreated specimens continue to keratinize. To investigate sequential stages of junctional assembly and growth, we examined thin sections and freeze-fracture replicas at daily intervals for 3 days. During the metaplastic process, tight junctions assemble in midepidermal and upper regions, beginning on day 1 and becoming maximal on day 3. Two tight-junctional patterns could be tentatively identified as contributing to the emergence of fully formed zonulae occludentes: (a) the formation of individual ridges along the margins of gap junctions; (b) de novo generation of continuous ramifying strands by fusion of short strand segments and linear particulate aggregates near cellular apices. Gap junction enlargement, already maximal at day 1, occurs primarily three to four cell layers deep. Growth appears to occur by annexation of islands of 20-40 8.5-nm particles into larger lattices of islands separated by particle-free aisles. Eventually, a single gap junction may occupy much of the exposed membrane face in freeze-fractured tissue, but during apical migration of the cells such junctions disappear. The vitamin- A chick-skin system is presented as a responsive model for the controlled study of junction assembly.     

Cell junctions in the cyst envelope in the silkworm testis,Bombyx mori linné

Summary The cell junctions of the cyst envelope in the testes of Bombyx mori were examined by electron microscopy utilizing a thin-sectioning technique following conventional fixation, tannic acid fixation and lanthanum tracer study, and also using a freeze-fracture technique. There are three kinds of junctions; septate junctions, gap junctions and tight junctions. Septate junctions are of the pleated type. Gap junctions are characterized by four electron-dense lines and three electronlucent lines in the reduced intercellular spaces seen by thin-sectioning. They are of the E type, having clusters of intramembraneous particles on the E-fracture face. The most striking finding is the frequent presence of tight junctions on the fracture planes, while focally fused outer leaflets of the junctional unit membranes are rarely detected on thin-sectioned preparations. Tight junctions are characterized by branching zigzag ridges on the P-fracture face and complementary grooves on the E-fracture face. It is proposed that tight junctions are new morphological evidence of blood-germ cell barrier in an insect. Acknowledgements: For helpful assistance the authors are indebted to their colleagues Miss N. Minemoto, Miss H. Kiyotake and Mr. Y. Goto     

Junctional diversity in two regions of the epidermis of Oikopleura dioica (Tunicata,Larvacea)

Summary A simple continuous epithelium surrounds the body of the pelagic larvacean. It consists of two zones of cells: oikoplast cells and flattened cells. The oikoplast cells are columnar and produce a thick extracellular house that ensheathes the body of the organism. These cells are joined laterally by wide tight junctions (zonulae occludentes). The tail of the animal is surrounded by exceedingly thin cells which are joined by narrow tight junctions under which lie intermediate junctions (zonulae adhaerentes) and gap junctions. A web of fibrous material inserts into the intermediate junctions. The transitional cells between the two epithelial zones have one lateral border with a wide tight junction, and the other lateral border with a narrow tight junction and a wide intermediate junction. In freeze-fracture replicas, the wide tight junction has a number of anastomosing ridges, in comparison with the narrow tight junction, which usually consists of only a single row of intramembranous particles. In replicas, the thin epithelial cells show unusual parallel arrays of particles in clusters on their apical plasma membranes. This simple epithelium, therefore, exhibits striking differences between the two cellular zones, in the structural characteristics of both the lateral borders and the apical membrane.     

Structure of occluding junctions in ileal epithelial cells of suckling rats

Summary Two kinds of occluding junctions are found between ileal epithelial cells of suckling rats: apical zonulae occludentes (ZO) and fasciae occludentes (FO) which are associated with the lateral plasma membranes of many epithelial cells. In unfixed preparations, glycerol treatment induces the further proliferation of extensive fasciae occludentes. Both kinds of junction have identical structural elements when visualized in freeze fracture replicas, although the arrangement of these elements differs. Zonulae occludentes consist of networks of branching and anastomosing linear ridges or rows of 10 nm particles with 20–30 nm spaces between the rows which form narrow belt-like structures around the apical region of adjacent cells. Fasciae occludentes, on the other hand, consist of similar linear ridges or rows of particles but the junction strands are often discontinuous, open ended and only occasionally intersect with each other. Several different fracture planes through the plasma membrane in the region of the occluding junctions have been observed and these provide further evidence that two components, one from each membrane, fused at the level of the extracellular space, form the junction sealing element. Furthermore, we present evidence which indicates a staggered rather than an in-register arrangement of these two components.This study was supported in part by National Institutes of Health Program Project No. NS10299 and National Institutes of Health Sciences Advancement Award No. RR06148 (J.D.R.) and by the Cancer Research Campaign (S.K.) and Medical Research Council (A.R.L.)     

Occluding-like junctions at mesaxons of central myelin in Anolis carolinensis are not 'tight'. A freeze-fracture-protein tracer analysis.

The junctional complexes of the myelin sheath of central nervous system axons in the American chameleon, Anolis carolinensis, exhibit an intramembrane ridge and groove construction in freeze-fracture replicas that has usually been interpreted in other organisms as evidence for an occluding or tight intercellular junction. Close examination of PF fracture face ridges, however, shows them to be made up of discontinuous rows of particles of variable length separated by frequent gaps of non-uniform width. Introduction of horseradish peroxidase into the intercellular milieu of the lizard central nervous system is followed by appearance of this protein in interlamellar spaces of the myelin sheath and in the intercellular spaces containing focal membrane fusions that correspond precisely in position and center-to-center spacing to the ridges and grooves in platinum replicas of the same tissue. Since the junctional ridges on PF fracture faces in these mesaxonal junctional complexes are conspicuously discontinuous and since the areas within the myelin sheath where these junctional complexes are located inner and outer mesaxons) are readily permeated by exogenous protein tracer, it is concluded that the junctional complexes of central myelin mesaxons, heretofore incorrectly interpreted as functionally tight, are actually very leaky and probably contribute only to the structural stability of the myelin sheath architecture.