Assembly of Gap Junctions During Amphibian Neurulation

105

Cell-to-cell communication was characterized in prefusion chick embryo myoblast cultures, and it was determined that the prefusion myoblasts can interact via gap junctions, ionic coupling, and metabolic coupling. The biological relevance of this communication was supported by the detection of gap junctions between myoblasts in embryonic muscle. Communication was also examined in fusionarrested cultures to determine its potential relationship to fusion competency. In cultures that were fusion arrested by treatment with either 1.8 mM ethyleneglycolbis-(fl-aminoethyl ether)N,N'-tetraacetic acid (EGTA), 3.3 x 10 -6 M 5-bromodeoxyuridine (BUdR), or 1 /~g/ml cycloheximide (CHX), both gap junctions and ionic coupling were present. Therefore, it is possible to conclude that cell communication is not a sufficient property by itself, to generate fusion between myoblasts. The potential role of communication in myogenesis is discussed with respect to these observations. KEY WORDS cell-to-cell communication junctions 9 ionic coupling metabolic coupling myoblast fusion myogenesis gapThe differentiation of skeletal muscle in vivo and in vitro (in culture) is accompanied by the fusion of mononucleated muscle cells (myoblasts) into multinucleated mature muscle fibers (58). This fusion event, in culture, is preceded by a period of cell multiplication and cellular interaction. During this time, the cells are closely apposed and aligned in multicellular strings; this stage of differentiation has been referred to as the "prefusion lag period" (61). Under standard culture conditions this lag period is quite constant, and only cells which have completed their prefusion changes participate in the formation of muitinucleated fibers (59). The fusion process that follows the lag period has been previously regarded as a critical event in myogenesis. However, at present, it appears that it is just one of the important events that contribute to the formation of a differentiated muscle fiber (1, 12. 16, 19, 26, 31, 34, 36, 41, 54, 55, 56, 57, 62). The actual fusion process requires a direct physical interaction of the plasma membranes from adjacent cells, and this had led to a series of investigations of the cell surface elements that may be involved in regulating fusion (5,14,20). Other studies have suggested that fusion is not necessarily regulated by cell surface components; it may, in fact, be under the direct control of intracellular elements (62). Recently, it has been reported that a specific type of cell contact, the gap junction (30), and low-resistance pathways (6) are present between muscle cells during amphibian myotome development in vivo. In both studies, it was suggested that gap junctional communication between the muscle cells is related to the efficient transfer of excitatory stimuli from somite to somite, and not to cell fusion. Data from studies on embryonic rat and chick skeletal muscle cells in culture indicate the possible existence of ionic coupling (measured indirectly) and gap junctionlike structures betwee...

Section: Ultrastructural Detection Of Gap Junctionssupporting

confidence: 56%

Cell-to-cell communication and myogenesis.

Kalderon¹,

Epstein²,

Gilula³

1977

105

“…Invagination, on the other hand, may represent a biological response to uncoupling and one of the means of gap junction disposal . In fact, it seems unlikely that invagination could reflect insertion of newly formed junctions on the cell surface, for there is evidence that (a) neoformation of gap junctions is not associated with presence of invaginated junctions (8,16), (b) invagination follows interruption of the blood supply (24), and (c) it may ultimately lead to junction internalization and digestion (19,24) .…”

Section: Rapid Communications Figures 8-10mentioning

confidence: 99%

Structure of rapidly frozen gap junctions.

Raviola¹,

Goodenough²,

Raviola³

1980

150

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

“…the literature indicate that three types of tight junction can be distinguished on the basis of the morphology of their junctional strands which appear as (a) continuous fibrils, e.g., in the epithelia of the gastro-intestinal tract (11,41), (b) bars, e.g., in the developing junctions (15,28,34) and in the mesothelium (present paper), or (c) rows of particles, e.g., in the arteriolar and capillary endothelium (37,38), stria vascularis (33), and among Sertoli cells (20). Forms transitional from one type to another are, however, occasionally encountered (1,20).…”

Section: S~mnescu and N Si~tonescu Cell Junctions In The Peritonementioning

confidence: 99%

Organization of cell junctions in the peritoneal mesothelium

Simionescu¹,

Simiónescu²

1977

104

Intercellular junctions in the mesothelium of the visceral (mesentery and omenturn), and parietal (diaphragm, pre-aortic, and iliac region) peritoneum were examined in rats and mice by using freeze-cleaved preparations. In addition to usual intercellular junctions (cell body junctions), special junctions are found between cell processes and the surface of the neighboring cell (cell process junctions). Cell body junctions are provided with tight junctions and communicating (gap) junctions. The former consist of one to two junctional strands which show a characteristic staggered arrangement, and focal discontinuities. In cell process junctions, the strands form loops or appear as short, free-ending elements; their polymorphism suggests considerable lability, probably in connection with their assembly and disassembly. The existence of free-ending strands indicates that such structures can be used as attachment devices without being concomitantly involved in the formation of occluding zonules. In both types of junctions, the strands can be resolved into bars, -80-100 nm long, frequently provided with terminal enlargements and intercalated particles which occur singly or in small clusters. These particles are morphologically similar to those present in communicating (gap) junctions. The mesothelium is also provided with isolated composite macular junctions. Throughout the mesothelium, the cleavage plane follows the outer contour of junctional strands and particles, suggesting that strand-to-strand interactions in the apposed membranes are weaker than interactions between each strand and underlying cytoplasmic structures. In their general geometry and cleavage characteristics, the mesothelial junctions resemble the junctions found in the venular endothelium.Earlier interest in the mechanisms of transport of solutes (3,5,6,8,21,9,32), particulate material (12, 10, 16), and cells across the mesothelium has been recently renewed after the success of peritoneal dialysis as a maintenance technique in chronically uremic patients (7, 23). Despite past investigations, current views concerning the structural substrate of the high permeability of the mesothelial membrane are still markedly divergent. According to a number of studies, water-soluble toolecules cross the mesothelium by passive diffusion presumably along intercellular junctions (5, 21,9, 12, 13, 10), whereas according to other investigations transport across the mesothelium is an active process (35, 32) effected primarily via vesicles (30,42,19,17). These divergent views have broader implications than are immediately apparent, since it has been postulated that the mesothelium, due to its being morphologically and functionally similar to the vascular endothelium, can