papillary cells during maturation of enamel.
Significant changes in cell-to-cell contacts also occur throughout all phases of enamel formation. They appear to be needed for the coordination of cellular activity and for controlling the compartmentation of the extracellular space. These requirements are met by the formation of gap junctions and zonula occludens junctions. The following sections provide brief reviews of the structure and function of these two junctions.
Gap junctions
Gap junctions provide hydrophilic passageways across adjacent cell membranes for the intercellular exchange of ions and small molecules (less than 1,000 Da).140,141 Special transmembrane gap junction proteins, called connexins (Fig 3-12), create the channel through the membrane. The connexin molecule has four transmembrane domains, two rather rigid extracellular domains, and two cytoplasmic domains.142 The carboxy terminal domain, larger than the amino terminal domain, contains amino acid sequences that regulate channel permeability.
Fig 3-12 Gap junction connexin protein from mammalian liver cells. The amino and carboxy terminals are located on the cytoplasmic surface. Two polypeptide loops of protein extend across the membrane to the external surface of the connexon.
Six connexin molecules aggregate in the membrane to form a supramolecular hemichannel, the connexon (Fig 3-13).143 In forming a connexon hemichannel, the gap junction proteins assemble with their hydrophobic surfaces facing the lipid phase of the plasma membrane and their hydrophilic surfaces oriented inward (toward each other) to delimit a central fluid-filled channel across the membrane. When connexons from two adjacent cells are connected across the narrow intercellular gap, and the connexons are open, an intercytoplasmic exchange of ions and small molecules may occur. Typical gap junctions are made up of a hundred or more connexons aggregated in complementary patches in the cell membranes of a pair of participating cells (Figs 3-13 and 3-14).
Fig 3-13 Gap junction in the (A) coupled and (B) uncoupled states, showing the association of connexons in two juxtaposed plasma membranes. In the open condition (A), ions and small molecules can move through a fluid-filled pore (green arrows) from cell to cell. When the gap junction is uncoupled (B), the connexons are constricted and the pore is closed (red arrows). (Adapted from Peracchia143 with permission from Kluwer Academic.)
Fig 3-14 Gap junction particles (arrows) are aggregated on the protoplasmic face (Pf) of the fractured plasma membrane. Pits (arrowhead) on the external face (Ef) of the membrane represent the position of the pore of the connexon particle. (Original magnification × 92,000.)
The flow of ions and small metabolites across gap junctions has been shown to be involved in coordinating and regulating cellular activity in groups of contiguous cells. For example, second messengers, such as cyclic adenosine monophosphate, calcium ions, and inositol triphosphate, have been shown to spread through gap junctions.144 In cardiac muscle, gap junctions, functioning as electrotonic synapses, coordinate the contraction of the heart. Gap junctions are needed during embryonic development to coordinate sequential differentiation of groups of cells.
Gap junction proteins are to some degree tissue specific. Lens, heart, and liver gap junction proteins have different molecular weights, suggesting that their respective connexons have tissue-specific physiologic functions in addition to common properties. The family of genes that encode connexins is made up of at least 12 members.141,145 Homotypic and heterotypic assembly of connexin proteins result in gap junctions with different physiologic properties.146 In addition, it is possible for a single cell type to form different types of connexons and to restrict each type to specific domains of the cell membrane.147
Participating cells are coupled when adjacent gap junction connexons are open. Various substances regulate the size of the pore opening and thereby control the degree of cell-to-cell coupling (reviewed by Bruzzone et al148). Cytosolic calcium, cellular pH, retinoic acid, and intracellular oxygen tension have been shown to influence coupling. Connexons close within minutes in response to increased intracellular calcium, acidification of the cytoplasm, and low intracellular oxygen tension. This decoupling represents an emergency shutdown mechanism to prevent a cell-to-cell spread of noxious stimuli.
The calcium-binding protein, calmodulin, has been shown to participate in regulating the action of calcium on connexon proteins. Cyclic adenosine monophosphate modulates the number of gap junctions by increasing the rate of connexon assembly. Since gap junction proteins have a half-life of about 6 hours, there is a constant turnover of connexons at the cell surface.
Gap junctions are present between all cells of the enamel organ, suggesting that intercellular communication is necessary during all phases of enamel development.11,12,96,149,150 Immunocytochemical studies have shown that connexin 43 localizes in the SI, IEE, and preameloblasts.151 Information transferred across gap junctions may control cell proliferation and coordinate the activation and subsequent regulation of protein matrix secretion.
Large gap junctions are formed during enamel maturation. This may indicate that a bidirectional flow of ions from ameloblasts to papillary cells is a significant component of cellular activity during enamel maturation. Annular gap junctions are especially conspicuous in papillary cells.150 The latter are believed to represent stages in the internalization and breakdown of gap junctions.
Gap junction proteins have a rapid turnover time of approximately 5 hours. The functional significance of an apparent high turnover of gap junctions during the maturation phase remains to be explored.
Tight junctional complexes
Epithelial cells that are closely juxtaposed may participate in forming zones of fusion (tight junctions) between adjacent plasma membranes. For tight junctions to form, specific proteins must migrate from cytoplasmic pools to the cell surface to be inserted into the plasma membrane at points of cell-to-cell contact. Tight junctional contacts occur either as spotlike macula occludens, larger sheetlike fascia occludens, or as beltlike zonula occludens specializations.
The exact significance of the macula and fascia occludens junctions is unclear. Although these junctions cannot compartmentalize an extracellular space, they might provide increased cell-to-cell adhesion, or they might act as intramembrane stabilizers to restrict the lateral diffusion of other integral membrane proteins. In contrast, because the zonula occludens seals the extracellular space in a beltlike zone around the entire circumference of the cell, it compartmentalizes the extracellular space.
The zonula occludens plays two important functions in the physiology of epithelial layers. It provides a variable permeability barrier in the intercellular space, thereby creating isolated compartments and delineating luminal spaces. Second, by preventing lateral diffusion of integral proteins in the plasma membrane, it maintains specific domains in the cell membrane, such as the basolateral and apical surfaces of polarized cells.152
The transmembrane protein responsible for creating the seal is called occludin. It binds additional proteins, zonula occludens 1 and zonula occludens 2, on its cytoplasmic domain. The zonula occludens proteins are kinases that may have signaling functions involved in regulating the degree of paracellular permeability.153
Structural analysis of the zonula occludens by electron microscopic observation of freeze-fracture replicas of the plasma membrane has shown that tight junctional proteins (occludin) assemble in linear strands or fibrils