Docking of the transport vesicle to the target membrane occurs when vesicle SNAREs (v-SNAREs) bind to their “target” membranes (t-SNAREs) (see Fig 2-14). Rab-GTP, a second type of monomeric guanosine triphosphatase, present in the vesicle membrane, functions as a monitor and stabilizer of the fit between the two types of SNARE molecules. A guanosine triphosphatase–activating protein in the target membrane causes ARF-GTP to hydrolyze GTP to GDP. In its GDP-binding state, ARF retracts its fatty acid anchor and detaches from the vesicle membrane. Simultaneously, the ARF-coatomer complex disassembles.
Fig 2-14 Formation, docking, and fusion of the transport vesicle. The coatomer-coated vesicle, with soluble N-ethylmaleimide–sensitive fusion attachment protein receptor (v-SNARE) molecules exposed beyond the coatomer coating, is available for binding (step 2, docking reaction) to the appropriate target membrane SNARE (t-SNARE) molecules. During the docking reaction, adenosine diphosphate ribosylation factor–guanosine triphosphate is hydrolyzed to adenosine diphosphate ribosylation factor–guanosine diphosphate and disassociates from the vesicle membrane. Coatomer proteins are also released. The attachment of v-SNARE to t-SNARE is monitored and stabilized by a second type of guanosine triphosphate, a Rab-GTP molecule present in the donor vesicle membrane. Fusion of the transport vesicle membrane to the target (step 3) is induced by a protein complex that includes N-ethylmaleimide–sensitive fusion protein (NSF-P) and soluble NSF attachment proteins (SNAPs).
For membrane fusion to occur, special fusion proteins are required to displace water molecules and to overcome the electrostatic repulsive forces between the two closely juxtaposed lipid membranes.198 The space between the adjacent membranes must be reduced to less than 1.5 nm. The v-SNARE to t-SNARE receptor-ligand docking reaction between the transport vesicle and its target membrane recruits fusion proteins to the site of attachment. N-ethylmaleimide– sensitive fusion (NSF) protein and soluble NSF attachment proteins (SNAPs) have been shown to carry out fusions in eukaryotic cells by interacting with SNAREs (see Fig 2-14). Conformational changes in the fusion protein, driven by ATP, destabilize the lipid membranes, leading to the formation of a fusion pore that rapidly expands to permit total fusion of the two membranes. Exactly how this machinery is assembled and how it functions during the fusion event is still speculative.
Application of this new knowledge of the regulation of cytoplasmic traffic must be applied to future studies of the odontoblastic Golgi complex to gain a clearer understanding of the secretion and retrieval of dentin matrix components.
From the trans-Golgi network, there are two basic pathways of secretion: the regulated pathway and the constitutive pathway.199,200 In the regulated pathway, secretory product is stored in vesicles or granules until secretion is triggered by an appropriate signal. Secretory granule formation involves condensation of the secretory product from larger condensing vacuoles (presecretory granules) considered to be part of the trans-Golgi network (see Fig 2-13).201 Budding of membrane from the condensing vacuole continues, until a smaller and denser secretory granule is formed. In the constitutive pathway, products are exported immediately after they are packaged in the Golgi apparatus.
Regulated secretion requires an intact microtubular system to transport granules to a specific region of the cell surface, usually the apical end of the cell. Constitutive secretion does not appear to be dependent on microtubules and may occur from many regions of the cell surface.
Microtubules form a radiating network, extending from the centrosome outward to the cell periphery. This network provides a structural pathway for the translocation of secretory granules. The energy source for granule transport is derived from the hydrolysis of ATP. Enzymatic motor proteins (mechanochemical ATPases) associated with the granule membrane hydrolyse ATP molecules when activated by contact with the microtubules.
Cyclical attachment and detachment produces movement of the granule along the length of the microtubule. The mechanochemical ATPase responsible for anterograde movement of secretory granules is a member of the kinesin family of motor proteins. Substances that interfere with microtubule assembly, such as antimitotic (antispindle) agents, and substances that deplete ATP or stop its production produce abnormalities in deposition of dentin, enamel, and bone matrices.
Microtubule-directed transport delivers secretory granules to the periphery of the secretory pole of the cell. At that point, further transport toward the plasma membrane is dependent on myosin. The final approach and fusion is both nonmicrotubule and nonmyosin dependent.202 Discussion of microtubules is continued in chapter 3.
A proposed pathway for secretion in the odontoblast, based on ultrastructural studies of the odontoblast and the current theories of Golgi organization, is outlined in Fig 2-15. Additional studies of odontoblast structure and function are needed to determine whether there are multiple forms of secretory granules in odontoblasts and to identify the signals that direct secretory granule discharge.
Fig 2-15 Odontoblast secretory pathway. Intermediate coated transport vesicles (CTV) bud from the rough endoplasmic reticulum (RER) and migrate to the cis-Golgi network (CGN), where they fuse with the outermost cisternae of the Golgi stack. Presecretory granules (PSG) form part of the trans-Golgi network (TGN). Concentration of the secretory product occurs by aggregation of proteins inside the PSGs and by the removal of fluid and membrane via budding of small vesicles. Vesicles containing membrane proteins and lipids are secreted in the constitutive pathway. Matrix secretory granules (SG) are transported via the regulated pathway, along microtubules, to the odontoblastic process.
Secondary, tertiary (reactive), and reparative dentin
Odontoblasts are nondividing cells with a long life span. During tooth development, they produce primary dentin at the rate of about 4 to 8 μm per day. Once the crown is completed and the apical length of the root has been established, odontoblasts produce secondary dentin at 1 to 2 μm per day. Histologic studies of human teeth have shown that all teeth contain secondary dentin. It is deposited throughout life as long as the pulp remains vital.
Although there are no reliable data on the estimated longevity of individual odontoblasts, the continued slow deposition of secondary dentin suggests that odontoblasts are long-lived cells. Odontoblasts lose nearly half of their RER and Golgi profiles following formation of primary dentin.203 Biochemical studies indicate that, on completion of primary dentin, there is an 80% reduction in alkaline phosphatase activity at the predentin-odontoblast region, and a concomitant reduction of ATPase activity in the odontoblasts. This reduced metabolic function is consistent with a slow production of secondary dentin.
Secondary dentin is characterized by a regular arrangement of dentinal tubules, usually in direct continuity with those of the primary dentin. Micro-hardness measurements indicate that secondary dentin is about 30% to 40% softer than primary dentin. The biochemical and matrix factors responsible for the decrease in mineral content have not been identified.
Tertiary or reactive dentin is produced in response to nonlethal irritation