Bioelectric signaling: Reprogrammable circuits underlying embryogenesis, regeneration, and cancer.

How are individual cell behaviors coordinated toward invariant large-scale anatomical outcomes in development and regeneration despite unpredictable perturbations? Endogenous distributions of membrane potentials, produced by ion channels and gap junctions, are present across all tissues. These bioelectrical networks process morphogenetic information that controls gene expression, enabling cell collectives to make decisions about large-scale growth and form. Recent progress in the analysis and computational modeling of developmental bioelectric circuits and channelopathies reveals how cellular collectives cooperate toward organ-level structural order. These advances suggest a roadmap for exploiting bioelectric signaling for interventions addressing developmental disorders, regenerative medicine, cancer reprogramming, and synthetic bioengineering.

A Neuro-hormonal Circuit for Paternal Behavior Controlled by a Hypothalamic Network Oscillation.

Parental behavior is pervasive throughout the animal kingdom and essential for species survival. However, the relative contribution of the father to offspring care differs markedly across animals, even between related species. The mechanisms that organize and control paternal behavior remain poorly understood. Using Sprague-Dawley rats and C57BL/6 mice, two species at opposite ends of the paternal spectrum, we identified that distinct electrical oscillation patterns in neuroendocrine dopamine neurons link to a chain of low dopamine release, high circulating prolactin, prolactin receptor-dependent activation of medial preoptic area galanin neurons, and paternal care behavior in male mice. In rats, the same parameters exhibit inverse profiles. Optogenetic manipulation of these rhythms in mice dramatically shifted serum prolactin and paternal behavior, whereas injecting prolactin into non-paternal rat sires triggered expression of parental care. These findings identify a frequency-tuned brain-endocrine-brain circuit that can act as a gain control system determining a species' parental strategy.

A Circadian Clock in the Blood-Brain Barrier Regulates Xenobiotic Efflux.

Endogenous circadian rhythms are thought to modulate responses to external factors, but mechanisms that confer time-of-day differences in organismal responses to environmental insults/therapeutic treatments are poorly understood. Using a xenobiotic, we find that permeability of the Drosophila "blood"-brain barrier (BBB) is higher at night. The permeability rhythm is driven by circadian regulation of efflux and depends on a molecular clock in the perineurial glia of the BBB, although efflux transporters are restricted to subperineurial glia (SPG). We show that transmission of circadian signals across the layers requires cyclically expressed gap junctions. Specifically, during nighttime, gap junctions reduce intracellular magnesium ([Mg2+]i), a positive regulator of efflux, in SPG. Consistent with lower nighttime efflux, nighttime administration of the anti-epileptic phenytoin is more effective at treating a Drosophila seizure model. These findings identify a novel mechanism of circadian regulation and have therapeutic implications for drugs targeted to the central nervous system.

The intercalated disc: a unique organelle for electromechanical synchrony in cardiomyocytes.

The intercalated disc (ID) is a highly specialized structure that connects cardiomyocytes via mechanical and electrical junctions. Although described in some detail by light microscopy in the 19th century, it was in 1966 that electron microscopy images showed that the ID represented apposing cell borders and provided detailed insight into the complex ID nanostructure. Since then, much has been learned about the ID and its molecular composition, and it has become evident that a large number of proteins, not all of them involved in direct cell-to-cell coupling via mechanical or gap junctions, reside at the ID. Furthermore, an increasing number of functional interactions between ID components are emerging, leading to the concept that the ID is not the sum of isolated molecular silos but an interacting molecular complex, an "organelle" where components work in concert to bring about electrical and mechanical synchrony. The aim of the present review is to give a short historical account of the ID's discovery and an updated overview of its composition and organization, followed by a discussion of the physiological implications of the ID architecture and the local intermolecular interactions. The latter will focus on both the importance of normal conduction of cardiac action potentials as well as the impact on the pathophysiology of arrhythmias.

Large-pore connexin hemichannels function like molecule transporters independent of ion conduction.

Connexin hemichannels were identified as the first members of the eukaryotic large-pore channel family that mediate permeation of both atomic ions and small molecules between the intracellular and extracellular environments. The conventional view is that their pore is a large passive conduit through which both ions and molecules diffuse in a similar manner. In stark contrast to this notion, we demonstrate that the permeation of ions and of molecules in connexin hemichannels can be uncoupled and differentially regulated. We find that human connexin mutations that produce pathologies and were previously thought to be loss-of-function mutations due to the lack of ionic currents are still capable of mediating the passive transport of molecules with kinetics close to those of wild-type channels. This molecular transport displays saturability in the micromolar range, selectivity, and competitive inhibition, properties that are tuned by specific interactions between the permeating molecules and the N-terminal domain that lies within the pore-a general feature of large-pore channels. We propose that connexin hemichannels and, likely, other large-pore channels, are hybrid channel/transporter-like proteins that might switch between these two modes to promote selective ion conduction or autocrine/paracrine molecular signaling in health and disease processes.

Dynamic electrical synapses rewire brain networks for persistent oscillations and epileptogenesis.

One of the very fundamental attributes for telencephalic neural computation in mammals involves network activities oscillating beyond the initial trigger. The continuing and automated processing of transient inputs shall constitute the basis of cognition and intelligence but may lead to neuropsychiatric disorders such as epileptic seizures if carried so far as to engross part of or the whole telencephalic system. From a conventional view of the basic design of the telencephalic local circuitry, the GABAergic interneurons (INs) and glutamatergic pyramidal neurons (PNs) make negative feedback loops which would regulate the neural activities back to the original state. The drive for the most intriguing self-perpetuating telencephalic activities, then, has not been posed and characterized. We found activity-dependent deployment and delineated functional consequences of the electrical synapses directly linking INs and PNs in the amygdala, a prototypical telencephalic circuitry. These electrical synapses endow INs dual (a faster excitatory and a slower inhibitory) actions on PNs, providing a network-intrinsic excitatory drive that fuels the IN-PN interconnected circuitries and enables persistent oscillations with preservation of GABAergic negative feedback. Moreover, the entities of electrical synapses between INs and PNs are engaged in and disengaged from functioning in a highly dynamic way according to neural activities, which then determine the spatiotemporal scale of recruited oscillating networks. This study uncovers a special wide-range and context-dependent plasticity for wiring/rewiring of brain networks. Epileptogenesis or a wide spectrum of clinical disorders may ensue, however, from different scales of pathological extension of this unique form of telencephalic plasticity.

Intercellular Mitochondrial Transfer: The Novel Therapeutic Mechanism for Diseases.

Mitochondria, the dynamic organelles responsible for energy production and cellular metabolism, have the metabolic function of extracting energy from nutrients and synthesizing crucial metabolites. Nevertheless, recent research unveils that intercellular mitochondrial transfer by tunneling nanotubes, tumor microtubes, gap junction intercellular communication, extracellular vesicles, endocytosis and cell fusion may regulate mitochondrial function within recipient cells, potentially contributing to disease treatment, such as nonalcoholic steatohepatitis, glioblastoma, ischemic stroke, bladder cancer and neurodegenerative diseases. This review introduces the principal approaches to intercellular mitochondrial transfer and examines its role in various diseases. Furthermore, we provide a comprehensive overview of the inhibitors and activators of intercellular mitochondrial transfer, offering a unique perspective to illustrate the relationship between intercellular mitochondrial transfer and diseases.

Cx31.1 can selectively intermix with co-expressed connexins to facilitate its assembly into gap junctions.

Connexins are channel-forming proteins that function to facilitate gap junctional intercellular communication. Here, we use dual cell voltage clamp and dye transfer studies to corroborate past findings showing that Cx31.1 (encoded by GJB5) is defective in gap junction channel formation, illustrating that Cx31.1 alone does not form functional gap junction channels in connexin-deficient mammalian cells. Rather Cx31.1 transiently localizes to the secretory pathway with a subpopulation reaching the cell surface, which is rarely seen in puncta reminiscent of gap junctions. Intracellular retained Cx31.1 was subject to degradation as Cx31.1 accumulated in the presence of proteasomal inhibition, had a faster turnover when Cx43 was present and ultimately reached lysosomes. Although intracellularly retained Cx31.1 was found to interact with Cx43, this interaction did not rescue its delivery to the cell surface. Conversely, the co-expression of Cx31 dramatically rescued the assembly of Cx31.1 into gap junctions where gap junction-mediated dye transfer was enhanced. Collectively, our results indicate that the localization and functional status of Cx31.1 is altered through selective interplay with co-expressed connexins, perhaps suggesting Cx31.1 is a key regulator of intercellular signaling in keratinocytes.

The epithelial polarity axis controls the resting membrane potential and Cl- co-transport in breast glandular structures.

The membrane potential (MP) controls cell homeostasis by directing molecule transport and gene expression. How the MP is set upon epithelial differentiation is unknown. Given that tissue architecture also controls homeostasis, we investigated the relationship between basoapical polarity and resting MP in three-dimensional culture of the HMT-3522 breast cancer progression. A microelectrode technique to measure MP and input resistance reveals that the MP is raised by gap junction intercellular communication (GJIC), which directs tight-junction mediated apical polarity, and is decreased by the Na+/K+/2Cl- (NKCC, encoded by SLC12A1 and SLC12A2) co-transporter, active in multicellular structures displaying basal polarity. In the tumor counterpart, the MP is reduced. Cancer cells display diminished GJIC and do not respond to furosemide, implying loss of NKCC activity. Induced differentiation of cancer cells into basally polarized multicellular structures restores widespread GJIC and NKCC responses, but these structures display the lowest MP. The absence of apical polarity, necessary for cancer onset, in the non-neoplastic epithelium is also associated with the lowest MP under active Cl- transport. We propose that the loss of apical polarity in the breast epithelium destabilizes cellular homeostasis in part by lowering the MP.

Gap junction-transported cAMP from the niche controls stem cell progeny differentiation.

The niche has been shown to control stem cell self-renewal in different tissue types and organisms. Recently, a separate niche has been proposed to control stem cell progeny differentiation, called the differentiation niche. However, it remains poorly understood whether and how the differentiation niche directly signals to stem cell progeny to control their differentiation. In the Drosophila ovary, inner germarial sheath (IGS) cells contribute to two separate niche compartments for controlling both germline stem cell (GSC) self-renewal and progeny differentiation. In this study, we show that IGS cells express Inx2 protein, which forms gap junctions (GJs) with germline-specific Zpg protein to control stepwise GSC lineage development, including GSC self-renewal, germline cyst formation, meiotic double-strand DNA break formation, and oocyte specification. Germline-specific Zpg and IGS-specific Inx2 knockdowns cause similar defects in stepwise GSC development. Additionally, secondary messenger cAMP is transported from IGS cells to GSCs and their progeny via GJs to activate PKA signaling for controlling stepwise GSC development. Therefore, this study demonstrates that the niche directly controls GSC progeny differentiation via the GJ-cAMP-PKA signaling axis, which provides important insights into niche control of stem cell differentiation and highlights the importance of GJ-transported cAMP in tissue regeneration. This may represent a general strategy for the niche to control adult stem cell development in various tissue types and organisms since GJs and cAMP are widely distributed.

Electrical Synapses Mediate Embryonic Hyperactivity in a Zebrafish Model of Fragile X Syndrome.

Although hyperactivity is associated with a wide variety of neurodevelopmental disorders, the early embryonic origins of locomotion have hindered investigation of pathogenesis of these debilitating behaviors. The earliest motor output in vertebrate animals is generated by clusters of early-born motor neurons (MNs) that occupy distinct regions of the spinal cord, innervating stereotyped muscle groups. Gap junction electrical synapses drive early spontaneous behavior in zebrafish, prior to the emergence of chemical neurotransmitter networks. We use a genetic model of hyperactivity to gain critical insight into the consequences of errors in motor circuit formation and function, finding that Fragile X syndrome model mutant zebrafish are hyperexcitable from the earliest phases of spontaneous behavior, show altered sensitivity to blockade of electrical gap junctions, and have increased expression of the gap junction protein Connexin 34/35. We further show that this hyperexcitable behavior can be rescued by pharmacological inhibition of electrical synapses. We also use functional imaging to examine MN and interneuron (IN) activity in early embryogenesis, finding genetic disruption of electrical gap junctions uncouples activity between mnx1 + MNs and INs. Taken together, our work highlights the importance of electrical synapses in motor development and suggests that the origins of hyperactivity in neurodevelopmental disorders may be established during the initial formation of locomotive circuits.

Human coronavirus OC43 infection remodels connexin 43-mediated gap junction intercellular communication in vitro.

ß-coronaviruses cause acute infection in the upper respiratory tract, resulting in various symptoms and clinical manifestations. OC43 is a human ß-coronavirus that induces mild clinical symptoms and can be safely studied in the BSL2 laboratory. Due to its low risk, OC43 can be a valuable and accessible model for understanding ß-coronavirus pathogenesis. One potential target for limiting virus infectivity could be gap junction-mediated communication. This study aims to unveil the status of cell-to-cell communications through gap junctions in human ß-coronavirus infection. Infection with OC43 leads to reduced expression of Cx43 in A549, a lung epithelial carcinoma cell line. Infection with this virus also shows a significant ER and oxidative stress increase. Internal localization of Cx43 is observed post-OC43 infection in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) region, which impairs the gap junction communication between two adjacent cells, confirmed by Lucifer yellow dye transfer assay. It also affects hemichannel formation, as depicted by the EtBr uptake assay. Impairment of Cx43 trafficking and the ability to form hemichannels and functional GJIC are hampered by virus-induced Golgi apparatus disruption. Altogether, these results suggest that several physiological changes accompany OC43 infection in A549 cells and can be considered an appropriate model system for understanding the differences in gap junction communication post-viral infections. This model system can provide valuable insights for developing therapies against human ß-coronavirus infections.IMPORTANCEThe enduring impact of the recent SARS-CoV-2 pandemic underscores the importance of studying human ß-coronaviruses, advancing our preparedness for future coronavirus infections. As SARS-CoV-2 is highly infectious, another human ß-coronavirus OC43 can be considered an experimental model. One of the crucial pathways that can be considered is gap junction communication, as it is vital for cellular homeostasis. Our study seeks to understand the changes in Cx43-mediated cell-to-cell communication during human ß-coronavirus OC43 infection. In vitro studies showed downregulation of the gap junction protein Cx43 and upregulation of the endoplasmic reticulum and oxidative stress markers post-OC43 infection. Furthermore, HCoV-OC43 infection causes reduced Cx43 trafficking, causing impairment of functional hemichannel and GJIC formation by virus-mediated Golgi apparatus disruption. Overall, this study infers that OC43 infection reshapes intercellular communication, suggesting that this pathway may be a promising target for designing highly effective therapeutics against human coronaviruses by regulating Cx43 expression.

HDAC3 promotes Sertoli cell maturation and maintains the blood-testis barrier dynamics.

Germ cell development depends on the capacity of somatic Sertoli cells to undergo differentiation into a mature state and establish a germ cell-specific blood-testis barrier (BTB). The BTB structure confers an immunological barrier for meiotic and postmeiotic germ cells, and its dynamic permeability facilitates a transient movement of preleptotene spermatocytes through BTB to enter meiosis. However, the regulatory factors involved in Sertoli cell maturation and how BTB dynamics coordinate germ cell development remain unclear. Here, we found a histone deacetylase HDAC3 abundantly expresses in Sertoli cells and localizes in both cytoplasm and nucleus. Sertoli cell-specific Hdac3 knockout in mice causes infertility with compromised integrity of blood-testis barrier, leading to germ cells unable to traverse through BTB and an accumulation of preleptotene spermatocytes in juvenile testis. Mechanistically, nuclear HDAC3 regulates the expression program of Sertoli cell maturation genes, and cytoplasmic HDAC3 forms a complex with the gap junction protein Connexin 43 to modulate the BTB integrity and dynamics through regulating the distribution of tight junction proteins. Our findings identify HDAC3 as a critical regulator in promoting Sertoli cell maturation and maintaining the homeostasis of the blood-testis barrier.

Genetically engineered human embryonic kidney cells as a novel vehicle for dual patch clamp study of human gap junction channels.

Mutations in more than half of human connexin genes encoding gap junction (GJ) subunits have been linked to inherited human diseases. Functional studies of human GJ channels are essential for revealing mechanistic insights into the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels.

Developmentally distinct CD4+ Treg lineages shape the CD8+ T cell response to acute Listeria infection.

SignificanceThe CD4+ Treg response following acute Listeria infection is heterogeneous and deploys two distinct modes of suppression coinciding with initial pathogen exposure and resolution of infection. This bimodal suppression of CD8+ T cells during priming and contraction is mediated by separate Treg lineages. These findings make a significant contribution to our understanding of the functional plasticity inherent within Tregs, which allows these cells to serve as a sensitive and dynamic cellular rheostat for the immune system to prevent autoimmune pathology in the face of inflammation attendant to acute infection, enable expansion of the pathogen-specific response needed to control the infection, and reestablish immune homeostasis after the threat has been contained.

Visual pigment-deficient cones survive and mediate visual signaling despite the lack of outer segments.

Rhodopsin and cone opsins are essential for light detection in vertebrate rods and cones, respectively. It is well established that rhodopsin is required for rod phototransduction, outer segment disk morphogenesis, and rod viability. However, the roles of cone opsins are less well understood. In this study, we adopted a loss-of-function approach to investigate the physiological roles of cone opsins in mice. We showed that cones lacking cone opsins do not form normal outer segments due to the lack of disk morphogenesis. Surprisingly, cone opsin-deficient cones survive for at least 12 mo, which is in stark contrast to the rapid rod degeneration observed in rhodopsin-deficient mice, suggesting that cone opsins are dispensable for cone viability. Although the mutant cones do not respond to light directly, they maintain a normal dark current and continue to mediate visual signaling by relaying the rod signal through rod-cone gap junctions. Our work reveals a striking difference between the role of rhodopsin and cone opsins in photoreceptor viability.

ERGIC2 and ERGIC3 regulate the ER-to-Golgi transport of gap junction proteins in metazoans.

The extremely dynamic life cycle of gap junction connections requires highly efficient intracellular trafficking system especially designed for gap junction proteins, but the underlying mechanisms are largely unknown. Here, we identified that the COPII-associated proteins ERGIC2 (ER-Golgi intermediate compartment) and ERGIC3 are specifically required for the efficient intracellular transport of gap junction proteins in both Caenorhabditis elegans and mice. In the absence of Ergic2 or Ergic3, gap junction proteins accumulate in the ER and Golgi apparatus and the size of endogenous gap junction plaques is reduced. Knocking out the Ergic2 or Ergic3 in mice results in heart enlargement and cardiac malfunction accompanied by reduced number and size of connexin 43 (Cx43) gap junctions. Invertebrates' gap junction protein innexins share no sequence similarity with vertebrates' connexins. However, ERGIC2 and ERGIC3 could bind to gap junction proteins in both worms and mice. Characterization of the highly specialized roles of ERGIC2 and ERGIC3 in metazoans reveals how the early secretory pathway could be adapted to facilitate the efficient transport for gap junction proteins in vivo.

Innexin-Mediated Adhesion between Glia Is Required for Axon Ensheathment in the Peripheral Nervous System.

Glia are essential to protecting and enabling nervous system function and a key glial function is the formation of the glial sheath around peripheral axons. Each peripheral nerve in the Drosophila larva is ensheathed by three glial layers, which structurally support and insulate the peripheral axons. How peripheral glia communicate with each other and between layers is not well established and we investigated the role of Innexins in mediating glial function in the Drosophila periphery. Of the eight Drosophila Innexins, we found two (Inx1 and Inx2) are important for peripheral glia development. In particular loss of Inx1 and Inx2 resulted in defects in the wrapping glia leading to disruption of the glia wrap. Of interest loss of Inx2 in the subperineurial glia also resulted in defects in the neighboring wrapping glia. Inx plaques were observed between the subperineurial glia and the wrapping glia suggesting that gap junctions link these two glial cell types. We found Inx2 is key to Ca2+ pulses in the peripheral subperineurial glia but not in the wrapping glia, and we found no evidence of gap junction communication between subperineurial and wrapping glia. Rather we have clear evidence that Inx2 plays an adhesive and channel-independent role between the subperineurial and wrapping glia to ensure the integrity of the glial wrap.SIGNIFICANCE STATEMENT Gap junctions are critical for glia communication and formation of myelin in myelinating glia. However, the role of gap junctions in non-myelinating glia is not well studied, yet non-myelinating glia are critical for peripheral nerve function. We found the Innexin gap junction proteins are present between different classes of peripheral glia in Drosophila. Here Innexins form junctions to facilitate adhesion between the different glia but do so in a channel-independent manner. Loss of adhesion leads to disruption of the glial wrap around axons and leads to fragmentation of the wrapping glia membranes. Our work points to an important role for gap junction proteins in mediating insulation by non-myelinating glia.

Human Cx50 Isoleucine177 prevents heterotypic docking and formation of functional gap junction channels with Cx43.

The human lens is an avascular organ, and its transparency is dependent on gap junction (GJ)-mediated microcirculation. Lens GJs are composed of three connexins with Cx46 and Cx50 being expressed in lens fiber cells and Cx43 and Cx50 in the epithelial cells. Impairment of GJ communication by either Cx46 or Cx50 mutations has been shown to be one of the main molecular mechanisms of congenital cataracts in mutant carrier families. The docking compatibility and formation of functional heterotypic GJs for human lens connexins have not been studied. Previous study on rodent lens connexins revealed that Cx46 can form functional heterotypic GJs with Cx50 and Cx43, but Cx50 cannot form heterotypic GJ with Cx43 due to its second extracellular (EL2) domain. To study human lens connexin docking and formation of functional heterotypic GJs, we developed a genetically engineered HEK293 cell line with endogenously expressed Cx43 and Cx45 ablated. The human lens connexins showed docking compatibility identical to those found in the rodent connexins. To reveal the structural mechanisms of the docking incompatibility between Cx50 and Cx43, we designed eight variants based on the differences between the EL2 of Cx50 and Cx46. We found that Cx50I177L is sufficient to establish heterotypic docking with Cx43 with some interesting gating properties. Our structure models indicate this residue is important for interdomain interactions within a single connexin, Cx50 I177L showed an increased interdomain interaction which might alter the docking interface structure to be compatible with Cx43.NEW & NOTEWORTHY The human lens is an avascular organ, and its transparency is partially dependent on gap junction (GJ) network composed of Cx46, Cx50, and Cx43. We found that human Cx46 can dock and form functional heterotypic GJs with Cx50 and Cx43, but Cx50 is unable to form functional heterotypic GJs with Cx43. Through mutagenesis and patch-clamp study of several designed variants, we found that Cx50 I177L was sufficient to form functional heterotypic GJs with Cx43.

A crystallin mutant cataract with mineral deposits.

Connexin mutant mice develop cataracts containing calcium precipitates. To test whether pathologic mineralization is a general mechanism contributing to the disease, we characterized the lenses from a nonconnexin mutant mouse cataract model. By cosegregation of the phenotype with a satellite marker and genomic sequencing, we identified the mutant as a 5-bp duplication in the γC-crystallin gene (Crygcdup). Homozygous mice developed severe cataracts early, and heterozygous animals developed small cataracts later in life. Immunoblotting studies showed that the mutant lenses contained decreased levels of crystallins, connexin46, and connexin50 but increased levels of resident proteins of the nucleus, endoplasmic reticulum, and mitochondria. The reductions in fiber cell connexins were associated with a scarcity of gap junction punctae as detected by immunofluorescence and significant reductions in gap junction-mediated coupling between fiber cells in Crygcdup lenses. Particles that stained with the calcium deposit dye, Alizarin red, were abundant in the insoluble fraction from homozygous lenses but nearly absent in wild-type and heterozygous lens preparations. Whole-mount homozygous lenses were stained with Alizarin red in the cataract region. Mineralized material with a regional distribution similar to the cataract was detected in homozygous lenses (but not wild-type lenses) by micro-computed tomography. Attenuated total internal reflection Fourier-transform infrared microspectroscopy identified the mineral as apatite. These results are consistent with previous findings that loss of lens fiber cell gap junctional coupling leads to the formation of calcium precipitates. They also support the hypothesis that pathologic mineralization contributes to the formation of cataracts of different etiologies.