Fine structure of the secretory and nonsecretory ameloblasts in the frog. I. Fine structure of the secretory ameloblasts.

Amelogenesis in the tooth germs of the frog Rana pipiens was examined by electron microscopy at different stages of tooth development. Cellular changes in secretory ameloblasts during this process showed many basic similarities to those in mammalian amelogenesis. Amelogenesis can be divided into three stages based on histological criteria such as thickness of enamel and the relative position of the tooth germ within the continuous succession of teeth. These stages are early, transitional and late. The fine structure of the enamel-secreting cells reflects the functional role of these ameloblasts as primarily secretory in the early stage, possibly transporting in the late stage and reorganizing between the two functions in the transitional stage. In early amelogenesis the cell exhibits well-developed granular endoplasmic reticulum, Golgi complex, microtubules, dense granules, smooth and coated vesicles, lysosome-like bodies in supranuclear and distal portions of the cell and mitochondria initially concentrated in the basal part of the cell. Numerous autophagic vacuoles are observed concomitant with the loss of some cell organelles at the transitional stage. During late amelogenesis the ameloblasts exhibit numerous vesicles, granules, convoluted cell membranes, junctional complexes and widely distributed mitochondria. Toward the end of amelogenesis, cells become oriented parallel to the enamel surface and the number of organelles is reduced. Amelogenesis in the frog is an extracellular process and mineralization seems to occur simultaneously with matrix formation.     

Immunocytochemical detection of estrogen receptors in a hormone-unresponsive mammary tumor

Summary A combination of two monoclonal antibodies and high resolution immunocytochemical technique was applied to label estrogen receptors in spontaneous mouse mammary tumors. Protein A-colloidal gold complex was used as an electron opaque marker. With this procedure estrogen receptors were labelled in the nuclei of cancer cells, predominantly over heterochromatin. In the cytoplasm a slight tagging of the rough endoplasmic reticulum was detected, apparently related with the sites of receptor biosynthesis. Other organelles and the mammary tumor viruses (MuMTV) were not stained immunocytochemically.The immunocytochemical procedure applied in this investigation allowed the detection of low levels of estrogen receptors in an estrogen-unresponsive mammary carcinoma. The presence of estrogen receptors with a specific distribution in estrogen-independent tumors suggests the need of a reevaluation of their capacity as indicators of hormone-dependence in mammary carcinomas.     

Immunocytochemical detection of estrogen receptors in a hormone-unresponsive mammary tumor

A combination of two monoclonal antibodies and high resolution immunocytochemical technique was applied to label estrogen receptors in spontaneous mouse mammary tumors. Protein A-colloidal gold complex was used as an electron opaque marker. With this procedure estrogen receptors were labelled in the nuclei of cancer cells, predominantly over heterochromatin. In the cytoplasm a slight tagging of the rough endoplasmic reticulum was detected, apparently related with the sites of receptor biosynthesis. Other organelles and the mammary tumor viruses (MuMTV) were not stained immunocytochemically. The immunocytochemical procedure applied in this investigation allowed the detection of low levels of estrogen receptors in an estrogen-unresponsive mammary carcinoma. The presence of estrogen receptors with a specific distribution in estrogen-independent tumors suggests the need of a reevaluation of their capacity as indicators of hormone-dependence in mammary carcinomas.     

Immunoglobulin heavy chain-binding protein binds to misfolded mutant insulin receptors with mutations in the extracellular domain.

Cell-surface proteins are transported through the endoplasmic reticulum and Golgi apparatus en route to the plasma membrane. Previously, we have identified three point mutations in the insulin receptor gene that impair transport of the mutant receptors to the cell surface: Asn15----Lys, His209----Arg, and Phe382----Val. Furthermore, these mutations impair post-translational processing steps that normally occur as the receptors are transported through the endoplasmic reticulum and Golgi apparatus. In this study, we have demonstrated that the unprocessed Arg209 and Val382 mutant proreceptors are bound to the immunoglobulin heavy chain-binding protein (BiP) in the endoplasmic reticulum. This was demonstrated by the fact that monoclonal anti-BiP antibody coimmunoprecipitated the mutant proreceptors. Moreover, when ATP was added to the immunoprecipitates, the mutant proreceptors were released from BiP. In contrast, neither the normal human insulin receptor nor the Lys15 mutant proreceptor was coimmunoprecipitated by anti-BiP antibody. It seems likely that the Lys15 receptor also binds BiP, but that the affinity was too low to resist dissociation during the stringent washing of the immunoprecipitate. In conclusion, these observation are consistent with the hypothesis that binding to BiP explains the impaired transport of mutant receptors through the endoplasmic reticulum and Golgi apparatus to the plasma membrane.     

Olfactory receptor trafficking involves conserved regulatory steps

Olfactory receptors are difficult to functionally express in heterologous cells. They are typically retained in the endoplasmic reticulum of cells commonly used for functional expression studies and are only released to the plasma membrane in mature cells of the olfactory receptor neuron lineage. A recently developed olfactory cell line, odora, traffics olfactory receptors to the plasma membrane when differentiated. We found that undifferentiated odora cells do not traffic olfactory receptors to their surface, even though they release the receptors to the Golgi apparatus and endosomes. This behavior differs from other cell lines tested thus far. Differentiated odora cells also properly traffic vomeronasal receptors of the VN1 type, which lack sequence similarity to olfactory receptors. ODR-4, a protein that is necessary for plasma membrane trafficking of a chemosensory receptor in nematodes, facilitates trafficking of rat olfactory receptor U131 in odora and Chinese hamster ovary cells. Olfactory receptor trafficking from the endoplasmic reticulum to the plasma membrane involves at least two steps whose regulation depends on the maturation state of cells in the olfactory receptor neuron lineage. These results also indicate that some components of the regulatory mechanism are conserved.     

Phosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes

beta-Hexosaminidase B purified from human fibroblast secretions was used as a ligand to study phosphomannosyl-enzyme receptors in membranes from rat tissues. Enzyme binding to rat liver membranes was saturable, competitively inhibited by mannose 6-phosphate, not dependent on calcium, and destroyed by prior treatment of the hexosaminidase with either alkaline phosphatase or endoglycosidase H. Most (90%) of the phosphomannosyl-enzyme receptors were found in endoplasmic reticulum, Golgi apparatus, and lysosomes; 9.5% in the plasma membrane, and less than 1% in nuclei and mitochondria. Receptors were vesicle-enclosed in all fractions except plasma membrane. Receptors in the endoplasmic reticulum apparently were occupied by endogenous ligands, but most receptors in lysosomes and plasma membrane were unoccupied. Most of the endogenous beta-hexosaminidase was in lysosomes and was released from vesicles by detergent treatment. Displacement of the residual receptor-bound endogenous beta-hexosaminidase (mostly in endoplasmic reticulum and Golgi apparatus) from detergent-treated membranes by mannose 6-phosphate released high uptake enzyme with properties expected for phosphomannosyl-enzymes. Mannose 6-phosphate-inhibitable enzyme receptor activity was found in nine rat organs and correlated roughly with their lysosomal enzyme content. These data support a general model for lysosomal enzyme transport in which the phosphomannosyl-enzyme receptor acts as a vehicle for delivery of newly synthesized acid hydrolases from the endoplasmic reticulum to lysosomes.     

Properties and biosynthetic connection of the nucleotide pyrophosphatases of rat liver plasma membrane and endoplasmic reticulum

The detergent-solubilized nucleotide pyrophosphatases of the rat liver plasma membrane and endoplasmic reticulum fractions were purified by lectin affinity chromatography. They have the same molecular mass of 148 000 dalton; their catalytic properties are also very similar and correspond to those of the trypsin-solubilized activities from the same membrane preparations. Pulse-chase experiments on isolated perfused livers using [3H]leucine indicated different labelling kinetics of the proteins isolated from plasma membrane and endoplasmic reticulum. The plasma membrane enzyme became only slightly labelled in the presence of 100 microM vinblastine. The data support a precursor-product relationship of the nucleotide pyrophosphatases from endoplasmic reticulum and plasma membrane.     

Intracellular distribution of Sindbis virus membrane proteins in BHK-21 cells infected with wild-type virus and maturation-defective mutants.

The association of Sindbis virus proteins with cellular membranes during virus maturation was examined by utilizing a technique for fractionating the membranes of BHK-21 cells into three subcellular classes, which were enriched for rough endoplasmic reticulum, smooth endoplasmic reticulum, and plasma membrane. Pulse-chase experiments with wild-type (strain SVHR) virus-infected cells showed that virus envelope proteins were incorporated initially into membranes of the rough endoplasmic reticulum and subsequently migrated to the smooth and plasma membrane fractions. Large amounts of capsid protein were associated with the plasma membrane fraction even at the earliest times postpulse, and relatively little was found associated with the other membranes, suggesting a rapid and preferential association of nucleocapsids with the plasma membrane. We also examined the intracellular processing of the proteins of two temperature-sensitive Sindbis virus mutants in pulse-chase experiments at the nonpermissive temperature. Labeled virus proteins of mutant ts-20 (complementation group E) first appeared in the rough endoplasmic reticulum and were then transported to the smooth and plasma membrane fractions, as in wild-type (strain SVHR) virus-infected cells. In cells infected with ts-23 (complementation group D), the pulse-labeled virus proteins appeared initially in the rough membrane fraction and were transported to the smooth membrane fraction, but only limited amounts reached the plasma membrane. Thus, in ts-23-infected cells, the transport of the virus-encoded proteins from the smooth membranes seemed to be defective. In both ts-20- and ts-23-infected cells the envelope precursor polypeptide PE2 was not processed to E2, and no label was incorporated into free virus at the nonpermissive temperature.     

Fine structural changes and lysosomal phosphatase cytochemistry of ameloblasts associated with the transitional stage of enamel formation in the rat incisor.

Trimetaphosphatase (TMPase) and cytidine-5'-monophosphatase (CMPase) were used as lysosomal markers in the transitional ameloblasts (TA) to investigate the distribution of lysosomal structures and to correlate the cytochemical findings with the ultrastructural features of these cells. Of particular interest were the cytochemical and morphological changes which occur as the ameloblasts approach the maturation stage of enamel formation. The sequence of changes observed provides a basis for designation of three regions of the transitional zone (early and late TA and modulating ameloblasts). In the early TA region, the cells decreased in height and contained phagic vacuoles as well as numerous TMPase and CMPase reactive structures. Late transitional ameloblasts had invaginations at their distal ends as well as membrane-bound structures, both filled with fine granular material. Dense bodies, phagic vacuoles, and other elements of the lysosomal system were enzyme reactive. Modulating ameloblasts lacked the phagic vacuoles but exhibited large numbers of multivesicular bodies, vesicles, and secretory granules. Their distal ends were morphologically altered indicating a change towards ruffle- or smooth-ended varieties of maturation ameloblast. In the former, increased granular material was observed within cell membrane invaginations and associated membrane-bound structures. In the latter, intercellular spaces widened and were filled with granular material. The present cytochemical findings of an extensive lyosomal system in transitional ameloblasts confirm the function of those cells in reducing the secretory ameloblast population and in the selective elimination of their protein-synthesizing organelles. Furthermore, this extensive lysosmal system and the present morphological findings are consistent with a potential role for transitional ameloblasts in contributing to the marked loss of enamel protein known to occur during maturation.     

Passage of viral membrane proteins through the Golgi complex

BHK-21 cells, infected with Semliki Forest virus, were treated with cycloheximide to stop further synthesis but not intracellular transport of the viral membrane proteins. These proteins were then localized in thin, frozen sections using specific antibodies labelled indirectly with ferritin or gold. Quantitation of the labelling on micrographs showed the movement of spike proteins from the rough endoplasmic reticulum and through the Golgi stacks. The spike proteins spent about 15 minutes in each of these intracellular organelles and their final destination was the plasma membrane. Parallel biochemical studies showed that most of the simple oligosaccharides on the viral spike proteins were modified to the complex form at the same time as these membrane proteins were passing through the Golgi stacks. Cell fractionation studies revealed the same pattern; the proteins passed from the rough endoplasmic reticulum to the plasma membrane via a vesicle fraction isolated according to its content of galactosyl transferase. Independent evidence that this fraction was derived at least in part from the Golgi complex in BHK cells was obtained by showing that it reacted specifically with an antibody raised to rat liver Golgi membranes.     

Immuno-gold localisation of arabinogalactan protein in the developing style of Nicotiana alata

Arabinogalactan-protein was localised ultrastructurally in the transmitting tissue of the Nicotinana alata Link & Otto style from the bud stage to anthesis using monoclonal antibody directed to terminal α-L-arabinosyl residues followed by goat anti-mouse IgG linked to 20 nm gold particles. Gold particles were localised on the Golgi apparatus, multivesicular bodies, cell wall and intercellular matrix with the highest concentration over multivesicular bodies. During flower development there was an increase in the total number of gold particles with a relative decrease on the multivesicular bodies and an increase on the intercellular matrix. There was also an increase during development in the proportion of intercellular matrix relative to all other components of the transmitting tissue. Vesiculation of the endoplasmic reticulum and the production of cup-shaped vesicles by the Golgi bodies were observed during development. It is concluded that secretion of arabinogalactan-protein from the intracellular to the extracellular location occurs via multivesicular bodies produced by the Golgi apparatus and possibly the endoplasmic reticulum.     

Ontogeny of insulin-like growth factor I and insulin receptor kinase activity in rat liver.

IGF-I and insulin receptors possess tyrosine-kinase enzymatic activity considered to be essential for signal transduction and thereby mediating the putative effects of these hormones on fetal growth and development. We investigated the ontogeny of IGF-I and insulin receptor tyrosine-kinase activity in at least 3 separate membrane preparations from liver of rats at 21 day of embryonic life (21ED), 1 and 5 day of postnatal life (1PD and 5PD respectively) and adult. Receptors purified by wheat germ agglutinin chromatography (WGA) were exposed to graded concentrations of IGF-I or insulin, and tyrosine-kinase activity was measured by quantifying incorporation of 32P into the exogenous substrate poly[Glu,Tyr; 4:1]. IGF-I stimulated tyrosine-kinase solely at 1 PD as documented by a maximal increase of 346 +/- 167% over basal kinase activity with 6.6 nmol/L IGF-I. While the lack of response in adult animals could be explained by a striking decrease in receptors at that age, 125I-IGF-I binding and affinity labelling of the WGA preparations indicated substantial IGF-I receptors were present in the liver at each of the perinatal ages. Furthermore, this dissociation between IGF-I binding and the tyrosine-kinase activity of these IGF-I receptors could not be attributed to the presence/absence of IGF-I binding proteins as judged by affinity labelling. In contrast, insulin-stimulated tyrosine-kinase activity was observed at all ages tested although it appeared greatest at 1PD. We conclude that (i) expression of IGF-I tyrosine-kinase activity is linked to developmental events and differs from that found for the insulin receptor tyrosine-kinase activity, (ii) during the perinatal period there is an apparent dissociation between ligand binding by the IGF-I receptor and receptor tyrosine-kinase activity. These observations suggest modulation of IGF-I receptor tyrosine-kinase activity may be an important regulator of IGF-I action during the perinatal period.     

Noninvasive imaging of 5-HT3 receptor trafficking in live cells: from biosynthesis to endocytosis

Sequential stages in the life cycle of the ionotropic 5-HT(3) receptor (5-HT(3)R) were resolved temporally and spatially in live cells by multicolor fluorescence confocal microscopy. The insertion of the enhanced cyan fluorescent protein into the large intracellular loop delivered a fluorescent 5-HT(3)R fully functional in terms of ligand binding specificity and channel activity, which allowed for the first time a complete real-time visualization and documentation of intracellular biogenesis, membrane targeting, and ligand-mediated internalization of a receptor belonging to the ligand-gated ion channel superfamily. Fluorescence signals of newly expressed receptors were detectable in the endoplasmic reticulum about 3 h after transfection onset. At this stage receptor subunits assembled to form active ligand binding sites as demonstrated in situ by binding of a fluorescent 5-HT(3)R-specific antagonist. After novel protein synthesis was chemically blocked, the 5-HT(3) R populations in the endoplasmic reticulum and Golgi cisternae moved virtually quantitatively to the cell surface, indicating efficient receptor folding and assembly. Intracellular 5-HT(3) receptors were trafficking in vesicle-like structures along microtubules to the cell surface at a velocity generally below 1 mum/s and were inserted into the plasma membrane in a characteristic cluster distribution overlapping with actin-rich domains. Internalization of cell surface 5-HT(3) receptors was observed within minutes after exposure to an extracellular agonist. Our orchestrated use of spectrally distinguishable fluorescent labels for the receptor, its cognate ligand, and specific organelle markers can be regarded as a general approach allowing subcellular insights into dynamic processes of membrane receptor trafficking.     

LOCALIZATION OF ANTIBODIES BY ELECTRON MICROSCOPY IN DEVELOPING ANTIBODY-PRODUCING CELLS

The development of antibody-producing cells in the early stages of the secondary or hyperimmune response has been studied with the electron microscope in lymph nodes of adult chinchilla rabbits immunized with ferritin or apoferritin. The intracellular distribution of antiferritin antibodies was determined in the lymph node cells at 1 to 5 days after a booster injection, employing the labelling technique previously used by the authors (12) to demonstrate the localization of antibodies in mature plasma cells. Antibodies were first detected at 48 hours in blasts; i.e., cells which have a poorly developed endoplasmic reticulum and a cytoplasm filled with many ribosomes grouped in clusters. The label was subsequently found in forms intermediate between blasts and plasma cells (plasmoblasts, immature plasma cells), in which the endoplasmic reticulum appeared progressively more developed. Antiferritin antibodies were also found in cells in mitosis. In all the above cell types, antigen-antibody precipitates were consistently found in the perinuclear space and in the cisternae of the granular endoplasmic reticulum, from an early stage in the development of the latter. Evidence was also obtained for the presence of antibody in the Golgi area. The results are discussed in relation to the possible cellular sites of antibody synthesis.     

Immunocytochemical localization of the intrinsic factor-cobalamin receptor in dog-ileum: distribution of intracellular receptor during cell maturation

Absorption of cobalamin is facilitated by the binding of the intrinsic factor-cobalamin complex (IF-cbl) to specific receptors in the ileum. The physical and biochemical characteristics of this ligand-receptor binding reaction have been extensively studied, but little is known about the cellular mechanisms or receptor synthesis, intracellular transport, and expression on the microvillus surface membrane. We attempted to delineate these mechanisms by using ultrastructural immunocytochemistry to localize the IF-cbl receptor in the crypt, mid-villus, and villus tip regions of mucosal biopsies obtained from the ileum of anesthetized dogs. Prior to initiating the ileal localization studies, the antisera to purified canine IF-cbl receptor that was employed in our studies was shown to have specificity for site (e.g., ileal enterocytes vs. other cells within the gastrointestinal tract) and immunohistochemical specificity. Receptor synthesis in endoplasmic reticulum begins in crypt enterocytes, but continues in cells throughout the villus. In the mid-villus region synthesized receptor translocates vectorially to the microvillus surface associated with membranous vesicles and then inserts into the microvillus pit. Receptor remains fixed to the microvillus pit and does not distribute uniformly over the brush border membrane. All villus tip enterocytes contained IF-cbl receptor in microvillus pits, vesicles, and endoplasmic reticulum, but in addition extensive perinuclear membrane staining was evident as well as re-internalized receptor associated with multivesicular bodies. Basolateral membranes contained no receptor at any level of the villus. These observations suggest that the IF-cbl receptor (a) translocates to the apical cell surface at the mid-villus region by transport in vesicles, (b) directly inserts into and then remains fixed in microvillus pits, (c) is elaborated on the luminal surface most extensively in villus tip cells, and (d) although reinternalized, does not move IF and/or cbl to the basolateral cell surface.     

The ultrastructure of human secretory ameloblasts

Summary The ultrastructure of secreting ameloblasts of deciduous teeth from a human foetus (crown-rump length 195 mm) was investigated. The ameloblasts demonstrate a formation of granules in a juxtanuclear Golgi complex. In the Tomes' process the granules are released either through the lateral plasma membrane into the intercellular space between the Tomes' processes or directly through the apical plasma membrane into the enamel.The human ameloblasts differ from non-human ameloblasts in having a non-oriented vesicular granular endoplasmic reticulum. Further, the majority of mitochondria are situated in the apical part of the ameloblast adjacent to the Tomes' process.We would like to thank chief-surgeon A. Christensen, Bispebjerg Hospital, Copenhagen for his help in acquiring foetal material. For technical assistance we would like to thank M. Balslev and U. Eberth, Anatomy Department A.     

The ultrastructure of macrogametes of Eimeria ferrisi Levine and Ivens 1965 in Mus musculus.

Macrogametes of Eimeria ferrisi occurred in epithelial cells of the cecum and colon of Mus musculus and were studied by electron microscopy. Young stages were identified as macrogamonts by the presence of wall-forming bodies. At first an outerlimiting membrane and remnants of the inner membrane complex of the former merozoite pellicle were present; the latter was later lost but in mature macrogametes 3 limiting membranes were observed. Type II wall-forming bodies appeared before type I; the former developed in expanded cisternae of the endoplasmic reticulum whereas the latter were smaller in size and appeared in the ground substance of the cytoplasm. After formation of the oocyst wall the bodies of the 2 types were no longer visible. The presenceodies of the 2 types were no longer visible. The persistence of micronemes in mature macrogametes and the presence of numerous layers of rough endoplasmic reticulum during wall formation have not been previously reported.     

Epoxide hydrolase is a marker for the smooth endoplasmic reticulum in rat liver.

Epoxide hydrolase (EH, EC 3.3.2.3) was chosen as a potential marker for smooth endoplasmic reticulum, because this enzyme is inducible by drugs such as phenobarbital. The hypothesis was verified in rat liver using immunochemical and immunocytochemical techniques. Antibodies were raised to the purified protein. These antibodies were affinity purified using the enzyme immobilized on Sepharose Ultrogel. The specificity of the antibodies was assayed by immunoelectrotransfer (Western blot). The labelling of rat liver thin frozen sections with protein A-gold particles demonstrated that the antibodies specifically recognised smooth endoplasmic reticulum membranes. Rough endoplasmic reticulum, other intracellular organelles and plasma membrane were unlabelled.     

Affinity cytochemical differentiation of glycoconjugates of small intestinal absorptive cells using Pisum sativum and Lens culinaris lectins

We studied the subcellular localization of glycoconjugates recognized by the garden pea and lentil lectins (Pisum sativum, PSA; Lens culinaris, LCA) in mature absorptive cells of duodenum and jejunum of fasted rats. PSA and LCA are mannose-, glucose-, and N-acetyl-glucosamine-recognizing lectins that bind with high affinity to fucosylated core regions of N-glycosidically linked glycans. The binding reactions were cytochemically demonstrated in a pre-embedment incubation system using peroxidase-labeled lectins. Both pea and lentil lectins bound with constituents of nuclear envelope and endoplasmic reticulum, cisternae of the Golgi apparatus, several Golgi-associated vesicles, lysosomes, and portions of the plasma membrane. PSA and LCA label was non-homogeneous in the endoplasmic reticulum; in the Golgi apparatus the reactions were most intense in the cis and medial cisternae of the stacks. For inhibition of the intense reactions apparent in the Golgi apparatus, in lysosomes, and at the plasma membrane, considerably higher concentrations of competitive sugars were necessary than for abolition of the endoplasmic reticulum label. This indicates that endoplasmic reticulum glycoconjugates bind at low affinities with pea and lentil lectins, and that high-affinity PSA/LCA-binding glycoconjugates, which may correspond to corefucosylated N-linked glycans, predominate in cis and medial Golgi cisternae, lysosomes, and at the plasma membrane.     

Cytochemical localization of Ca2+-Mg2+ adenosine triphosphatase in rat incisor ameloblasts during enamel secretion and maturation

A modified Wachstein-Meisel medium containing lead or cerium as capturing ions was used to localize Ca2+-Mg2+ adenosine triphosphatase (ATPase; EC 3.6.1.3) in rat incisor ameloblasts during enamel formation. Sections representing different developmental stages were processed for electron microscopic cytochemistry. Distribution and intensity of the observed reaction product, which was almost exclusively associated with cell membranes, varied according to the stage of enamel formation. During the secretory stage, intense reaction product was evident along the entire plasma membrane of ameloblasts and papillary cells. The early transitional ameloblasts showed reaction product on their proximal and lateral cell membranes, but not distally. In late transitional (pre-absorptive) ameloblasts, distal cell membranes exhibited intense reaction product. During enamel maturation, smooth-ended ameloblasts showed reaction product proximally and laterally, but not distally. Ruffle-ended maturative ameloblasts exhibited intense reaction product along their lateral and distal membranes. The intensity of the latter was decreased but not eliminated by levamisole. In the transition from smooth-ended to ruffle-ended cells, the reaction product became evident distally, concomitant with the appearance of cell membrane invaginations. These data are consistent with a possible role for Ca2+-Mg2+ ATPase in controlling calcium availability at the enamel mineralization front.