Research

Ion transport in enamel

Rodrigo Lacruz has worked on enamel and teeth for over 15 years first detailing changes in daily growth lines in enamel in teeth of human ancestors and then investigating ion transporters in enamel cells. His previous research focused on pH regulation in enamel forming cells (ameloblasts) including investigating the role of bicarbonate exchangers (NBCe1) and carbonic anhydrases (Fig 1).

Model for transepithelial ion transport in maturation stage ameloblasts: Ameloblasts are represented here during the ruffled-border stage in maturation, when cells increase their transport functions. The cell at left depicts the main components of Ca2+ handling, whereas the cells at right represents proteins involved in the maintenance of pH balance in the extracellular space. The Ca2+ handling toolkit in ameloblasts includes ORAI1 as the main Ca2+ influx channel, activated by STIM1 upon depletion of ER Ca2+ stores. Activation of STIM1 likely involves the IP3 receptors and ER refilling is provided by SERCA. Our unpublished data also suggest that the mitochondrial Ca2+ uniporter (MCU) might be involved in buffering SOCE. Ca2+ extrusion is handled largely by NCKX4, with NCX and PMCA being associated with a housekeeping role in the outward movement of Ca2+. Bicarbonate transport and pH regulation (cell at right) are important functions in enamel cells. HCO3- uptake is mediated by NBCe1 at the basal pole and is also generated within the cells by CA2. HCO3- transfer outside the cell is provided by a number of exchangers including AE2 and several members of the SLC26a gene family (SAT1, DRA, Pendrin, PAT1, SUT1). Potentially, the CFTR outward transport of Cl- may work in tandem with the SLC26a proteins which would extrude HCO3- in exchange for Cl-. H+ generated by mitochondrial respiration and the activity of CA2 are transferred outside the cell by NHE. Mg2+ extrusion is likely mediated by CNNM4, with TRPM7 possibly mediating its uptake distally, although the localization of TRPM7 is yet to be elucidated in detail. Likewise, the proposal that NaPi plays a role in the outward transfer of Pi, requires additional testing.

_{Figure 1: Paine ML, Boyde A, Lacruz RS (in press) Transport functions of ectoderm epithelial cells forming dental enamel. Hamilton K and Devor D. C. (Eds). Physiology in Health and Diseases Series. APS/Springer-Verlag.}

Calcium

As a result of a gene screening of enamel cells reported in 2013 by Lacruz and collaborators, multiple Ca2+elements were found to be upregulated during the process of enamel mineralization (aka maturation). This work formed the basis for subsequent work on Ca2+ channels including the CRAC channels mediated by the Ca2+sensors STIM1 and STIM2 found in the membrane of the endoplasmic reticulum, and ORAI proteins (ORAI1,2,3) which form the pore of the channel in the cell membrane. The work by the Lacruz Lab identified the expression of CRAC genes in ameloblasts and functionally described the involvement of SOCE as the main Ca2+influx channel in enamel (Fig.1)

New research avenues

Lacruz was recently awarded a multi-PI NIH R01 grant with a colleague at the NYU School of Medicine to study the role of SOCE in the etiology of an autoimmune disease known as Sjogren’s syndrome that affects the function of salivary gland leading to hyposalivation.

His recent research includes investigations into the role of fluoride in enamel cells. This work will also be expanded to include other cell types that are known to be affected in fluorosis.

Facial growth

Lacruz’s training in palaeontolgy and palaeoanthropology was important to pursue scientific questions that merged development with function or skeletal form. His interest in growth a development resulted in several studies that reported changes in facial development in early hominins, including Neanderthals (Fig.3).

Experimental tools and approaches

We study Ca2+ signaling and mitochondrial function in live cells and use several murine models.

• Calcium imaging: We use a NIKON TIE2 inverted microscope fully automated with filters for various wavelengths capable of imaging 3-4 channels simultaneously (e.g. imaging cytosolic, ER and mitochondrial calcium simultaneously), and a Flexstation plate reader
• Imaging: We use confocal and imaging to evaluate subcellular localization of several proteins
• We use several probes and Seahorse Flux Analyzer to study mitochondrial function as well as reporter mice with cytosolic and mitochondrial fluorescent probes to measure ROS
• We use electron microscopy to visualize surface details in enamel crystals by SEM and TEM to evaluate cell ultrastructure
• Electrophysiology approaches to study channel properties in cells