Feedback Regulation of Intracellular Hydrostatic Pressure in Surface Cells of the Lens

In wild-type lenses from various species, an intracellular hydrostatic pressure gradient goes from ∼340 mmHg in central fiber cells to 0 mmHg in surface cells. This gradient drives a center-to-surface flow of intracellular fluid. In lenses in which gap-junction coupling is increased, the central pressure is lower, whereas if gap-junction coupling is reduced, the central pressure is higher but surface pressure is always zero. Recently, we found that surface cell pressure was elevated in PTEN null lenses. This suggested disruption of a feedback control system that normally maintained zero surface cell pressure. Our purpose in this study was to investigate and characterize this feedback control system. We measured intracellular hydrostatic pressures in mouse lenses using a microelectrode/manometer-based system. We found that all feedback went through transport by the Na/K ATPase, which adjusted surface cell osmolarity such that pressure was maintained at zero. We traced the regulation of Na/K ATPase activity back to either TRPV4, which sensed positive pressure and stimulated activity, or TRPV1, which sensed negative pressure and inhibited activity. The inhibitory effect of TRPV1 on Na/K pumps was shown to signal through activation of the PI3K/AKT axis. The stimulatory effect of TRPV4 was shown in previous studies to go through a different signal transduction path. Thus, there is a local two-legged feedback control system for pressure in lens surface cells. The surface pressure provides a pedestal on which the pressure gradient sits, so surface pressure determines the absolute value of pressure at each radial location. We speculate that the absolute value of intracellular pressure may set the radial gradient in the refractive index, which is essential for visual acuity.     

Connections between connexins, calcium, and cataracts in the lens

There is a good deal of evidence that the lens generates an internal micro circulatory system, which brings metabolites, like glucose, and antioxidants, like ascorbate, into the lens along the extracellular spaces between cells. Calcium also ought to be carried into the lens by this system. If so, the only path for Ca2+ to get out of the lens is to move down its electrochemical gradient into fiber cells, and then move by electrodiffusion from cell to cell through gap junctions to surface cells, where Ca-ATPase activity and Na/Ca exchange can transport it back into the aqueous or vitreous humors. The purpose of the present study was to test this calcium circulation hypothesis by studying calcium homeostasis in connexin (Cx46) knockout and (Cx46 for Cx50) knockin mouse lenses, which have different degrees of gap junction coupling. To measure intracellular calcium, FURA2 was injected into fiber cells, and the gradient in calcium concentration from center to surface was mapped in each type of lens. In wild-type lenses the coupling conductance of the mature fibers was approximately 0.5 S/cm2 of cell to cell contact, and the best fit to the calcium concentration data varied from 700 nM in the center to 300 nM at the surface. In the knockin lenses, the coupling conductance was approximately 1.0 S/cm2 and calcium varied from approximately 500 nM at the center to 300 nM at the surface. Thus, when the coupling conductance doubled, the concentration gradient halved, as predicted by the model. In knockout lenses, the coupling conductance was zero, hence the efflux path was knocked out and calcium accumulated to approximately 2 microM in central fibers. Knockout lenses also had a dense central cataract that extended from the center to about half the radius. Others have previously shown that this cataract involves activation of a calcium-dependent protease, Lp82. We can now expand on this finding to provide a hypothesis on each step that leads to cataract formation: knockout of Cx46 causes loss of coupling of mature fiber cells; the efflux path for calcium is therefore blocked; calcium accumulates in the central cells; at concentrations above approximately 1 microM (from the center to about half way out of a 3-wk-old lens) Lp82 is activated; Lp82 cleaves cytoplasmic proteins (crystallins) in central cells; and the cleaved proteins aggregate and scatter light.     

Dominant cataracts result from incongruous mixing of wild-type lens connexins

Gap junctions are composed of proteins called connexins (Cx) and facilitate both ionic and biochemical modes of intercellular communication. In the lens, Cx46 and Cx50 provide the gap junctional coupling needed for homeostasis and growth. In mice, deletion of Cx46 produced severe cataracts, whereas knockout of Cx50 resulted in significantly reduced lens growth and milder cataracts. Genetic replacement of Cx50 with Cx46 by knockin rescued clarity but not growth. By mating knockin and knockout mice, we show that heterozygous replacement of Cx50 with Cx46 rescued growth but produced dominant cataracts that resulted from disruption of lens fiber morphology and crystallin precipitation. Impedance measurements revealed normal levels of ionic gap junctional coupling, whereas the passage of fluorescent dyes that mimic biochemical coupling was altered in heterozygous knockin lenses. In addition, double heterozygous knockout lenses retained normal growth and clarity, whereas knockover lenses, where native Cx46 was deleted and homozygously knocked into the Cx50 locus, displayed significantly deficient growth but maintained clarity. Together, these findings suggest that unique biochemical modes of gap junctional communication influence lens clarity and lens growth, and this biochemical coupling is modulated by the connexin composition of the gap junction channels.     

Cell to cell communication and pH in the frog lens

Fiber cells of the lens are electrically and diffusionally interconnected through extensive gap junctions. These junctions allow fluxes of small solutes to move between inner cells and peripheral cells, where the majority of transmembrane transport takes place. We describe here a method utilizing two intracellular microelectrodes to measure the cell to cell resistance between fiber cells at any given distance into the intact lens. We also use ion-sensitive microelectrodes to record intracellular pH at various depths in the intact lens. We find that gap junctions connecting inner fiber cells differ in pH sensitivity as well as normal coupling resistance from those connecting peripheral cells. The transition occurs in a zone between 500 and 650 microns into the lens. Fiber cells peripheral to this zone have a specific coupling resistance of 1.1 omega cm2, whereas those inside have a specific coupling resistance of 2.7 omega cm2. However, when the cytoplasm of fiber cells is acidified by bubbling with CO2, peripheral cells uncouple and the cell to cell resistance goes up more than 40-fold, whereas junctions inside this zone are essentially unaffected by changes in intracellular pH. In a normal frog lens, the intracellular pH in fiber cells near the lens surface is 7.02, a value significantly alkaline to electrochemical equilibrium. Our data suggest that Na/H exchange and perhaps other Na gradient-dependent mechanisms in the peripheral cells maintain this transmembrane gradient. Deep in the lens, the fiber cell cytoplasm is significantly more acidic (pHi 6.81) due to influx of hydrogen across the inner fiber cell membranes and production of H+ by the inner fiber cells. Because of the normally acid cytoplasm of interior fiber cells, their loss of gap junctional sensitivity to pH may be essential to lens survival.     

Epithelial water transport in a balanced gradient system.

The relationship between epithelial fluid transport, standing osmotic gradients, and standing hydrostatic pressure gradients has been investigated using a perturbation expansion of the governing equations. The assumptions used in the expansion are: (a) the volume of lateral intercellular space per unit volume of epithelium is small; (b) the membrane osmotic permeability is much larger than the solute permeability. We find that the rate of fluid reabsorption is set by the rate of active solute transport across lateral membranes. The fluid that crosses the lateral membranes and enters the intercellular cleft is driven longitudinally by small gradients in hydrostatic pressure. The small hydrostatic pressure in the intercellular space is capable of causing significant transmembrane fluid movement, however, the transmembrane effect is countered by the presence of a small standing osmotic gradient. Longitudinal hydrostatic and osmotic gradients balance such that their combined effect on transmembrane fluid flow is zero, whereas longitudinal flow is driven by the hydrostatic gradient. Because of this balance, standing gradients within intercellular clefts are effectively uncoupled from the rate of fluid reabsorption, which is driven by small, localized osmotic gradients within the cells. Water enters the cells across apical membranes and leaves across the lateral intercellular membranes. Fluid that enters the intercellular clefts can, in principle, exit either the basal end or be secreted from the apical end through tight junctions. Fluid flow through tight junctions is shown to depend on a dimensionless parameter, which scales the resistance to solute flow of the entire cleft relative to that of the junction. Estimates of the value of this parameter suggest that an electrically leaky epithelium may be effectively a tight epithelium in regard to fluid flow.     

Junctions between lens cells in differentiating cultures: structure, formation, intercellular permeability, and junctional protein expression

We previously described cultures of chick embryo lens cells which displayed a marked degree of differentiation. In this report, the junctions found between the lens fiber-like cells in the differentiated "lentoids" are characterized in several ways. Thin-section methods with electron microscopy first demonstrated that numerous, large junctions between lentoid cells accompanied the other differentiated features of these cells. Freeze-fracture techniques, including quantitative analysis, then revealed that (a) junctional particles were loosely arranged as is typical of fiber cells, (b) the population of individual junctional areas in culture was indistinguishable from that found in 10- to 12-day chick embryo lenses, and (c) apparent junction formation occurred during the development of the lens cells, with lacy arrays of particles being associated with fiber-like junctions. In addition, gap junctions with hexagonally packed particles, typical of lens epithelial cells, largely disappeared during the course of differentiation. Injection of tracer dyes into lentoid cells resulted in rapid intercellular movement of dye, consistent with functional cell-to-cell channels connecting lentoid cells. During the development of the lens cells in culture, as junction formation occurred, an increase of approximately eight-fold in MP28 protein was observed within the cells. These combined results indicate that (a) extensive lens fiber junctions and functional cell-to-cell channels are found between differentiated lentoid lentoid cells in vitro, (b) lens fiber junctions appear to form during the course of lens cell differentiation in culture, (c) a significant increase occurs in the putative junctional protein before the cultures are highly developed, (d) the increased levels of MP28 and junction formation may be required for the full expression of the differentiated state in the lens fiber cell, and (e) this culture system should prove to be valuable for additional experiments on lens junctions and for other studies requiring the development of lens fiber cells in vitro.     

The Effects of GPX-1 Knockout on Membrane Transport and Intracellular Homeostasis in the Lens

Glutathione peroxidase-1 (GPX-1) is an enzyme that protects the lens against H₂O₂-mediated oxidative damage. The purpose of the present study was to determine the effects of GPX-1 knockout (KO) on lens transport and intracellular homeostasis. To investigate these lenses we used (1) whole lens impedance studies to measure membrane conductance, resting voltage and fiber cell gap junction coupling conductance; (2) osmotic swelling of fiber cell membrane vesicles to determine water permeability; and (3) injection of Fura2 and Na⁺-binding benzofuran isophthalate (SBFI) into fiber cells to measure [Ca²⁺] _i and [Na⁺] _i , respectively, in intact lenses. These approaches were used to compare wild-type (WT) and GPX-1 KO lenses from mice around 2 months of age. There were no significant differences in clarity, size, resting voltage, membrane conductance or fiber cell membrane water permeability between WT and GPX-1 KO lenses. However, in GPX-1 KO lenses, coupling conductance was 72% of normal in the outer shell of differentiating fibers and 45% of normal in the inner core of mature fibers. Quantitative Western blots showed that GPX-1 KO lenses had about 50% as much labeled Cx46 and Cx50 protein as WT, whereas they had equivalent labeled AQP0 protein as WT. Both Ca²⁺ and Na⁺ accumulated significantly in the core of GPX-1 KO lenses. In summary, the major effect on lens transport of GPX-1 KO was a reduction in gap junction coupling conductance. This reduction affected the lens normal circulation by causing [Na⁺] _i and [Ca²⁺] _i to increase, which could increase cataract susceptibility in GPX-1 KO lenses.     

Biochemical and immunological approaches to the study of gap junctional communication

Studies on gap junctions isolated from rat liver by a procedure that avoids exogenous proteolysis (Hertzberg, E. L.; Gilula. N. B.; J. Biol. Chem. 254: 2138-2147; 1979) are described. The original isolation procedure was modified to increase the yield and has been extended to the preparation of gap junctions from mouse and bovine liver. Peptide map studies showed that the 27,000-dalton polypeptides present in liver gap junction preparations from all three sources are homologous and are not derived from other polypeptides of higher molecular weight that are observed in cruder preparations. Similar studies with lens fiber junctions demonstrated no homology between liver and lens junction polypeptides. Antibodies to the lens junction polypeptides did not cross-react with the liver gap junction polypeptide, further supporting this conclusion.     

Diverse gap junctions modulate distinct mechanisms for fiber cell formation during lens development and cataractogenesis

Different mutations of alpha3 connexin (Cx46 or Gja8) and alpha8 connexin (Cx50 or Gja8), subunits of lens gap junction channels, cause a variety of cataracts via unknown mechanisms. We identified a dominant cataractous mouse line (L1), caused by a missense alpha8 connexin mutation that resulted in the expression of alpha8-S50P mutant proteins. Histology studies showed that primary lens fiber cells failed to fully elongate in heterozygous alpha8(S50P/+) embryonic lenses, but not in homozygous alpha8(S50P/S50P), alpha8-/- and alpha3-/- alpha8-/- mutant embryonic lenses. We hypothesized that alpha8-S50P mutant subunits interacted with wild-type alpha3 or alpha8, or with both subunits to affect fiber cell formation. We found that the combination of mutant alpha8-S50P and wild-type alpha8 subunits specifically inhibited the elongation of primary fiber cells, while the combination of alpha8-S50P and wild-type alpha3 subunits disrupted the formation of secondary fiber cells. Thus, this work provides the first in vivo evidence that distinct mechanisms, modulated by diverse gap junctions, control the formation of primary and secondary fiber cells during lens development. This explains why and how different connexin mutations lead to a variety of cataracts. The principle of this explanation can also be applied to mutations of other connexin isoforms that cause different diseases in other organs.     

Steady-state voltages, ion fluxes, and volume regulation in syncytial tissues.

Equations are developed that describe the steady-state relationships among ion fluxes, solute fluxes, water flow, voltage, concentration of solute, and hydrostatic pressure in a spherically symmetrical syncytial tissue. Each cell of the syncytium is assumed to have membrane channels for Na, K, and Cl, a membrane pump for Na/K, and some concentration of intracellular protein of net negative charge. However, the surface cells and inner cells of the tissue are assumed to have different distributions of membrane transport properties, hence there is a radial circulation of fluxes and a radial distribution of forces. Some reasonable approximations are made that allow analytic solutions of the nonlinear differential equations. These solutions are used to analyze data from the frog lens and are shown to account for the known steady-state properties of this tissue. Moreover, these solutions are used to make predictions on other steady-state properties, which have not been directly measured, and graphical results on the circulation of water, ions and solute through the frog lens are presented.     

Comparative analysis of the major polypeptides from liver gap junctions and lens fiber junctions

Gap junctions from rat liver and fiber junctions from bovine lens have similar septilaminar profiles when examined by thin-section electron microscopy and differ only slightly with respect to the packing of intramembrane particles in freeze-fracture images. These similarities have often led to lens fiber junctions being referred to as gap junctions. Junctions from both sources were isolated as enriched subcellular fractions and their major polypeptide components compared biochemically and immunochemically. The major liver gap junction polypeptide has an apparent molecular weight of 27,000, while a 25,000-dalton polypeptide is the major component of lens fiber junctions. The two polypeptides are not homologous when compared by partial peptide mapping in SDS. In addition, there is not detectable antigenic similarity between the two polypeptides by immunochemical criteria using antibodies to the 25,000-dalton lens fiber junction polypeptide. Thus, in spite of the ultrastructural similarities, the gap junction and the lens fiber junction are comprised of distinctly different polypeptides, suggesting that the lens fiber junction contains a unique gene product and potentially different physiological properties.     

Gap junctional coupling between progenitor cells at the retinal margin of adult goldfish

We prepared living slice preparations of the peripheral retina of adult goldfish to examine electrical membrane properties of progenitor cells at the retinal margin. Cells were voltage-clamped near resting potential and then stepped to either hyperpolarizing or depolarizing test potentials using whole-cell voltage-clamp recordings. Electrophysiologically examined cells were morphologically identified by injecting both Lucifer Yellow (LY) and biocytin. All progenitor cells examined (n = 37) showed a large amount of passively flowing currents of either sign under suppression of the nonjunctional currents flowing through K(+) and Ca(2+) channels in the cell membrane. They did not exhibit any voltage-gated Na(+) currents. Cells identified by LY fills were typically slender. As the difference between the test potential and the resting potential increased, 13 out of 37 cells exhibited symmetrically voltage- and time-dependent current decline on either sign at the resting potential. The symmetric current profile suggests that the current may be driven and modulated by the junctional potential difference between the clamping cell and its neighbors. The remaining 24 cells did not exhibit voltage dependency. A gap junction channel blocker, halothane, suppressed the currents. A decrease in extracellular pH reduced coupling currents and its increase enhanced them. Dopamine, cAMP, and retinoic acid did not influence coupling currents. Injection of biocytin into single progenitor cells revealed strong tracer coupling, which was restricted in the marginal region. Immature ganglion cells closely located to the retinal margin exhibited voltage-gated Na(+) currents. They did not reveal apparent tracer coupling. These results demonstrate that the marginal progenitor cells couple with each other via gap junctions, and communicate biochemical molecules, which may subserve or interfere with cellular differentiation.     

Reconstitution of native-type noncrystalline lens fiber gap junctions from isolated hemichannels

Gap junctions contain numerous channels that are clustered in apposed membrane patches of adjacent cells. These cell-to-cell channels are formed by pairing of two hemichannels or connexons, and are also referred to as connexon pairs. We have investigated various detergents for their ability to separately solubilize hemichannels or connexon pairs from isolated ovine lens fiber membranes. The solubilized preparations were reconstituted with lipids with the aim to reassemble native-type gap junctions and to provide a model system for the characterization of the molecular interactions involved in this process. While small gap junction structures were obtained under a variety of conditions, large native-type gap junctions were assembled using a novel two-step procedure: in the first step, hemichannels that had been solubilized with octylpolyoxyethylene formed connexon pairs by dialysis against n-decyl-beta-D-maltopyranoside. In the second step, connexon pairs were reconstituted with phosphatidylcholines by dialysis against buffer containing Mg2+. This way, double-layered gap junctions with diameter < or = 300 nm were obtained. Up to several hundred channels were packed in a noncrystalline arrangement, giving these reconstituted gap junctions an appearance that was indistinguishable from that of the gap junctions in the lens fiber membranes.     

The structural organization and protein composition of lens fiber junctions

The structural organization and protein composition of lens fiber junctions isolated from adult bovine and calf lenses were studied using combined electron microscopy, immunolocalization with monoclonal and polyclonal anti-MIP and anti-MP70 (two putative gap junction-forming proteins), and freeze-fracture and label-fracture methods. The major intrinsic protein of lens plasma membranes (MIP) was localized in single membranes and in an extensive network of junctions having flat and undulating surface topologies. In wavy junctions, polyclonal and monoclonal anti-MIPs labeled only the cytoplasmic surface of the convex membrane of the junction. Label-fracture experiments demonstrated that the convex membrane contained MIP arranged in tetragonal arrays 6-7 nm in unit cell dimension. The apposing concave membrane of the junction displayed fracture faces without intramembrane particles or pits. Therefore, wavy junctions are asymmetric structures composed of MIP crystals abutted against particle-free membranes. In thin junctions, anti-MIP labeled the cytoplasmic surfaces of both apposing membranes with varying degrees of asymmetry. In thin junctions, MIP was found organized in both small clusters and single membranes. These small clusters also abut against particle-free apposing membranes, probably in a staggered or checkerboard pattern. Thus, the structure of thin and wavy junctions differed only in the extent of crystallization of MIP, a property that can explain why this protein can produce two different antibody-labeling patterns. A conclusion of this study is that wavy and thin junctions do not contain coaxially aligned channels, and, in these junctions, MIP is unlikely to form gap junction-like channels. We suggest MIP may behave as an intercellular adhesion protein which can also act as a volume-regulating channel to collapse the lens extracellular space. Junctions constructed of MP70 have a wider overall thickness (18-20 nm) and are abundant in the cortical regions of the lens. A monoclonal antibody raised against this protein labeled these thicker junctions on the cytoplasmic surfaces of both apposing membranes. Thick junctions also contained isolated clusters of MIP inside the plaques of MP70. The role of thick junctions in lens physiology remains to be determined.     

LAMP, a new imaging assay of gap junctional communication unveils that Ca2+ influx inhibits cell coupling

Using a new class of photo-activatible fluorophores, we have developed a new imaging technique for measuring molecular transfer rates across gap junction connexin channels in intact living cells. This technique, named LAMP, involves local activation of a molecular fluorescent probe, NPE-HCCC2/AM, to optically label a cell. Subsequent dye transfer through gap junctions from labeled to unlabeled cells was quantified by fluorescence microscopy. Additional uncagings after prior dye transfers reached equilibrium enabled multiple measurements of dye transfer rates in the same coupled cell pair. Measurements in the same cell pair minimized variation due to differences in cell volume and number of gap junctions, allowing us to track acute changes in gap junction permeability. We applied the technique to study the regulation of gap junction coupling by intracellular Ca(2+) ([Ca(2+)](i)). Although agonist or ionomycin exposure can raise bulk [Ca(2+)](i) to levels higher than those caused by capacitative Ca(2+) influx, the LAMP assay revealed that only Ca(2+) influx through the plasma membrane store-operated Ca(2+) channels strongly reduced gap junction coupling. The noninvasive and quantitative nature of this imaging technique should facilitate future investigations of the dynamic regulation of gap junction communication.     

Axo-axonal coupling. a novel mechanism for ultrafast neuronal communication

We provide physiological, pharmacological, and structural evidence that axons of hippocampal principal cells are electrically coupled, with prepotentials or spikelets forming the physiological substrate of electrical coupling as observed in cell somata. Antidromic activation of neighboring axons induced somatic spikelet potentials in neurons of CA3, CA1, and dentate gyrus areas of rat hippocampal slices. Somatic invasion by these spikelets was dependent on the activation of fast Na(+) channels in the postjunctional neuron. Antidromically elicited spikelets were suppressed by gap junction blockers and low intracellular pH. Paired axo-somatic and somato-dendritic recordings revealed that the coupling potentials appeared in the axon before invading the soma and the dendrite. Using confocal laser scanning microscopy we found that putative axons of principal cells were dye coupled. Our data thus suggest that hippocampal neurons are coupled by axo-axonal junctions, providing a novel mechanism for very fast electrical communication.     

Connexin 46 (Cx46) Gap Junctions Provide a Pathway for the Delivery of Glutathione to the Lens Nucleus

Maintenance of adequate levels of glutathione (GSH) in the lens nucleus is critical for protection of lens proteins from the effects of oxidative stress and for lens transparency. How GSH is transported to the nucleus is unknown. We show that GSH diffuses to the nucleus from the outer cortex, where a high concentration of the anti-oxidant is established by synthesis or uptake, via the network of gap junctions. Using electrophysiological measurements, we found that channels formed by Cx46 and Cx50, the two connexin isoforms expressed in the lens, were moderately cation-selective (P_Na/P_Cl ∼5 for Cx46 and ∼3 for Cx50). Single channel permeation of the larger GSH anion was low but detectable (P_Na/P_GSH ∼12 for Cx46 and ∼8 for Cx50), whereas permeation of divalent anion glutathione disulfide (GSSG) was undetectable. Measurement of GSH levels in the lenses from connexin knock-out (KO) mice indicated Cx46, and not Cx50, is necessary for transport of GSH to the core. Levels of GSH in the nucleus were markedly reduced in Cx46 KO, whereas they were unaffected by Cx50 KO. We also show that GSH delivery to the nucleus is not dependent on the lens microcirculation, which is believed to be responsible for extracellular transport of other nutrients to membrane transporters in the core. These results indicate that glutathione diffuses from cortical fiber cells to the nucleus via gap junction channels formed by Cx46. We present a model of GSH diffusion from outer cells to inner fiber cells through gap junctions.     

Sharing signals: connecting lung epithelial cells with gap junction channels

Gap junction channels enable the direct flow of signaling molecules and metabolites between cells. Alveolar epithelial cells show great variability in the expression of gap junction proteins (connexins) as a function of cell phenotype and cell state. Differential connexin expression and control by alveolar epithelial cells have the potential to enable these cells to regulate the extent of intercellular coupling in response to cell stress and to regulate surfactant secretion. However, defining the precise signals transmitted through gap junction channels and the cross talk between gap junctions and other signaling pathways has proven difficult. Insights from what is known about roles for gap junctions in other systems in the context of the connexin expression pattern by lung cells can be used to predict potential roles for gap junctional communication between alveolar epithelial cells.     

Quantifying changes in gap junction structure as a function of lens fiber cell differentiation

The ocular lens is an ideal model system for studying gap junction structure-function relationships. Here we apply novel methods to quantitatively compare connexin expression over macroscopic distances while simultaneously resolving the intracellular distribution of gap junctions in sub-micron detail. Our approach has identified three distinct zones of connexin density and allowed changes in gap junction plaque size, number and dispersion to be quantified. Our analysis is the first to precisely correlate changes in gap junction plaque structure with the reported changes in gap junction function that occur as a consequence of fiber cell differentiation.     

Quantifying Changes in Gap Junction Structure as a Function of Lens Fiber Cell Differentiation

The ocular lens is an ideal model system for studying gap junction structure-function relationships. Here we apply novel methods to quantitatively compare connexin expression over macroscopic distances while simultaneously resolving the intracellular distribution of gap junctions in sub-micron detail. Our approach has identified three distinct zones of connexin density and allowed changes in gap junction plaque size, number and dispersion to be quantified. Our analysis is the first to precisely correlate changes in gap junction plaque structure with the reported changes in gap junction function that occur as a consequence of fiber cell differentiation.