Figs 3-15a and 3-15b Freeze-fracture replica of the distal plasma membrane of a ruffle-ended maturation ameloblast (MAb). (a) Low magnification reveals the ruffled border (RB), components of the zonula occludens (ZO), and a gap junction (GJ). (Original magnification × 17,000.) (b) Higher magnification of the protoplasmic face (Pf). The tight junctional strands (S) of the zonula occludens are visible, as are depressions (D) created by the strands in the external face (Ef). (Original magnification × 80,000.)
Physiologic studies of epithelial permeability have shown that there is no clear correlation between the number and arrangement of tight junctional strands and the degree of intercellular occlusion. Some zonula occludens act as total barriers, while others (leaky tight junctions) permit the flow of ions and solutes through the paracellular space. Modulation of the contraction of the actomyosin ring (terminal web) associated with the zonula occludens and zonula adherens has been proposed as an explanation for differences in tight junctional permeability. Contraction of the actomyosin ring mediated by myosin light-chain kinase exerts tension on components of the zonula occludens, thereby altering the permeability of the paracellular space.154
The presence of a zonula occludens at the distal end of the secretory ameloblast, just proximal to Tomes process, suggests that the space into which the enamel matrix is deposited is isolated from the intercellular spaces of the enamel organ. The enamel mineralization compartment is bounded below by mineralized dentin and above by the ameloblasts joined together by zonula occludens junctions. Analysis of the fluid contained in this compartment indicates that it has a different composition than the general extracellular fluid and serum.
In addition to creating an intercellular barrier, the zonula occludens of the secretory ameloblast may stabilize the secretory domain of Tomes process (analogous to the development of a luminal membrane compartment in other polarized secretory cells). The zonula occludens of the ruffle-ended ameloblast may have a similar role in maintaining the ruffled border and sealing the intercellular space of the enamel organ from the enamel compartment.
Microtubules and motor proteins in secretion
Microtubules (MTs) form a key component of the cytoskeleton in all cells. They provide a scaffold on which organelles, vesicles, and secretory granules are translocated by the action of motor proteins. In addition, MTs act as rigid struts involved in maintaining cell shape. During mitosis, MTs assemble to form the spindle apparatus required for chromosomal segregation.
Each MT is a hollow cylinder constructed of 13 protofilaments of tubulin. Tubulin protofilaments are assembled from heterodimers of α and β tubulin molecules (Fig 3-16).155 The addition and removal of tubulin heterodimers takes place at opposite ends of an MT. The positive (+) end of an MT is the growing end, while the negative (–) end is the point of removal of tubulin. The removal of subunits at the negative end of an MT is slower than the rate of addition of new subunits at the positive end.
Fig 3-16 Microtubule assembly by parallel association of tubulin protofilaments. Each protofilament forms by binding heterodimers of α and β tubulin at the positive end of the microtubule. Heterodimers are added when they are in the guanosine triphosphate (GTP)–bound state.
Both α and β tubulins are guanosine triphosphate (GTP)-binding proteins. In the GTP-bound state, the β tubulin subunit has a high binding affinity, thereby favoring rapid addition of subunits at the growing end of the elongating protofilament.156 Hydrolysis of GTP on the β tubulin subunits destabilizes the protofilament structure, causing rapid depolymerization of the MT (Fig 3-17). Microtubules continue to grow as long as the rate of addition of GTP tubulin is faster than the rate of GTP hydrolysis.
Fig 3-17 Hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP). Hydrolysis of GTP on the β tubulin subunit destabilizes the protofilaments, leading to rapid depolymerization of microtubules.
Initiation of the polymerization of an MT requires the action of the microtubular organizing center (MTOC). The composition and mechanism of action of the MTOC is poorly understood. A third form of tubulin, γ tubulin, is found in MTOCs, where it performs a nucleating function. The most prominent MTOC is associated with the centrioles. Numerous MTOCs are located in the cytoplasm (the pericentriolar matrix) surrounding each pair of centrioles (Fig 3-18).
Fig 3-18 Microtubule organizing centers. Numerous microtubule organizing centers are located in the centrosomal matrix associated with the centrioles. Each microtubule organizing center nucleates the development of a microtubule and stabilizes the microtubule by capping the negative end.
The negative end of the growing MT is stabilized by components of the MTOC. This arrangement permits the polarized growth of MTs away from the MTOC and toward the peripheral cytoplasm (Fig 3-19). Microtubules radiate from the centrosome outward toward the plasma membrane, where the positive end of each MT is capped by special proteins.
Fig 3-19 Centrosome, consisting of a pair of centrioles and associated microtubule organizing centers. The centrosome regulates the polarization of the cellular microtubule network. The positive ends of the microtubules are located in the peripheral cytoplasm.
Microtubules are stabilized by interaction with capping proteins, microtubule-associated proteins, and by detyrosination (removal of tyrosine from the carboxy terminal of tubulin). Detyrosinated MTs constitute a small percentage of the total microtubular complement of the cell. They have a life span of about 2 hours. Most MTs are unstable. Unstable MTs are dynamic structures whose average life span is about 10 minutes.
Microtubules serve as conduits for the transport of organelles and vesicles.157–159 Transport requires the action of microtubule-associated motor proteins (motor MAPs) and ATP. The most widely studied motor MAPs are the kinesin and dynein families of motor proteins. Classic kinesin is composed of two heavy chains and two light chains.160 The heavy chain contains a large N-terminal globular head group with binding sites for ATP and tubulin. The tail portions, stabilized in a helical conformation, contain binding sites for various integral membrane proteins that are contained in the limiting membranes of organelles, vesicles, and granules (Fig 3-20). Dynein is a multimeric complex of heavy, intermediate, and light chains.
Motor MAPs transform the chemical energy released by the hydrolysis of ATP to adenosine diphosphate into mechanical displacement of the motor protein and its cargo along the surface of the MT (see Fig 3-20). It is unclear whether the movement of the motor protein and its cargo is caused by a conformational change (rachet power stroke) in the motor protein or by some form of biased diffusion along the MT surface. In general, kinesin transports cargo from the centrosome toward the peripheral cytoplasm, while dynein transports cargo in the opposite direction. For example, dynein has been shown to