seminars in CELL BIOLOGY Vol 3, 1992: pp 49-58
The crystalline lens. A system networked by gap junctional intercellular communication Daniel A. Goodenough The vertebrate eye lens is a solid cyst of cells which grows throughout life by addition of new cells at the surface. The older cells) buried by thenewergenerations) differentiate into long) prismaticfibers) losing their cellular organelles and jilling their cytoplasms with high concentrations of soluble proteins) thecrystallins. The long-lived lensfibers areinterconnected by gapjunctions) both with themselves and with an anterior layer of simple cuboidal epithelial cells at thelens surface. This network of gap junctions joins the lens cells into a syncytium with respect to small molecules, permitting metabolic co-operation: intercellular diffusion of ions) metabolites) and water. In contact with nutrients at the lens surface) theepithelial cells retain their cellular organelles) and are able to provide the metabolic energy to maintain correct ion and metabolite concentrations within the lens fiber cytoplasms, such that the crystallins remain in solution and do not aggregate (cataract). Gapjunctions areformed by a family of integral membrane channel-formingproteins called connexins. Gap junctions between lens epithelial cells are composed of a connexin which is common between marry different cell types, notably myocardial cells and connective tissuefibroblasts. The gapjunctions between epithelial cells and lens fibers havenotyet been biochemically characterized. The gapjunctionsformed between lensfibers are composed of at least two different connexins, one of which has not been detected between other celltypes. The unusualphysiology and longeviry of the lens fibers may require the special set of connexins which arefound joining these cells.
become the lens placode. The placode then invaginates into the embryo to eventually pinch off from the ectoderm as a hollow sphere of cells, the lens vesicle. Subsequently, the posterior cells of the lens vesicle elongate anteriorly, obliterating the lumen of the lens vesicle, coming into apposition to, and making gap junctions with, the anterior lens epithelium. At the end of this process, the early lens is a solid cyst of cells, with an epithelium anteriorly and elongated cells, the lens fibers, posteriorly (Figure 1). The epithelium and fibers are continuous with each other at the edges, or bow region, of the lens. In this bow region, a population of undifferentiated stem cells persists throughout life, continuously dividing and adding new, elongating lens fibers equatorially at the organ surface. 1•2 The lens floats in the aqueous humor, suspended posterior to the iris by zonular fibers which attach it to the ciliary body. The lens has no blood supply and no connective tissue, save a thick capsule, the enlarged original basement membrane of the embryonic ectoderm, composed in part of type IV collagen. The stem cell population of the lens continues to divide throughout life generating daughter cells which undergo cytodifferentiation: massive synthesis of tissue-specific proteins, the soluble crystallins, and loss of the nucleus and other organelles.I The crystallins are gene products unique to the lens, and due to the isolation of the lens during early embryonic development from both the blood supply and the process of self-recognition by the immune system, these unique gene products are never programmed as 'self and can elicit an autoimmune reaction following eye injury. The purpose of the accumulation of high concentrations of soluble proteins and loss of organelles is to allow the lens to achieve both a high refractive index and transparency.t The presence of organelles in the lens would result in image-degrading lightscattering.V' In order not to view the world through a cytochrome-tinted haze, the differentiated lens cells avoid oxidative phosphorylation," and the organ derives the bulk of its energy from anaerobic glycolysis, with lactate its chief excretory product.
Key words: lens / gap junctions / intercellular communication / connexins
The lens is a cellular system, interconnected by gap junctions During development, an outgrowth of neurectoderm, the optic vesicle, becomes apposed to, and 'induces' the overlying embryonic ectoderm to thicken and From the Department of Cellular Biology andAnatomy) Harvard Medital School, 220 Longwood Avenue, Boston, .MA 02115, USA ©1992 Academic Press Ltd 1013·4682/92/010049 + 10S5. 00/0 49
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Bow
Figure 1. A diagram outlines the junctional interactions between the lens fibers and epithelial cells. The lens fibers are drawn as straight, connecting the anteriorly disposed epithelial cells with the lens posterior surface. In early stages of lens development, the fibers are disposed in this fashion. In older lenses, the fibers wrap concentrically, creating a large population of internalized cells which are not in contact with the lens surface. The boxed area shows an enlarged portion of the epithelium-to-fiber interface. The gap junctions are represented by spots where the outlines of the cells come into contact. Three types of interaction are shown: epithelium/epithelium (E/E), epithelium/fiber (ElF), and fiber/fiber (FIF). The shaded circles and ellipses represent cellular nuclei, which become pyknotic and disappear as the fibers age. The extracellular matrix of the capsule is indicated in the inset but not the main figure. The fibers in the center of the lens, since they have neither blood supply nor organelles, are uniquely dependent on communication with cells at the lens surface. Electrophysiological data8- 14 have established that the inner lens fibers have typical cellular membrane potentials ( - 70 m V), and that they are joined into a syncytium with respect to ions. This syncytium is achieved by an enormous number of gap junctions between adjacent lens cells, and perhaps some frank fiber-fiber membrane fusions.P which permit ions, and fluorescent dyes l6-19 as well as small, transported metabolites-" to diffuse between adjacent cells. The lens fiber membranes have no active transport channels and very low passive permeability to ions.I!
The anatomy and physiology of the lens partially explain how the internal lens fibers, which live the lifetime of the individual, can maintain active resting potentials and not need a continuing supply of newly synthesized transport proteins: the epithelial cells at the lens surface, known to contain the bulk of the N a + - K + ATPase activity in the lens,22-25 pump ions for the remainder of the lens fibers to which they are connected by gap junctions. This is the classic 'pump-leak' model of Kinsey,26 which explains the potential difference across the organy·29 While the locations of the various pumps and leaks are not known with precision, extracellular vibrating probe studies with the rat lens demonstrate clear circulating current flow through complex pathways in the
The crystalline lens
lens. 30 An attractive hypothesis has suggested that the ion (mostly sodium) and accompanying passive fluid flow are radially inward through the extracellular spaces between the lens fibers, and radially back outward through the cytoplasms of the lens fibers, through the gap junctions to the epithelial cells, where active transport occurs. H,21 This flow would not only serve to circulate fluids through the lens, but also might create a negative hydrostatic pressure in the intercellular spaces between lens fibers, assuring that these spaces remain collapsed and non-light scattering, even during movements of the lens during accommodation. This miniature circulatory system has been presented in a model by Mathias-! which predicts both the intracellular and extracellular voltage gradients measurable in the lens. H,32 To reduce the workload of the epithelial cells as much as possible, the lens fiber membranes are constructed so as to have very large specific resistances, on the order of 1 Mil em - 2, thus reducing the passive ion leak into these cells which must be pumped out by the epithelial cells. 21,33 ,34 In addition to ions, metabolites and key metabolic products, such as glutathione,35,36 in the cytoplasms of the lens epithelial cells, can diffuse into the lens fibers through gap junctions joining the two different cell types. 20 This means, then, that the whole lens depends on the healthy functioning of the surface epithelial cells, and that pathology of these cells may well lead to pathology of the whole organ. Morphologically, gap junctions have been described joining lens epithelial cells with each other, joining lens fibers to each other, and joining epithelial cells to fibers,37-39 creating a networked system (Figure 1). As the lens becomes larger with age, the biology of the earliest lens fibers, contained in the lens nucleus, becomes increasingly difficult to study.40 It is clear there are age-related changes which occur within the nuclear fibers, but the functional sequellae of these changes are not known. The remainder of this paper will describe measurable phenomena in the lens cortex, and in smaller, embryological lenses, mindful that these data may not adequately describe physiological mechanisms within the enigmatic lens nucleus.
Lens fibers are joined by a structurally and functionally unique class of gap junctions Like the trunk of a tree, the lens grows .at its equatorial circumference throughout life, continuously
51
differentiating new lens fibers on top of older ones . The anucleate, organelle-free lens fibers, some of which date back to the embryological lens, must express a set of structural proteins, including those forming gap junctional channels, which can be maintained throughout the lifetime of the individual in the absence of protein synthesis and replacement. Gap junctional channel proteins in other tissues turn over with rapid half-lives . The connexin 32 molecules comprising part of the gap junctions in the mouse liver turn over with surprising rapidity,41-44 with a half-life of about 5 h. In tissue culture, the half-life of connexin 43, a gap junction protein found between many cell types in vivo, turns over with a 2-3 h half-life,45 although organ measurements suggest that the half-life is longer in lens epithelium.tf The function of this rapid turnover in communication biology is not known, but in other systems, rapid turnover offers cells a level of transcriptional or translational control of cellular functions. The rapid turnover of connexins 32 and 43 gap junction proteins is not compatible with lens fiber longevity. It follows that different connexins, with different turnover patterns, must be expressed by these highly differentiated cells, and that this different protein composition may be reflected in different structures observable in the electron microscope. Freeze-fracture electron microscopy demonstrated that lens fiber junctions are composed of connexons which do not display the tendency to crystallize in the plane of the junctional membranes,38,47,48 as is seen in gap junctions from other tissues, including lens epithelium,20,49 although these observations enjoy a dose of healthy controversy. 50-52 In contrast, intra-epithelial gap junctions are composed of connexons which do crystallize following chemical fixat ion . 2o,37-39 In addition, thin section electron microscopy reveals that the lens fiber junctions do not have the 20 A gap between the apposed junctional membranes, characteristic of gap junctions in other tissues. 38,47,48,53,54On the basis of these observations, an hypothesis was tendered which suggested that the intercellular channels between lens fibers were composed of a novel set of gap junction proteins which were .designed to remain open under conditions where other gap junction channels showed strong gating or regulation by rapid turnover.s''
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Experimental studies of intercellular communication in the lens Schuetze and Goodenough'f demonstrated that during lens development there is a rapid switch at stage 14 in 'chick embryos when gap junctions with the unique morphology of lens fiber junctions make their first appearance between the posterior future lens fibers of the lens vesicle. Concomitant with this morphological switch, the dye transfer between these cells becomes insensitive to superfusion of the lens with medium equilibrated with 90% CO 2 , a condition which has been shown to acidify the cytoplasm of other cell types and close gap junctional intercellular channels. 55 ,56 Thus, the morphologically unique lens fiber gap junctions may be temporally correlated with the acquisition of pH insensitivity. While pH resistant, the lens fiber communication pathways may be closed by other uncoupling conditions; for example, Rae et al 57 has shown that DNP will electrically uncouple lens fibers in the frog. In later stage chick embryos, Miller and Goodenoughl? have shown that lens epithelial cell intercellular communication is sensitive to cytoplasmic acidification, while the fiber-fiber communication is insensitive. Thus, in the lens, gap junctions composed of connexons which show a tendency to crystallize are pH sensitive, while those junctions which do not crystallize are pH resistant. By chick embryonic stage 23,. the fibers and lens epithelium have interacted to form heterotypic cell junctions. Morphologically, these heterotypic junctions are composed of connexons which do not crystallize. Consistent with the correlation developed by Schuetze and Goodenough.I'' the epithelium-tofiber communication pathways are insensitive to acidification. 19 Thus, the versatile lens epithelial cell can make two morphologically and physiologically distinguishable classes of gap junctions: a homotypic junction within the epithelium and a heterotypic junction with subjacent lens fibers. These ideas have been challenged by Brown et al58 who were able to find only rare epithelial/fiber gap junctions in 2-month-old adult chick lenses, and these junctions showed the crystalline morphology. These authors also reported the presence of numerous clathrin-containing coated vesicles, and they postulated ions and metabolites were likely to be exchanged across the epithelial/fiber interface by endocytosis. 59 Receptor-mediated endocytosis is an unusual way to exchange small metabolites and ions
D.A. Goodenough
between cells, given that receptors for these molecules are unknown, and that most cells, including lens epithelial cells, have well-characterized membrane transport channels. 6o -62 While the resolution of the differences between the morphological data of Brown et a1 58 , Miller and Goodenough-? and Goodenough et al 20 must await further study, junctional communication between epithelium and lens fibers has been directly demonstrated physiologically in the chicken and frog,19,63 on a time scale and using fluorescent molecules which are incompatible with endocytic and exocytic events.
Connexins in the lens As reviewed by Gilula in this issue, gap junctions in many different organs and organisms are comprised of a family of proteins, with conserved and variable domains in their amino acid sequences, called connexins. Beyer et al64 cloned eDNA coding for connexin 43, a connexin prototypically found in gap junctions joining myocardial cells. Antisera generated against synthetic oligopeptides predicted by connexin 43 eDNA stained gap junctions joining lens epithelial cells at both the light65 and electron microscope levels. 46 These antisera did not recognize the fiber/fiber junctions. In 1985, Kistler et al 66 developed a monoclonal antibody which produced a junction-like staining pattern on ovine lens fibers by immunofluorescence, and which reacted with a 70 kDa polypeptide on SDS gels, prompting the naming of this protein MP70 as suggested by Bloemendal et al. 68 The protein was shown to be cleaved to lower molecular weight forms as a natural consequence of lens fiber aging. Immunocytochemistry revealed that the anti-MP70 monoclonal reacted with lens fiber junctions.s? providing a candidate for a structural protein. Kistler et a[7o obtained N-terminal amino acid sequence data from ovine MP70 and its degradation products, and demonstrated that this protein, which had been localized to lens fiber junctions by immunocytochemistry, was homologous to connexins 32 and 43 and was a new member of the connexin family. Two different but related N-termini in MP70 were found, suggesting the possibility of two different proteins. Together with connexin 43 in the lens epithelium, it appeared as though molecular components contained in at least two of the three junctional interactions in the lens were known.
53
The crystalline lens
Starting with low-stringency hybridization oflens eDNA libraries, Beyer et al,71 and Paul et al 72 isolated a eDNA which codes for a connexin with a predicted size of 46 kDa, named connexin 46. This connexin is not unique to the lens; Northern analysis of whole organ RNA has revealed the presence of connexin 46 in both myocardium and, to a lesser degree, in kidney, although RNase protection assays of mRNA fractions will undoubtedly reveal this connexin being transcribed in other locations as well. Comparison of the N-terminal amino acid sequences reveals an exact amino acid match between one of the possible combinations from the MP70 Edman data and connexin 46. Immunoblot experiments, however, reveal that MP70 and connexin 46 are different proteins. For example, parallel immunoblots of rat lens membranes using Kistler et al's66 monoclonal antibody shows that MP70 in the rat has an M, of 64 kDa, while polyclonal antisera raised against synthetic oligopeptides predicted for different cytoplasmic domains of connexin 46 react with a 54 kDa protein. While the rat lens connexin 46 migrates on SDS gels at about 54 kDa, connexin 46 synthesized in reticulocyte lysate migrates at M, at 46 kDa, indicating that the lens fiber is capable of considerable post-translational modification of the primary translation product. The nature of this modification is not known. The anti-connexin 46 antisera stain lens fiber junctions and do not stain lens fiber non-junctional plasma membranes. This pattern of staining is similar to that seen with the anti-MP70 monoclonal. At the electron microscope level, anti-MP70 and anti-connexin 46 colocalize to the same junctional maculae, but due to the limited resolution using goldlabeled secondary antibodies, it is not possible to tell if MP70 and connexin 46 form heterohexameric connexons. Connexin 46 cRNA has been injected into Xenopus oocytes to study whether this connexin can direct intercellular communication in the oocyte-pair system, and if so, if this communication is pH insensitive, mirroring dye transfer experiments between lens fibers in situ. 18 ,19 The oocytes are able to synthesize immunoprecipitable connexin 46, but the protein is not processed from 46 kDa to the 54 kDa size seen in the mature lens. Following synthesis of connexin 46 in the 46 kDa form, the oocytes loose their membrane potentials and lyse. The oocytes may be protected from lysis by incubating them in 5 % Ficoll; under these conditions the cells
can be shown to be permeable to Lucifer Yellow CH, a 457 Dalton fluorescent molecule known to be permeable through gap junction channel~.73 Voltage clamping of oocytes at different potentials reveals that voltage-gated channels can be opened in the oocyte membrane of cells injected with connexin 46 cRNA when the cells are clamped positive to -10mV. Taken together, these data suggest that the connexin 46 is either directly forming or indirectly inducing open channels in the oocyte plasma membrane. These channels depolarize the cells, and the resulting influx of water either directly, or by stimulating the opening of endogenous stretchactivated channels, causes the cells to lyse. Since mature lens fiber membranes have very low conductances ,(vide supra), these open channels are presumably not formed in the lens fiber nonjunctional plasma membrane, where the protein is posttranslationally processed. The relationships between MP70 and connexin 46 are not understood. Different connexins are known to colocalize to the same junctional maculae in the liver,74,75 but the functional consequences of this intermingling are not yet clear..The molecular components of the epithelial/fiber communicating pathways are also not known. While antisera specific for MP70 and connexin 46 appear to concentrate to the epithelial/fiber interface at the light microscope level, the data are not yet adequate to define their localization in heterotypic gap junctions. Nor is it possible in the light microscope to observe staining of both surfaces of the junctions; electron microscopy is essential to determine both if the epithelial/fiber junctions contain MP70 and connexin 46, and if these proteins are found on both sides of these heterotypic intercellular interactions.
MP26, a lens fiber membrane protein In addition to connexins, other lens fiber integral membrane proteins, some of which are gene products unique to the lens, have been suggested to have a structural and functional role in intercellular communication. The lens fibers are unusually rich in a 26 kDa integral membrane 'protein, called the major intrinsic polypeptide (MIP) or MP26. Due to the non-crystallizing nature of the connexons, lens fiber gap junctions are not resistant to solubilization by detergents, and hence cannot be isolated using protocols developed for other tissues.7 6 ,77 In the late 1970s, appropriate control of
54 proteolysis revealed that isolated liver gap junctions were enriched in a 27 kDa protein. 78-80 The similarity in molecular weight between MP26 and the liver 27 kDa protein, together with the large numbers of lens fiber junctions which appeared to correspond to the preponderance of MP26 in lens fiber membrane fractions, prompted the hypothesis that MP26 was a principal structural component of lens fiber junctions. 38 ,81-84
MP26 is not a member of the connexin family D espite the similarity in electrophoretic mobility between liver and lens fiber data, there were reasons to suspect that MP26 and the liver 27 kDa proteins might be different. Hertzberg et a(i35 demonstrated that MP26 and liver 27 kDa are non-identical when compared by partial peptide mapping. They also prepared an antiserum to MP26, and were unable to demonstrate any cross-reactivity to the liver protein. This comparison was extended to amino acid sequence 'analysis, and there seemed to be no homology between liver 27 kDa and lens MP26 within the first 50 amino acid residues. 86 ,87 Nicholson et a(i38 showed using sequencing analysis that gap junctions formed between hepatocytes and myocardial cells were composed of different, but related proteins, giving the first biochemical indication that gap junctions were composed of a family of related proteins, and that MP26, if it participated in intercellular communication, was not a member of this family. The lack of homology between MP26, liver 27 kDa arid heart gap junction protein held true for the complete amino acid sequences predicted from cDNA clones. Gorin et a(i39 isolated a cDNA coding for MP26 . Paul 90 and Kumar and Gilula'l isolated cDNAs coding connexin 32, the liver 27 kDa protein, and Beyer et a(S4 isolated cDNA coding for connexin 43, a heart gap junction protein. Comparison of the amino acid sequence of MP26 with those of connexins 32 and 43 reveals that MP26 is a completely unrelated protein, indicating that if MP26 is a junctional protein, it represents a parallel evolutionary solution to the structure of a communicating channel. Ehring et aP2 have proposed an innovative model wherein channels composed of MP26 facilitate intercellular communication via a highly resistive intercellular space and function to minimize extracellular fluids between lens fibers (vide infra).
D .A. Goodenough
Immunolocalization of MP26 in lens fiber membranes Several laboratories have attempted to localize MP26 in the lens fiber junctions using immunocytochemistry. In some studies, antisera directed against lens MP26 stain lens fiber gap junctions viewed in the electron microscope, in addition to staining nonjunctional lens fiber plasma membranes. 93 -95 However, Paul and Goodenough'f found that a wide variety of anti-MP26 antisera would not stain lens fiber junctions, but rather stained only the lens fiber plasma membranes and a 'thin' pentalaminar structure of unknown function . The 'thin: pentalaminar structures were stained only on one side, demonstrating an asymmetric protein distribution. These findings have been confirmed by Zampighi et al,97 who have correlated the asymmetric protein distribution with a freeze-fracture analysis of the 'thin' junctions which show intramembrane particles in only one of the two membranes. The differences in immunocytochemical data obtained by different laboratories has not yielded to exchanges of antisera, membranes or both, implicating subtle differences in specimen preparation as being responsible for these rather substantive differences in antibody localization. It is thus probable that immunocytochemistry will not of itself answer questions either about the function ofMP26 or its role in intercellular communication.
The functions of MP26 The functions of MP26 are not currently understood. Some have argued that it serves a structural role 98,99 for the lens fiber membrane. Others have reconstituted MP26 into planar lipid bilayers lOO and lipid vesicles'P! and have shown the protein to have channel activity. Channel activity in single membranes is not proof of function in intercellular communication. MP26 cRNA was injected into a paired Xenopus oocyte system developed by Werner et al102 under conditions where cRNAs coding for liver (connexin 32) myocardial (connexin 43), and embryonic gap junctions were able to direct the assembly of functional junctions. 103 ,104 The MP26 translated in these cells was neither able to migrate to the site of cell-cell apposition nor able to induce junctional conductances above background. While the function of MP26 in lens fiber biology and a direct role for MP26 in intercellular channel
The crystalline lens
formation remains to be clearly demonstrated, Ehring et al92 have measured voltage-sensitive single channel conductances in planar lipid bilayers reconstituted with HPLC-purified MP26. The channels are large (380 and 160 pS in 0.1 M KCI), show symmetrical voltage dependence, and are insensitive to Ca2 + and H + ion concentrations. The vesicles used for the insertion of the channels into the planar bilayers were studied by freezefracture analysis across a broad range oflipid-protein concentration ratios. At the higher protein concentrations, these studies demonstrated crystalline arrays within the vesicle membranes indistinguishable from those arrays correlated with the 'thin' junctional profiles mentioned above. On the basis of these studies, Ehring et al 92 have proposed an innovative function for the MP26 channels wherein the channels in the interior of the lens would be open, permitting intercellular communication via a narrow extracellular space, and also functioning to keep water from accumulating in the intercellular spaces since intercellular water would tend to enter the cytoplasm of the osmotically active lens fibers. Given that the lens fiber intercellular spaces must be kept narrow to avoid light scattering, this may prove to be an essential function for MP26 channel activity; Other homologous proteins to MP26 have been identified in diverse systems which also have channel activit yl05.106 which may provide additional clues ofMP26 function . Recently, yet another protein has been localized to the lens fiber junctions by immunocytochemistry. A 20 kDa protein, MP20, has been carefully studied and shown to localize at lens fiber junctions and to be unrelated to either MP26 or the connexin family by both antigenic and N-terminal sequence criteria.l07.108 MP20 has been shown to be a phosphoprotein, phosphorylated by both cAMPdependent protein kinase and protein kinase C in oitro, and to bind calmodulin, both properties shared with MP26. 107 The picture emerges, then, that the crystalline lens is an organ whose physiology and homeostasis depends centrally on gap junction mediated intercellular communication. The molecular nature of the gap junctions joining lens cells is not yet fully understood. Additional connexins may be found in the lens' repertoire, in addition to connexins 43, 46 and MP70. MP26 and MP20 have been localized to lens fiber junctions in several laboratories, and while these are not members of the connexin family of proteins, their potential roles in cell-cell communication remain an intriguing possibility.
55 Given the availability of lens-specific promoters, which have been shown to drive expression of exogenous proteins both in lens cultures and in transgenic animals,109,11O there are possible experimental approaches to study some of these unknowns.
Acknowledgements The author gratefully acknowledges the collaboration and friendship of Dr David L. Paul, and research support from grant EY02430 from the National Institutes of Health.
References 1. Bloemendal H (1977) The vertebrate eye lens . Science 197:127-138 2. Farnsworth PN, Burke-Gadomski P, Kulyk T, Mauriello jA, Cinotti AA (1976) Surface ultrastructure of the epithelial cells of the mature human lens. Exp Eye Res 22:615-62ol 3. Vrensen GFJM, GrawJ, DeWolf A (1991) Nuclear breakdown during terminal differentiation of primary lens fibers in mice: a transmission electron microscope study. Exp Eye Res 52:6H-659 ol. Bettelheim FA (1985) Physical basis of lens transparency, in The Ocular Lens: Structure, Function and Pathology (Meisel H, ed), pp 265-300. Marcel Dekker, New York 5. Benedek GB (1971) Theory of transparency of the eye. Appl Optics 1O:459·H3 6. Benedek GB, Clarke JI, Serallach EN, Young CY, Mengel L, Sanke T, Bagg A, Benedek K (1979) Light scattering and reversible cataracts in the calf and human lens . Philos Trans R Soc Lond Ser B 293:329-3ol0 T, Kinsey VE, Frohman CE (1951) Distribution of cytochrome, total riboflavin, lactate and pyruvate and its metabolic significance. Arch Ophthal NY ol6:536-5H 8. Duncan G (1969) Relative permeabilities of the lens membranes to sodium and potassium. Exp Eye Res 8:315-325 9. Duncan G (1969) The site of ion restricting membranes in the toad lens . Exp Eye Res 8:406-412 10. Duncan G (1969) Kinetics of potassium movement across amphibian lens membranes. Exp Eye Res 8:ol13-420 11. Eisenberg RS, RaeJL (1976) Current-voltage relationships in the crystalline lens. J Physiol 262:285-300 12. RaeJL (1973) The potential dilTerenceof the frog lens. Exp Eye Res 15:ol85-494 13. RaeJL (1979) The electrophysiology of the crystalline lens. Curr Top Eye Res 1:37-90 H. Mathias RT, Rae JL (1985) Transport properties of the lens . Am J PhysioI2ol9:CI81-CI90 15. KuszakJR, Macsai MS, Bloom KJ, RaeJL, Weinstein RS (1985) Cell-to-cell fusion oflens fiber cells in situ: correlative light, scanning electron microscopic, and freeze-fracture studies. J Ultrastruct Res 93: 144-160 16. Rae JL (1974) The movement of procion dye in the crystalline lens. Invest Ophthal Vis Sci 13:H7-150 17. RaeJL, Stacey T (1979) Lanthanum and 'proeion yellow as extracellular markers in the crystalline lens of the rat. Exp Eye Res 28:1-21
56 18. Schuetze SM, Goodenough DA (1982) Dye transfer between cells of the embryonic chick lens becomes less sensitive to CO 2-treatment with development. J Cell BioI 92:694--705 19. Miller TM, Goodenough DA (1986) Evidence for two physiologically distinct gap junctions expressed by the chick lens epithelial cell. J Cell BioI 102:194--199 20. Goodenough DA, Dick JSB, II, Lyons JE (1980) Lens metabolic cooperation: a study of mouse lens transport and permeability visualized with freeze-substitution autoradiography and electron microscopy. J Cell BioI 86:576-589 21. Mathias RT, Rae JL, Ebihara L, McCarthy RT (1985) The localization of transport properties in the frog lens. Biophys J 4-8:4-23-4-3422. Kinsey VE, Reddy DVN (1965) Studies on the crystalline lens. XI. The relative role of the epithelium and capsule in transport. Invest Ophthalmol Vis Sci 4-:104--116 23. Palva M, Palkama A (1974-) Histochemically demonstrable sodium-potassium-activated adenosine triphosphatase (Na-K-ATPase) activity in the rat lens. Exp Eye Res 19:117-123 24-. Neville MC, Patterson CA, Hamilton PM (1978) Evidence for two sodium pumps in the crystalline lens of the rabbit eye. Exp Eye Res 27:1-12 24-. Gorthy WC, Anderson JW (1980) Special characteristics of the polar regions of the rat lens: morphology and phosphatase histochemistry. Invest Ophthalmol Vis Sci 19:1038-1052 26. Kinsey VE (1965) The pump-leak concept of transport in the ocular lens, .in The Structure of the Eye (Rohen JW, ed), pp 384--394-.~ Schattauer, Stuttgart 27. Candia OA, Bentley PJ, Mills CD, Toyofuku H (1970) Asymmetrical distribution of the potential difference in the toad lens. Nature 227:852-853 28. Candia OA, Bentley PJ, Mills CD (1971) Short-circuit current and active transport across isolated lens of the toad. Am J Physiol 220:558-56429. Delamere NA, Duncan G (1979) The properties of bovine lens membranes measured by a conventional doublechamber method. J Physiol (Lond) 295:24-1-24-9 30. Robinson KR, PattersonJW (1983) Localization of steadystate currents in the lens. Curr Eye Res 2:84-3-84-7 31. Mathias RT (1985) Steady-state voltages, ions fluxes and volume regulation in syncitial tissues. BiophysJ 4-8:4-35-H8 32. RaeJL (1974-) Voltage compartments in the lens. Exp Eye Res 19:235-24-2 33. Mathias RT, Rae JL, Eisenberg RS (1979) Electrical properties of structural components of the crystalline lens. Biophys J 25:181-201 34-. Mathias RT, Rae JL (1989) Cell to cell communication in the lens, in Cell Interactions and Gap Junctions (Sperelakis N, Cole WC, eds), pp 29-50. CRC Press, Boca Raton, FL 35. Kern HL, Ho C-K (1973) Transport of L-glutamic acid and L-glutamine and their incorporation into lenticular glutathione. Exp Eye Res 17:455-462 36. Calvin HI, Medvedovsky C, David JC, Broglio TM, HessJL, Fu S-CJ, Worgul BV (1991) Rapid deterioration of lens fibers in GSH-depleted mouse pups. Invest Ophthalmol Vis Sci 32: 1916-1924 37. Peracchia C (1978) Calcium effects on gap junction structure and cell coupling. Nature 271:669-671 38. Goodenough DA (1979) Lens gap junctions: a structural hypothesis for nonregulated low-resistance intercellular pathways. Invest Ophthalmol Vis Sci 18:1104--1122 39. Miller TM, Goodenough DA (1985) Gap junction structures after experimental alteration of junctional channel conductance. J Cell BioI 101:1741-174-8
D.A. Goodenough 40. Itoi M, Kodama R, Takayama S, Eguchi G (1991) Ultrastructural observations of typical gap junctions in human foetal lens nucleus. Curr Eye Res 10: 1-9 41. Yee AG, Revel JP (1978) Loss and reappearance of gap junctions in regenerating liver. J Cell BioI 78:554--564 42. Fallon RF, Goodenough DA (1981) Five hour half-life of mouse liver gap-junction protein. J Cell BioI 90:521-526 43. Yancey SB, Nicholson BJ, RevelJP (1981) The dynamic state ofliver gap junctions. J Supramol Struct Cell Biochem 16:221-232 H. Traub 0, Druge M, Willecke K (1983) Degradation and resynthesis of gap junction protein in plasma membranes of regenerating liver after partial hepatectomy Drcholestasis. Proc Natl Acad Sci USA 80:755-759 4-5. Musil LS, Cunningham BA, Edelman GM, Goodenough DA (1990) Differential phosphorylation of the gap junction protein connexin 43 in junctional communication-competent and -deficient cell lines. J Cell BioI 111 :2077-2088 46. Musil LS, Beyer EC, Goodenough DA (1990) Expression of the gap junction protein connexin 43 in embryonic chick lens: molecular cloning, ultrastructural localization, and post-translational phosphorylation. J Mernbr BioI 116: 163-175 47. Benedetti EL, Dunia I, Bloemendal H (1974) Development of junctions during differentiation oflens fibers. Proc Natl Acad Sci USA 71:5073-5077 48. Benedetti EL, Dunia I, Bentzel CJ, Vermorken AJM, Kibbelaar M, Bloemendal H (1976) A portrait of plasma membrane specializations in eye lens epithelium and fibers. Biochim Biophys Acta 457:353-384 49. Goodenough DA, Gilula NB (1974) The splitting of hepatocyte gap junctions and zonulae occludentes with hypertonic disaccharides. J Cell BioI 61:575-590 50. Peracchia C, Peracchia LL (1980) Gapjunction dynamics: reversible effects of divalent cations. J Cell BioI 87:708-718 51. Peracchia C, Peracchia LL (1980) Gap junction dynamics: reversible effects of hydrogen ions.J Cell BioI 87:719-727 52. KistlerJ, Bullivant S (1980) The connexon order in isolated lens gap junctions. J Ultrastruct Res 72:27-38 53. Revel JP, Karnovsky MJ (1967) Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J Cell BioI 33:c7-c10 54-. Goodenough DA, RevelJP (1970) A fine structural analysis of intercellular junctions in the mouse liver. J Cell BioI 45:272-290 55. Turin L, Warner A (1977) Carbon dioxide reversibly abolishes ionic communication between cells of early amphibian embryos. Nature 270:56-57 56. Spray DC, Harris AL, Bennett MVL (1981) Gapjunctional conductance is a simple and sensitive function of intracellular pH. Science 211:712-715 57. RaeJL, Thompson RO, Eisenberg RS (1982) The effect of 2,-4 dinitrophenol on cell to cell communication in the frog lens. Exp Eye Res 35:597 58. Brown HG, Pappas GD, Ireland ME, KuszakJR (1990) Ultrastructural, biochemical, and immunologic evidence of receptor mediated endocytosis in the crystalline lens. Invest Ophthalmol Vis Sci 31:2579-2592 59. KuszakJR, SivakJG, WeerheimJA (1991) Lens optical quality is a direct function oflens sutural architecture. Invest Ophthalmol Vis Sci 32:2119-2129 60. Kern HL, Ho C-K (1973) Localization and specificity of the transport system for sugars in the calf lens. Exp Eye Res 15:751-765 61. Kern HL, Ho C-K; Ostrove SA (1977) Comparison of transport at the anterior and posterior surfaces of the calf lens. Exp Eye Res 24-:559-570 62. Kern HL (1979) Transport of organic solutes in the lens. Curr Top Eye Res 1:217-24-0
The crystalline lens 63. RaeJL, KuszakJR (1983) The electrical coupling of epithelium and fibers in the frog lens. Exp Eye Res 36:317-326 61. Beyer EC, Paul DL, Goodenough DA (1987) Connexin13: a protein from rat heart homologous to a gap junction protein from liver. J Cell BioI 105:2621-2629 65. Beyer EC, Kistler J, Paul DL, Goodenough DA (1989) Antisera directed against connexin13 peptides react with a 13-kD protein localized to gap junctions in myocardium and other tissues. J Cell BioI 108:595-605 66. Kistler J, Kirkland B, BuIIivant S (1985) Identification of a 70,000-D protein in lens membrane junctional domains. J Cell BioI 101:28-35 67. Bloemendal H, Vermorken A, Kibbelaar M, Dunia I, Benedetti EL (1977) Nomenclature for the polypeptide chains oflens plasma membrane. Exp Eye Res 21:113-115 69. Gruijters WTM, Kistler J, BuIIivant S, Goodenough DA (1987) Immunolocalization ofMP70 in lens fiber 16-17 nm intercellular junctions. J Cell BioI 101:565-572 70. Kistler J, Christie D, BuIIivant S (1988) Homologies between gap junction proteins in lens, heart and liver. Nature 331:721-723 71. Beyer EC, Goodenough DA, Paul DL (1988) The connexins, a family of related gap junction proteins, in Gap Junctions (Hertzberg EL and Johnson RG eds), pp 167-175. Alan R, Liss, New York 72. Paul DL, Ebihara L, Takemoto LJ, Swenson KI, Goodenough DA (1191) Connexin16, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes. J Cell BioI 115:1077-1089 73. Stewart WW (1978) Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer. Cell 11:711-759 71. Nicholson BJ, Derrnietzel R, Teplow D, Traub 0, WiIIecke K, Revel j-r (1987) Two homologous protein components of hepatic gap junctions. Nature 329:732-731 75. Zhang J-T, Nicholson B (1989) Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its cDNA.J Cell BioI 109:3391-3101 76. Goodenough DA, Stoeckenius W (1972) The isolation of mouse hepatocyte gap junctions. Preliminary chemical characterization and X-ray diffraction. J Cell BioI 51:616-656 77. Kensler RW, Goodenough DA (1980) Isolation of mouse myocardial gap junctions. J Cell BioI 86:755-761 78. DuguidJR, RevelJ-P (1976) The protein components of the gap junction. Cold Spring Harb Symp Quant BioI 10:15-17 79. Hertzberg EL, Gilula NB (1979) Isolation and characterization of gap junctions from rat liver. J BioI Chem 251:2138-2117 80. Henderson D, Eibl H, Weber K (1979) Structure and biochemistry of mouse hepatic gap junctions. J Mol BioI 132:193-218 81. AlcalaJ, Lieska N, Maisel H (1975) Protein composition of bovine lens cortical fiber cell membranes. Exp Eye Res 21:581-589 82. Broekhuyse R, Kuhlman E, Stols A (1976) Lens membranes II. Isolation and characterization ofthe main intrinsic polypeptide (MIP) of bovine lens fiber membranes. Exp Eye Res 23:365-371 83. Maisel H (1977) The nature of the urea-insoluble material of the human lens. Exp Eye Res 21:117-119 81. Kuszak J, Maisel H, Harding CV (1978) Gap junctions of chick lens fiber cells. Exp Eye Res 27:195-198 85. Hertzberg EL, Anderson DJ, Friedlander M, Gilula NB (1982) Comparative analysis of the major polypeptides from liver gap junctions and lens fibers junctions. J Cell BioI 92:53-59
57 86. Nicholson BJ, Hunkapiller MW, Grim LB, Hood LE, Revel JP (1981) Rat liver gap junction protein: properties and partial sequence. Proc Nat! Acad Sci USA 78:7591-7598 87. Nicholson BJ, Takemoto LJ, Hunkapillar LE, Hood LE, Revel JP (1983) Differences between liver gap junction protein and lens MP26 from rat: implications for tissue specificity of gap junctions. Cell 32:967-978 88. Nicholson BJ, Gros DB, Kent SBH, Hood LE, RevelJ-P (1985) The Me 28,000 "gap junction proteins from rat heart and liver are different but related. J BioI Chern 260:6511-6517 89. Gorin MB, Yancey SB, ClineJ, RevelJ-P, HorwitzJ (1981) The major intrinsic protein (MIP) of the bovine lens fiber membrane: characterization and structure based on cDNA cloning. Cell 39:19-59 90. Paul DL (1986) Molecular cloning of eDNA for rat liver gap junction protein. J Cell BioI 103:123-131 91. Kumar NM, Gilula NB (1986) Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein. J Cell BioI 103:767-776 92. Ehring GR, Zampighi G, Horwitz J, Bok D, Hall JE (1990) Properties of channels reconstituted from the major intrinsic protein of lens fiber membranes. J Gen Physiol 96:631-661 93. Bok D, Dockstader J, Horwitz J (1982) Immunocytochemical localization of the lens main intrinsic polypeptide (MIP) in communicating junctions. J Cell BioI 92:213-220 91. FitzGerald PG, Bok D, Horwitz J (1983) Immunocytochemical localization of the main intrinsic polypeptide (MIP26) in ultrathin frozen sections of rat lens. J Cell BioI 97:1191-1199 95. Sas DF, Sas MJ,Johnson KR, Menko AS, Johnson RG (1985)Junctions between lens fiber cells are labeled with a monoclonal antibody shown to be specific for MP26. J Cell BioI 100:216-225 96. Paul DL, Goodenough DA (1983) Preparation, characterization, and localization of antisera against bovine MP26, an integral protein from lens fiber plasma membrane. J Cell BioI 96:625-632 97. Zampighi G, HaIlJE, EhringGR, Simon SA (1989) The structural organization and protein composition of lens fiber junctions. J Cell BioI 108:2255-2275 98. Costello MJ, McIntosh TJ, RobertsonJD (1989) Distribution of gap junctions and square array junctions in the mammalian lens. Invest Ophthalmol Vis Sci 30:975-989 99. Gruijters WTM (1989) A non-connexon protein (MIP) is involved in eye lens gap-junction formation. J Cell Sci 93:509-513 100. Zampighi GA, HaIlJE, Kreman M (1985) Purified lens junctional protein forms channels in planar lipid films. Proc Natl Acad Sci USA 82:8168-8172 101. Peracchia C, Girsch SJ (1985) Functional modulation of cell coupling: evidence for a calmodulin-driven channel gate. Am J Physiol 218:H765-H782 102. Werner R, Miller T, Azarnia R, Dahl G (1985) Translation and functional expression of cell-cellchannel mRNA in Xenopus oocytes. J Membrane BioI 87:253-268 103. Swenson KI, Jordan JR, Beyer EC, Paul DL (1989) Formation of gap junctions by expression of connexins in Xenopus oocyte pairs. Cell 57:115-155 101. Ebihara L, Beyer EC, Swenson KI, Paul DL, Goodenough DA (1989) Cloning and expression of a Xenopus embryonic gap junction protein. Science 213:1191-1195 105. Baker ME, Saier MHJr (1990) A common ancestor for bovine lens fiber major intrinsic protein, soybean nodulin226 protein, and E. coli glycerol facilitator. Cell 60:185-186
58 106. Van Aelst L, Hohmann S, Zimmermann FK, Jans AWH, Thevelein JM (1991) A yeast homologue of the bovine lens fibre MIP gene family complements the growth defect of a Saccharomyces cereoisiae mutant on fermentable sugars but not its defect in glucoseinduced RAS-mediated cAMP signalling. EMBO J 10:2095-2104 107. Galvan A , Lampe PO, Hur KC, Howard JB, Eccleston ED, Arneson M, Louis CF (1989) Structural organization of the lens fiber cell plasma membrane protein MPI8. J Bioi Chern 264:19974-19978 108. Louis CF, Hur KC , Galvan AC, TenBroek EM, Jarvis LJ, Eccleston ED, Howard JB (1989) Identification of an
D.A. Goodenough 18,000 -Dalton protein in mammalian lens fibers. J Bioi Chern 264:19967-19973 109. Chepelinsky AB, King CR. Zelenka PS, Piatigorsky J (1985) Lens-specific expression of the choramphenicol acetyltransferase gene promoted by 5' flanking sequences of the murine aA-crystallin gene in explanted chicken lens epithelia. Proc Nat! Acad Sci USA 82:2334-2338 110. Overbeek PA, Chepelinsky AB, KhilIanJS, PiatigorskyJ, Westphal H (1985) Lens-specific expression and developmental regulation of the bacterial chloramphenicol acetyltransferase gene driven by the murine aA-crystallin promoter in transgenic mice. Proc Natl Acad Sci USA 82:7815-7819
The crystalline lens. A system networked by gap junctional intercellular communication Daniel A. Goodenough The vertebrate eye lens is a solid cyst of cells which grows throughout life by addition of new cells at the surface. The older cells) buried by thenewergenerations) differentiate into long) prismaticfibers) losing their cellular organelles and jilling their cytoplasms with high concentrations of soluble proteins) thecrystallins. The long-lived lensfibers areinterconnected by gapjunctions) both with themselves and with an anterior layer of simple cuboidal epithelial cells at thelens surface. This network of gap junctions joins the lens cells into a syncytium with respect to small molecules, permitting metabolic co-operation: intercellular diffusion of ions) metabolites) and water. In contact with nutrients at the lens surface) theepithelial cells retain their cellular organelles) and are able to provide the metabolic energy to maintain correct ion and metabolite concentrations within the lens fiber cytoplasms, such that the crystallins remain in solution and do not aggregate (cataract). Gapjunctions areformed by a family of integral membrane channel-formingproteins called connexins. Gap junctions between lens epithelial cells are composed of a connexin which is common between marry different cell types, notably myocardial cells and connective tissuefibroblasts. The gapjunctions between epithelial cells and lens fibers havenotyet been biochemically characterized. The gapjunctionsformed between lensfibers are composed of at least two different connexins, one of which has not been detected between other celltypes. The unusualphysiology and longeviry of the lens fibers may require the special set of connexins which arefound joining these cells.
become the lens placode. The placode then invaginates into the embryo to eventually pinch off from the ectoderm as a hollow sphere of cells, the lens vesicle. Subsequently, the posterior cells of the lens vesicle elongate anteriorly, obliterating the lumen of the lens vesicle, coming into apposition to, and making gap junctions with, the anterior lens epithelium. At the end of this process, the early lens is a solid cyst of cells, with an epithelium anteriorly and elongated cells, the lens fibers, posteriorly (Figure 1). The epithelium and fibers are continuous with each other at the edges, or bow region, of the lens. In this bow region, a population of undifferentiated stem cells persists throughout life, continuously dividing and adding new, elongating lens fibers equatorially at the organ surface. 1•2 The lens floats in the aqueous humor, suspended posterior to the iris by zonular fibers which attach it to the ciliary body. The lens has no blood supply and no connective tissue, save a thick capsule, the enlarged original basement membrane of the embryonic ectoderm, composed in part of type IV collagen. The stem cell population of the lens continues to divide throughout life generating daughter cells which undergo cytodifferentiation: massive synthesis of tissue-specific proteins, the soluble crystallins, and loss of the nucleus and other organelles.I The crystallins are gene products unique to the lens, and due to the isolation of the lens during early embryonic development from both the blood supply and the process of self-recognition by the immune system, these unique gene products are never programmed as 'self and can elicit an autoimmune reaction following eye injury. The purpose of the accumulation of high concentrations of soluble proteins and loss of organelles is to allow the lens to achieve both a high refractive index and transparency.t The presence of organelles in the lens would result in image-degrading lightscattering.V' In order not to view the world through a cytochrome-tinted haze, the differentiated lens cells avoid oxidative phosphorylation," and the organ derives the bulk of its energy from anaerobic glycolysis, with lactate its chief excretory product.
Key words: lens / gap junctions / intercellular communication / connexins
The lens is a cellular system, interconnected by gap junctions During development, an outgrowth of neurectoderm, the optic vesicle, becomes apposed to, and 'induces' the overlying embryonic ectoderm to thicken and From the Department of Cellular Biology andAnatomy) Harvard Medital School, 220 Longwood Avenue, Boston, .MA 02115, USA ©1992 Academic Press Ltd 1013·4682/92/010049 + 10S5. 00/0 49
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Bow
Figure 1. A diagram outlines the junctional interactions between the lens fibers and epithelial cells. The lens fibers are drawn as straight, connecting the anteriorly disposed epithelial cells with the lens posterior surface. In early stages of lens development, the fibers are disposed in this fashion. In older lenses, the fibers wrap concentrically, creating a large population of internalized cells which are not in contact with the lens surface. The boxed area shows an enlarged portion of the epithelium-to-fiber interface. The gap junctions are represented by spots where the outlines of the cells come into contact. Three types of interaction are shown: epithelium/epithelium (E/E), epithelium/fiber (ElF), and fiber/fiber (FIF). The shaded circles and ellipses represent cellular nuclei, which become pyknotic and disappear as the fibers age. The extracellular matrix of the capsule is indicated in the inset but not the main figure. The fibers in the center of the lens, since they have neither blood supply nor organelles, are uniquely dependent on communication with cells at the lens surface. Electrophysiological data8- 14 have established that the inner lens fibers have typical cellular membrane potentials ( - 70 m V), and that they are joined into a syncytium with respect to ions. This syncytium is achieved by an enormous number of gap junctions between adjacent lens cells, and perhaps some frank fiber-fiber membrane fusions.P which permit ions, and fluorescent dyes l6-19 as well as small, transported metabolites-" to diffuse between adjacent cells. The lens fiber membranes have no active transport channels and very low passive permeability to ions.I!
The anatomy and physiology of the lens partially explain how the internal lens fibers, which live the lifetime of the individual, can maintain active resting potentials and not need a continuing supply of newly synthesized transport proteins: the epithelial cells at the lens surface, known to contain the bulk of the N a + - K + ATPase activity in the lens,22-25 pump ions for the remainder of the lens fibers to which they are connected by gap junctions. This is the classic 'pump-leak' model of Kinsey,26 which explains the potential difference across the organy·29 While the locations of the various pumps and leaks are not known with precision, extracellular vibrating probe studies with the rat lens demonstrate clear circulating current flow through complex pathways in the
The crystalline lens
lens. 30 An attractive hypothesis has suggested that the ion (mostly sodium) and accompanying passive fluid flow are radially inward through the extracellular spaces between the lens fibers, and radially back outward through the cytoplasms of the lens fibers, through the gap junctions to the epithelial cells, where active transport occurs. H,21 This flow would not only serve to circulate fluids through the lens, but also might create a negative hydrostatic pressure in the intercellular spaces between lens fibers, assuring that these spaces remain collapsed and non-light scattering, even during movements of the lens during accommodation. This miniature circulatory system has been presented in a model by Mathias-! which predicts both the intracellular and extracellular voltage gradients measurable in the lens. H,32 To reduce the workload of the epithelial cells as much as possible, the lens fiber membranes are constructed so as to have very large specific resistances, on the order of 1 Mil em - 2, thus reducing the passive ion leak into these cells which must be pumped out by the epithelial cells. 21,33 ,34 In addition to ions, metabolites and key metabolic products, such as glutathione,35,36 in the cytoplasms of the lens epithelial cells, can diffuse into the lens fibers through gap junctions joining the two different cell types. 20 This means, then, that the whole lens depends on the healthy functioning of the surface epithelial cells, and that pathology of these cells may well lead to pathology of the whole organ. Morphologically, gap junctions have been described joining lens epithelial cells with each other, joining lens fibers to each other, and joining epithelial cells to fibers,37-39 creating a networked system (Figure 1). As the lens becomes larger with age, the biology of the earliest lens fibers, contained in the lens nucleus, becomes increasingly difficult to study.40 It is clear there are age-related changes which occur within the nuclear fibers, but the functional sequellae of these changes are not known. The remainder of this paper will describe measurable phenomena in the lens cortex, and in smaller, embryological lenses, mindful that these data may not adequately describe physiological mechanisms within the enigmatic lens nucleus.
Lens fibers are joined by a structurally and functionally unique class of gap junctions Like the trunk of a tree, the lens grows .at its equatorial circumference throughout life, continuously
51
differentiating new lens fibers on top of older ones . The anucleate, organelle-free lens fibers, some of which date back to the embryological lens, must express a set of structural proteins, including those forming gap junctional channels, which can be maintained throughout the lifetime of the individual in the absence of protein synthesis and replacement. Gap junctional channel proteins in other tissues turn over with rapid half-lives . The connexin 32 molecules comprising part of the gap junctions in the mouse liver turn over with surprising rapidity,41-44 with a half-life of about 5 h. In tissue culture, the half-life of connexin 43, a gap junction protein found between many cell types in vivo, turns over with a 2-3 h half-life,45 although organ measurements suggest that the half-life is longer in lens epithelium.tf The function of this rapid turnover in communication biology is not known, but in other systems, rapid turnover offers cells a level of transcriptional or translational control of cellular functions. The rapid turnover of connexins 32 and 43 gap junction proteins is not compatible with lens fiber longevity. It follows that different connexins, with different turnover patterns, must be expressed by these highly differentiated cells, and that this different protein composition may be reflected in different structures observable in the electron microscope. Freeze-fracture electron microscopy demonstrated that lens fiber junctions are composed of connexons which do not display the tendency to crystallize in the plane of the junctional membranes,38,47,48 as is seen in gap junctions from other tissues, including lens epithelium,20,49 although these observations enjoy a dose of healthy controversy. 50-52 In contrast, intra-epithelial gap junctions are composed of connexons which do crystallize following chemical fixat ion . 2o,37-39 In addition, thin section electron microscopy reveals that the lens fiber junctions do not have the 20 A gap between the apposed junctional membranes, characteristic of gap junctions in other tissues. 38,47,48,53,54On the basis of these observations, an hypothesis was tendered which suggested that the intercellular channels between lens fibers were composed of a novel set of gap junction proteins which were .designed to remain open under conditions where other gap junction channels showed strong gating or regulation by rapid turnover.s''
52
Experimental studies of intercellular communication in the lens Schuetze and Goodenough'f demonstrated that during lens development there is a rapid switch at stage 14 in 'chick embryos when gap junctions with the unique morphology of lens fiber junctions make their first appearance between the posterior future lens fibers of the lens vesicle. Concomitant with this morphological switch, the dye transfer between these cells becomes insensitive to superfusion of the lens with medium equilibrated with 90% CO 2 , a condition which has been shown to acidify the cytoplasm of other cell types and close gap junctional intercellular channels. 55 ,56 Thus, the morphologically unique lens fiber gap junctions may be temporally correlated with the acquisition of pH insensitivity. While pH resistant, the lens fiber communication pathways may be closed by other uncoupling conditions; for example, Rae et al 57 has shown that DNP will electrically uncouple lens fibers in the frog. In later stage chick embryos, Miller and Goodenoughl? have shown that lens epithelial cell intercellular communication is sensitive to cytoplasmic acidification, while the fiber-fiber communication is insensitive. Thus, in the lens, gap junctions composed of connexons which show a tendency to crystallize are pH sensitive, while those junctions which do not crystallize are pH resistant. By chick embryonic stage 23,. the fibers and lens epithelium have interacted to form heterotypic cell junctions. Morphologically, these heterotypic junctions are composed of connexons which do not crystallize. Consistent with the correlation developed by Schuetze and Goodenough.I'' the epithelium-tofiber communication pathways are insensitive to acidification. 19 Thus, the versatile lens epithelial cell can make two morphologically and physiologically distinguishable classes of gap junctions: a homotypic junction within the epithelium and a heterotypic junction with subjacent lens fibers. These ideas have been challenged by Brown et al58 who were able to find only rare epithelial/fiber gap junctions in 2-month-old adult chick lenses, and these junctions showed the crystalline morphology. These authors also reported the presence of numerous clathrin-containing coated vesicles, and they postulated ions and metabolites were likely to be exchanged across the epithelial/fiber interface by endocytosis. 59 Receptor-mediated endocytosis is an unusual way to exchange small metabolites and ions
D.A. Goodenough
between cells, given that receptors for these molecules are unknown, and that most cells, including lens epithelial cells, have well-characterized membrane transport channels. 6o -62 While the resolution of the differences between the morphological data of Brown et a1 58 , Miller and Goodenough-? and Goodenough et al 20 must await further study, junctional communication between epithelium and lens fibers has been directly demonstrated physiologically in the chicken and frog,19,63 on a time scale and using fluorescent molecules which are incompatible with endocytic and exocytic events.
Connexins in the lens As reviewed by Gilula in this issue, gap junctions in many different organs and organisms are comprised of a family of proteins, with conserved and variable domains in their amino acid sequences, called connexins. Beyer et al64 cloned eDNA coding for connexin 43, a connexin prototypically found in gap junctions joining myocardial cells. Antisera generated against synthetic oligopeptides predicted by connexin 43 eDNA stained gap junctions joining lens epithelial cells at both the light65 and electron microscope levels. 46 These antisera did not recognize the fiber/fiber junctions. In 1985, Kistler et al 66 developed a monoclonal antibody which produced a junction-like staining pattern on ovine lens fibers by immunofluorescence, and which reacted with a 70 kDa polypeptide on SDS gels, prompting the naming of this protein MP70 as suggested by Bloemendal et al. 68 The protein was shown to be cleaved to lower molecular weight forms as a natural consequence of lens fiber aging. Immunocytochemistry revealed that the anti-MP70 monoclonal reacted with lens fiber junctions.s? providing a candidate for a structural protein. Kistler et a[7o obtained N-terminal amino acid sequence data from ovine MP70 and its degradation products, and demonstrated that this protein, which had been localized to lens fiber junctions by immunocytochemistry, was homologous to connexins 32 and 43 and was a new member of the connexin family. Two different but related N-termini in MP70 were found, suggesting the possibility of two different proteins. Together with connexin 43 in the lens epithelium, it appeared as though molecular components contained in at least two of the three junctional interactions in the lens were known.
53
The crystalline lens
Starting with low-stringency hybridization oflens eDNA libraries, Beyer et al,71 and Paul et al 72 isolated a eDNA which codes for a connexin with a predicted size of 46 kDa, named connexin 46. This connexin is not unique to the lens; Northern analysis of whole organ RNA has revealed the presence of connexin 46 in both myocardium and, to a lesser degree, in kidney, although RNase protection assays of mRNA fractions will undoubtedly reveal this connexin being transcribed in other locations as well. Comparison of the N-terminal amino acid sequences reveals an exact amino acid match between one of the possible combinations from the MP70 Edman data and connexin 46. Immunoblot experiments, however, reveal that MP70 and connexin 46 are different proteins. For example, parallel immunoblots of rat lens membranes using Kistler et al's66 monoclonal antibody shows that MP70 in the rat has an M, of 64 kDa, while polyclonal antisera raised against synthetic oligopeptides predicted for different cytoplasmic domains of connexin 46 react with a 54 kDa protein. While the rat lens connexin 46 migrates on SDS gels at about 54 kDa, connexin 46 synthesized in reticulocyte lysate migrates at M, at 46 kDa, indicating that the lens fiber is capable of considerable post-translational modification of the primary translation product. The nature of this modification is not known. The anti-connexin 46 antisera stain lens fiber junctions and do not stain lens fiber non-junctional plasma membranes. This pattern of staining is similar to that seen with the anti-MP70 monoclonal. At the electron microscope level, anti-MP70 and anti-connexin 46 colocalize to the same junctional maculae, but due to the limited resolution using goldlabeled secondary antibodies, it is not possible to tell if MP70 and connexin 46 form heterohexameric connexons. Connexin 46 cRNA has been injected into Xenopus oocytes to study whether this connexin can direct intercellular communication in the oocyte-pair system, and if so, if this communication is pH insensitive, mirroring dye transfer experiments between lens fibers in situ. 18 ,19 The oocytes are able to synthesize immunoprecipitable connexin 46, but the protein is not processed from 46 kDa to the 54 kDa size seen in the mature lens. Following synthesis of connexin 46 in the 46 kDa form, the oocytes loose their membrane potentials and lyse. The oocytes may be protected from lysis by incubating them in 5 % Ficoll; under these conditions the cells
can be shown to be permeable to Lucifer Yellow CH, a 457 Dalton fluorescent molecule known to be permeable through gap junction channel~.73 Voltage clamping of oocytes at different potentials reveals that voltage-gated channels can be opened in the oocyte membrane of cells injected with connexin 46 cRNA when the cells are clamped positive to -10mV. Taken together, these data suggest that the connexin 46 is either directly forming or indirectly inducing open channels in the oocyte plasma membrane. These channels depolarize the cells, and the resulting influx of water either directly, or by stimulating the opening of endogenous stretchactivated channels, causes the cells to lyse. Since mature lens fiber membranes have very low conductances ,(vide supra), these open channels are presumably not formed in the lens fiber nonjunctional plasma membrane, where the protein is posttranslationally processed. The relationships between MP70 and connexin 46 are not understood. Different connexins are known to colocalize to the same junctional maculae in the liver,74,75 but the functional consequences of this intermingling are not yet clear..The molecular components of the epithelial/fiber communicating pathways are also not known. While antisera specific for MP70 and connexin 46 appear to concentrate to the epithelial/fiber interface at the light microscope level, the data are not yet adequate to define their localization in heterotypic gap junctions. Nor is it possible in the light microscope to observe staining of both surfaces of the junctions; electron microscopy is essential to determine both if the epithelial/fiber junctions contain MP70 and connexin 46, and if these proteins are found on both sides of these heterotypic intercellular interactions.
MP26, a lens fiber membrane protein In addition to connexins, other lens fiber integral membrane proteins, some of which are gene products unique to the lens, have been suggested to have a structural and functional role in intercellular communication. The lens fibers are unusually rich in a 26 kDa integral membrane 'protein, called the major intrinsic polypeptide (MIP) or MP26. Due to the non-crystallizing nature of the connexons, lens fiber gap junctions are not resistant to solubilization by detergents, and hence cannot be isolated using protocols developed for other tissues.7 6 ,77 In the late 1970s, appropriate control of
54 proteolysis revealed that isolated liver gap junctions were enriched in a 27 kDa protein. 78-80 The similarity in molecular weight between MP26 and the liver 27 kDa protein, together with the large numbers of lens fiber junctions which appeared to correspond to the preponderance of MP26 in lens fiber membrane fractions, prompted the hypothesis that MP26 was a principal structural component of lens fiber junctions. 38 ,81-84
MP26 is not a member of the connexin family D espite the similarity in electrophoretic mobility between liver and lens fiber data, there were reasons to suspect that MP26 and the liver 27 kDa proteins might be different. Hertzberg et a(i35 demonstrated that MP26 and liver 27 kDa are non-identical when compared by partial peptide mapping. They also prepared an antiserum to MP26, and were unable to demonstrate any cross-reactivity to the liver protein. This comparison was extended to amino acid sequence 'analysis, and there seemed to be no homology between liver 27 kDa and lens MP26 within the first 50 amino acid residues. 86 ,87 Nicholson et a(i38 showed using sequencing analysis that gap junctions formed between hepatocytes and myocardial cells were composed of different, but related proteins, giving the first biochemical indication that gap junctions were composed of a family of related proteins, and that MP26, if it participated in intercellular communication, was not a member of this family. The lack of homology between MP26, liver 27 kDa arid heart gap junction protein held true for the complete amino acid sequences predicted from cDNA clones. Gorin et a(i39 isolated a cDNA coding for MP26 . Paul 90 and Kumar and Gilula'l isolated cDNAs coding connexin 32, the liver 27 kDa protein, and Beyer et a(S4 isolated cDNA coding for connexin 43, a heart gap junction protein. Comparison of the amino acid sequence of MP26 with those of connexins 32 and 43 reveals that MP26 is a completely unrelated protein, indicating that if MP26 is a junctional protein, it represents a parallel evolutionary solution to the structure of a communicating channel. Ehring et aP2 have proposed an innovative model wherein channels composed of MP26 facilitate intercellular communication via a highly resistive intercellular space and function to minimize extracellular fluids between lens fibers (vide infra).
D .A. Goodenough
Immunolocalization of MP26 in lens fiber membranes Several laboratories have attempted to localize MP26 in the lens fiber junctions using immunocytochemistry. In some studies, antisera directed against lens MP26 stain lens fiber gap junctions viewed in the electron microscope, in addition to staining nonjunctional lens fiber plasma membranes. 93 -95 However, Paul and Goodenough'f found that a wide variety of anti-MP26 antisera would not stain lens fiber junctions, but rather stained only the lens fiber plasma membranes and a 'thin' pentalaminar structure of unknown function . The 'thin: pentalaminar structures were stained only on one side, demonstrating an asymmetric protein distribution. These findings have been confirmed by Zampighi et al,97 who have correlated the asymmetric protein distribution with a freeze-fracture analysis of the 'thin' junctions which show intramembrane particles in only one of the two membranes. The differences in immunocytochemical data obtained by different laboratories has not yielded to exchanges of antisera, membranes or both, implicating subtle differences in specimen preparation as being responsible for these rather substantive differences in antibody localization. It is thus probable that immunocytochemistry will not of itself answer questions either about the function ofMP26 or its role in intercellular communication.
The functions of MP26 The functions of MP26 are not currently understood. Some have argued that it serves a structural role 98,99 for the lens fiber membrane. Others have reconstituted MP26 into planar lipid bilayers lOO and lipid vesicles'P! and have shown the protein to have channel activity. Channel activity in single membranes is not proof of function in intercellular communication. MP26 cRNA was injected into a paired Xenopus oocyte system developed by Werner et al102 under conditions where cRNAs coding for liver (connexin 32) myocardial (connexin 43), and embryonic gap junctions were able to direct the assembly of functional junctions. 103 ,104 The MP26 translated in these cells was neither able to migrate to the site of cell-cell apposition nor able to induce junctional conductances above background. While the function of MP26 in lens fiber biology and a direct role for MP26 in intercellular channel
The crystalline lens
formation remains to be clearly demonstrated, Ehring et al92 have measured voltage-sensitive single channel conductances in planar lipid bilayers reconstituted with HPLC-purified MP26. The channels are large (380 and 160 pS in 0.1 M KCI), show symmetrical voltage dependence, and are insensitive to Ca2 + and H + ion concentrations. The vesicles used for the insertion of the channels into the planar bilayers were studied by freezefracture analysis across a broad range oflipid-protein concentration ratios. At the higher protein concentrations, these studies demonstrated crystalline arrays within the vesicle membranes indistinguishable from those arrays correlated with the 'thin' junctional profiles mentioned above. On the basis of these studies, Ehring et al 92 have proposed an innovative function for the MP26 channels wherein the channels in the interior of the lens would be open, permitting intercellular communication via a narrow extracellular space, and also functioning to keep water from accumulating in the intercellular spaces since intercellular water would tend to enter the cytoplasm of the osmotically active lens fibers. Given that the lens fiber intercellular spaces must be kept narrow to avoid light scattering, this may prove to be an essential function for MP26 channel activity; Other homologous proteins to MP26 have been identified in diverse systems which also have channel activit yl05.106 which may provide additional clues ofMP26 function . Recently, yet another protein has been localized to the lens fiber junctions by immunocytochemistry. A 20 kDa protein, MP20, has been carefully studied and shown to localize at lens fiber junctions and to be unrelated to either MP26 or the connexin family by both antigenic and N-terminal sequence criteria.l07.108 MP20 has been shown to be a phosphoprotein, phosphorylated by both cAMPdependent protein kinase and protein kinase C in oitro, and to bind calmodulin, both properties shared with MP26. 107 The picture emerges, then, that the crystalline lens is an organ whose physiology and homeostasis depends centrally on gap junction mediated intercellular communication. The molecular nature of the gap junctions joining lens cells is not yet fully understood. Additional connexins may be found in the lens' repertoire, in addition to connexins 43, 46 and MP70. MP26 and MP20 have been localized to lens fiber junctions in several laboratories, and while these are not members of the connexin family of proteins, their potential roles in cell-cell communication remain an intriguing possibility.
55 Given the availability of lens-specific promoters, which have been shown to drive expression of exogenous proteins both in lens cultures and in transgenic animals,109,11O there are possible experimental approaches to study some of these unknowns.
Acknowledgements The author gratefully acknowledges the collaboration and friendship of Dr David L. Paul, and research support from grant EY02430 from the National Institutes of Health.
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