Developmental regulation of the direct interaction between the intracellular loop of connexin 45.6 and the C terminus of major intrinsic protein (aquaporin-0)

The eye lens is dependent upon a network of gap junction-mediated intercellular communication to facilitate its homeostasis and development. Three gap junction-forming proteins are expressed in the lens of which two are in lens fibers, namely connexin (Cx) 45.6 and 56. Major intrinsic protein (MIP), also known as aquaporin-0 (AQP0), is the most abundant membrane protein in lens fibers. However, its role in the lens is not clear. Our previous studies show that MIP(AQP0) associates with gap junction plaques formed by Cx45.6 and Cx56 during the early stages of embryonic chick lens development but not in late embryonic and adult lenses. We report here that MIP(AQP0) directly interacts with Cx45.6 but not with Cx56. We further identified the intracellular loop of Cx45.6 as the interacting domain for the MIP(AQP0) C terminus. Surface plasmon resonance experiments indicated that the C-terminal domain of MIP(AQP0) interacts with two binding sites within the intracellular loop region of Cx45.6 with a K(D(app)) of 7.5 and 10.3 microm, respectively. The K(D(app)) for the full-length loop region is 7.7 microm. The cleavage at the intracellular loop of Cx45.6 was observed during lens development, and the C terminus of MIP(AQP0) did not interact with the loop-cleaved form of Cx45.6. Thus, the dissociation between these two proteins that occurs in the mature fibers of late lens development is likely caused by this cleavage. Finally this interaction had no impact on Cx45.6-mediated intercellular communication, suggesting that the Cx45.6-MIP(AQP0) interaction plays a novel unidentified role in lens fibers.     

Molecular cloning, functional analysis, and RNA expression analysis of connexin45.6: a zebrafish cardiovascular connexin

In the vertebrate cardiovascular system, gap junctions function in intercellular communication essential for both the coordinated propagation of the heartbeat and the control of vasomotor responses in the vascular system. Connexins, the protein subunits of gap junctions, are coded by a multigene family. In this study, a connexin gene (zfCx45.6), which exhibits 53% amino acid identity to chick Cx42, was cloned from zebrafish genomic DNA. With the use of the LN54 radiation hybrid panel, zfCx45.6 was mapped to zebrafish linkage group 9. Northern blots and RT-PCR revealed the presence of zfCx45.6 mRNA in the embryo before 2 h postfertilization (hpf) and then again beginning at about 12 hpf, after which time no major changes in relative expression levels were detected. In the adult, zfCx45.6 mRNA continued to be detected in the heart, as well as the brain, liver, and ovary, but not the lens. Whole mount in situ hybridization revealed zfCx45.6 mRNA was expressed at high levels in the major vessels of the entire embryo and in both the atrium and ventricle of the adult heart. Expression of zfCx45.6 channels in paired Xenopus oocytes produced high levels of intercellular coupling that was voltage sensitive. With the previous isolation of zebrafish Cx43 and Cx43.4, zebrafish orthologues have now been isolated for three of the four connexins expressed in the mammalian cardiovascular system.     

Regulation of lens connexin 45.6 by apoptotic protease,caspase-3

Gap junctions are important in maintaining lens homeostasis. Here we report that connexin 45.6 (Cx45.6) was partially truncated to a 46 kDa fragment during chicken lens development. This specific truncation initiated during embryonic days and the truncated fragment accumulated towards the later developmental stages. When membranes of the embryonic lens were subjected to caspase-3 treatment, the 46 kDa fragment of Cx45.6 was reproduced, suggesting apoptotic protease caspase-3 is a potential protease involved. The COOH-terminus of Cx45.6 in GST-fusion protein was also cleaved by caspase-3, confirming that Cx45.6 is a direct substrate of caspase-3. Induction of apoptosis in lens primary cultures regenerated the 46 kDa fragment and this cleavage was blocked by a caspase-3 inhibitor. Alteration of amino acid residue Asp364 or Glu367 to Ala prevented Cx45.6 from cleavage by caspase-3, suggesting that the cleavage site of Cx45.6 is likely to be between Glu367 and Gly361. Phosphorylation of Ser363, a known substrate for casein kinase II (CKII) in vivo, inhibited the cleavage of Cx45.6 by caspase-3. Thus, this study demonstrates that a lens connexin can be a direct target of caspase-3 and the cleavage by caspase-3 leads to the development-associated truncation of Cx45.6. Finally, caspase-3 mediated truncation can be modulated by the specific connexin phosphorylation.     

The development-associated cleavage of lens connexin 45.6 by caspase-3-like protease is regulated by casein kinase II-mediated phosphorylation.

Gap junctions are important in maintaining lens transparency and metabolic homeostasis. In this paper, we report that the gap junction-forming protein, connexin (Cx) 45.6, was specifically truncated during lens development and that the majority of the truncated fragments were located in the differentiated lens fibers. When isolated lens membranes were treated by caspase-3, the truncated fragments of Cx45.6 were reproduced, and this truncation occurred at the COOH terminus of Cx45.6. Moreover, when primary lens cells were treated with apoptosis-inducing reagents, Cx45.6 was cleaved similarly as the in vitro treatment by caspase-3, and this cleavage was blocked by a caspase-3 inhibitor. These results suggest that caspase-3 is responsible for the development-associated cleavage of Cx45.6. The cleavage site of Cx45.6 was identified between amino acid residues Glu(367) and Gly(368). We have shown previously that Ser(363) is an in vivo phosphorylated site by casein kinase II, and this specific phosphorylation leads to a rapid turnover of Cx45.6. Interestingly, we found here that when Ser(363) was phosphorylated by casein kinase II, the cleavage of Cx45.6 catalyzed by caspase-3 was inhibited. This study, for the first time, demonstrates that a connexin can be a direct target of an apoptotic protease and that cleavage by caspase-3-like protease leads to the development-associated truncation of a lens connexin. Finally, caspase-3-mediated cleavage can be regulated by casein kinase II-mediated phosphorylation, suggesting that Cx45.6 turnover and specific cleavage by caspase-3-like protease is alternatively modulated.     

Regulation of Lens Connexin 45.6 by Apoptotic Protease,Caspase-3

Gap junctions are important in maintaining lens homeostasis. Here we report that connexin 45.6 (Cx45.6) was partially truncated to a 46 kDa fragment during chicken lens development. This specific truncation initiated during embryonic days and the truncated fragment accumulated towards the later developmental stages. When membranes of the embryonic lens were subjected to caspase-3 treatment, the 46 kDa fragment of Cx45.6 was reproduced, suggesting apoptotic protease caspase-3 is a potential protease involved. The COOH-terminus of Cx45.6 in GST-fusion protein was also cleaved by caspase-3, confirming that Cx45.6 is a direct substrate of caspase-3. Induction of apoptosis in lens primary cultures regenerated the 46 kDa fragment and this cleavage was blocked by a caspase-3 inhibitor. Alteration of amino acid residue Asp³⁶⁴or Glu³⁶⁷ to Ala prevented Cx45.6 from cleavage by caspase-3, suggesting that the cleavage site of Cx45.6 is likely to be between Glu³⁶⁷ and Gly³⁶⁸. Phosphorylation of Ser³⁶³, a known substrate for casein kinase II (CKII) in vivo, inhibited the cleavage of Cx45.6 by caspase-3. Thus, this study demonstrates that a lens connexin can be a direct target of caspase-3 and the cleavage by caspase-3 leads to the development-associated truncation of Cx45.6. Finally, caspase-3 mediated truncation can be modulated by the specific connexin phosphorylation.     

Expression of the gap junction protein connexin43 in embryonic chick lens: Molecular cloning,ultrastructural localization,and post-translational phosphorylation

Summary Lens epithelial cells are physiologically coupled to each other and to the lens fibers by an extensive network of intercellular gap junctions. In the rat, the epithelial-epithelial junctions appear to contain connexin43, a member of the connexin family of gap junction proteins. Limitations on the use of rodent lenses for the study of gap junction formation and regulation led us to examine the expression of connexin43 in embryonic chick lenses. We report here that chick connexin43 is remarkably similar to its rat counterpart in primary amino acid sequence and in several key structural features as deduced by molecular cDNA cloning. The cross-reactivity of an anti-rat connexin43 serum with chick connexin43 permitted definitive immunocytochemical localization of chick connexin43 to lens epithelial gap junctional plaques and examination of the biosynthesis of connexin43 by metabolic radiolabeling and immunoprecipitation. We show that chick lens cells synthesize connexin43 as a single, 42-kD species that is efficiently posttranslationally converted to a 45-kD form. Metabolic labeling of connexin43 with³²P-orthophosphate combined with dephosphorylation experiments reveals that this shift in apparent molecular weight is due solely to phosphorylation. These results indicate that embryonic chick lens is an appropriate system for the study of connexin43 biosynthesis and demonstrate for the first time that connexin43 is a phosphoprotein.     

Retrovirus-mediated gene expression during chick visual system development

The chick embryo is an excellent model for studying eye morphogenesis, retinal cell fate determination, and retinotectal projections due to its accessibility and the available molecular tools. Avian replication-competent retroviruses allow efficient infection of proliferating cells and stable integration of the viral genome, including up to 2.3kb of foreign cDNA, into the host chromosome. High-titer retroviruses are produced by transient transfection of avian DF-1 cells followed by centrifugation of the culture medium. Targeted infection of the optic vesicle, the lens vesicle, the retina and pigmented epithelium, the periocular mesenchyme, and the tectum can be performed at different developmental stages in ovo. In addition, retroviruses can be used to transduce genes of interest into various ocular tissue explants or cells in vitro. Virus-mediated gene expression can be detected within 12h of infection. Therefore, avian replication-competent retroviruses serve as powerful tools to misexpress wild-type and mutant gene products and to study molecular mechanisms underlying vertebrate visual system development.     

Casein kinase II phosphorylates lens connexin 45.6 and is involved in its degradation

Connexin (Cx) 45.6, an avian counterpart of rodent Cx50, is phosphorylated in vivo, but the sites and function of the phosphorylation have not been elucidated. Our peptide mapping experiments showed that the Ser(363) site in the carboxyl (COOH) terminus of Cx45.6 was phosphorylated and that this site is within casein kinase (CK) II consensus sequence, although showing some similarity to CKI sequence. The peptide containing Ser(363) could be phosphorylated in vitro by CKII, but not by CKI. Furthermore, CKII phosphorylated Cx45.6 in embryonic lens membrane and the fusion protein containing the COOH terminus of Cx45.6. Two-dimensional peptide mapping experiments showed that one of the Cx45.6 peptides phosphorylated in vivo migrated to the same spot as one of those phosphorylated by CKII in vitro. Furthermore, CKII activity could be detected in lens lysates. To assess the function of this phosphorylation event, exogenous wild type and mutant Cx45.6 (Ser(363) --> Ala) were expressed in lens primary cultures by retroviral infection. The mutant Cx45.6 was shown to be more stable having a longer half-life compared with wild type Cx45.6. Together, the evidence suggests that CKII is likely a kinase responsible for the Ser(363) phosphorylation, leading to the destablization and degradation of Cx45.6. The connexin degradation induced by phosphorylation has a broad functional significance in the regulation of gap junctions in vivo.     

Lens fiber connexin turnover and caspase-3-mediated cleavage are regulated alternately by phosphorylation

Lens connexins are phosphorylated in vivo; however, the function and regulation of the phosphorylation remain largely unknown. We have previously identified an in vivo phosphorylation site, Ser(364), at the COOH terminus of lens connexin (Cx) Cx45.6 and phosphorylation appears to regulate connexin protein turnover. To assess the specific mechanism of Ser(364) phosphorylation in Cx45.6, exogenous wild type and Ser(364) mutant Cx45.6 were expressed in primary lens cultures through retroviral infection. Cx45.6 turnover was attenuated primarily by proteasomal inhibitors and to a lesser extent by lysosomal inhibitors. Furthermore, the level of Cx45.6 protein in ubiquitin co-expressed cells was significantly reduced as compared to the cells expressing Cx45.6 alone. Moreover, overexpression of ubiquitin led to a more significant decrease in wild type Cx45.6 than Cx45.6(S364A), a mutant deficient of phosphorylation site at Ser(364), although we did not detect any difference in the levels of ubiquitination between wild type and mutant Cx45.6. Interestingly, the mutant mimicking constitutive phosphorylation, Cx45.6(S364D), partially prevented the cleavage of Cx45.6 by caspase-3. Together, our data suggest that phosphorylation of Cx45.6 at Ser(364) appears to stimulate Cx45.6 turnover primarily through proteasome pathway and this phosphorylation inhibits the cleavage of Cx45.6 by caspase-3. These findings provide further insights into regulatory mechanism of the specific phosphorylation of connexins in the lens.     

Disruption of Gja8 (alpha8 connexin) in mice leads to microphthalmia associated with retardation of lens growth and lens fiber maturation.

The development of the vertebrate lens utilizes a sophisticated cell-cell communication network via gap junction channels, which are made up of at least three connexin isoforms, alpha8 (Cx50), alpha3 (Cx46) and alpha1 (Cx43), and which are encoded by three different genes. In a previous study, we reported that, with a disruption of Gja3 (alpha3 connexin), mice developed nuclear cataracts with a normal sized lens. We show that Gja8tm1 (alpha8-/-) mice develop microphthalmia with small lenses and nuclear cataracts, while the alpha8 heterozygous (+/-) mice have relatively normal eyes and lenses. A comparative study of these alpha3 and alpha8 knockout mice showed that the protein levels of both alpha3 and alpha8 were independently regulated and there was no compensation for either the alpha3 or alpha8 protein from the wild-type allele when the other allele was disrupted. More interestingly, western blotting data indicated that the presence of alpha8 in the lens nucleus is dependent on alpha3 connexin, but not vice versa. The staining of the knock-in lacZ reporter gene showed the promoter activity of alpha8 connexin is much higher than that of alpha3 connexin in embryonic lenses and in adult lens epithelium. More importantly, a delayed denucleation process was observed in the interior fibers of the alpha8-/- lenses. Therefore, alpha8 connexin is required for proper fiber cell maturation and control of lens size.     

Developmental changes in proteins of purified membranes of chicken lenses and evidence for contamination by cytoplasmic delta-crystallin.

Urea-washed membranes from embryonic chick lenses (15 days old) and from the cortical and nuclear regions of adult chicken lenses (1 year) have been prepared by repeated centrifugation through discontinuous density gradients. The protein components of the isolated membranes have been examined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and urea. Proteins with molecular weights of 75 000, 56 000, 54 000, 48 000, 34 000, 32 000, 25 000, and 22 000 were present in all the membrane preparations, although their proportions changed during development. One additional protein, molecular weight 70 000, was seen only in the embryonic lens membranes. The greatest developmental change was the increase in 25 000 molecular weight protein from 12% in the embryonic lens to about 45% in the adult lens. Since it has been suggested that this protein is associated with gap junctions, its increase during development may reflect a corresponding increase in the number of gap junctions in the lens. The 50 000 molecular weight protein of embryonic lens membranes and membranes of adult nuclear lens fibers consisted at least partly of delta-crystallin, since delta-crystallin peptides could be identified in tryptic peptide maps of the isolated protein after in vitro radioiodination. Peptide maps of the 50 000 molecular weight protein of cortical lens fiber membranes contained no identifiable delta-crystallin peptides, although it is possible that modified delta-crystallin peptides may be present. The level of cytoplasmic contamination of the membrane fraction was estimated by preparing lens membranes in the presence of added delta-[35S]crystallin. The results indicated that cytoplasmic contamination contributes significantly to the presence of delta-crystallin in lens membrane preparations.     

The mouse gap junction gene connexin29 is highly expressed in sciatic nerve and regulated during brain development

A novel mouse gap junction gene, coding for a presumptive protein of 258 amino acids (molecular mass: 28 981 Da), has been designated connexin29. This single copy gene was mapped to distal mouse chromosome 5 and shows 75% sequence identity to a human connexin30.2 sequence in the database. Connexin29 mRNA (4.4 kb) is highly expressed in mouse sciatic nerve and less abundant in spinal cord as well as in adult brain, where it increased 12-fold between day 7 and 14 post partum. Our expression data suggest that the new connexin gene is active in myelin-forming glial cells.     

Identification, localization, and primary structure of CAP-23, a particle-bound cytosolic protein of early development

We report the identification of CAP-23, a novel particle-bound cytosolic protein associated with developing cells in both mammalian and avian tissues. CAP-23 was a substrate for purified protein kinase C (PKC) in vitro, and the protein was phosphorylated in a PMA-sensitive manner in cultured cells, indicating that it is a PKC substrate in situ. cDNA coding for chick CAP-23 was isolated. The deduced sequence revealed an unusual amino acid composition that strikingly resembled that of rat GAP-43, a growth-associated neuron-specific PKC substrate. Further predicted features of CAP-23 included a PKC phosphorylation site at Ser-6, and the presence of basic NH2- and COOH-terminal domains. CAP-23 was encoded by an mRNA of approximately 1.5 kb, whose distribution during chick development resembled that of the corresponding protein. Southern blot analysis revealed the presence of a single main hybridizing species in the chick genome. The distribution of CAP-23 during development was analyzed with Western blots and by immunofluorescence on tissue sections. In cultured cells the protein appeared to be distributed in a regular spotted pattern below the entire cell surface. In early chick embryos (E2), CAP-23 was present in most if not all cells. The protein then became progressively restricted to only some developing tissues and to only certain cells in these tissues. In most tissues CAP-23 levels fell below detection limits between E15 and E19. Highest levels of the protein were found in the nervous system, where CAP-23 levels peaked around E18, and where elevated levels were still detectable at birth.     

Organization of delta-crystallin genes in the chicken.

Double-stranded DNA was synthesized from delta-crystallin mRNA prepared from lens fibers of 15-day-old chick embryos and cloned at the Pst I site of the plasmid pBR322. Using the cloned cDNA and single-stranded cDNA as hybridization probes, a number of genomic DNA fragments containing delta-crystallin gene sequences have been cloned from the partial and complete EcoRI digests of chick brain DNA. One of the clones from the partial digests contains a DNA fragment that consists of four EcoRI fragments of 7.6 kb, 4.0 kb, 2.6 kb, and 0.8 kb. The gene sequences reside in the (5')7.6 kb - 0.8 kb - 4.0 kb (3') fragments. Electron microscopy has provided evidence that the cloned DNA fragment includes the entire gene sequences complementary to delta-crystallin mRNA except for the 3' terminal poly(A) tail, and that the delta-crystallin gene is interrupted by at least 13 intervening sequences. Another clone contains a genomic fragment that consists of two EcoRI fragments of 3.0 kb and 11 kb. The DNA fragment in the latter clone represents a different delta-crystallin gene, as judged by restriction endonuclease mapping and by electron microscopy.     

Developmental changes in proteins of purified membranes of chicken lenses and evidence for contamination by cytoplasmic δ-crystallin

Urea-washed membranes from embryonic chick lenses (15 days old) and from the cortical and nuclear regions of adult chicken lenses (1 year) have been prepared by repeated centrifugation through discontinuous density gradients. The protein components of the isolated membranes have been examined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and urea. Proteins with molecular weights of 75 000, 56 000, 54 000, 48 000, 34 000, 32 000, 25 000, and 22 000 were present in all the membrane preparations, although their proportions changed during development. One additional protein, molecular weight 70 000, was seen only in the embryonic lens membranes. The greatest developmental change was the increase in 25 000 molecular weight protein from 12% in the embryonic lens to about 45% in the adult lens. Since it has been suggested that this protein is associated with gap junctions, its increase during development may reflect a corresponding increase in the number of gap junctions in the lens.The 50 000 molecular weight protein of embryonic lens membranes and membranes of adult nuclear lens fibers consisted at least partly of δ-crystallin, since δ-crystallin peptides could be identified in tryptic pepetide maps of the isolated protein after in vitro radioiodination. Peptide maps of the 50 000 molecular weight protein of cortical lens fiber membranes contained no identifiable δ-crystallin peptides, although it is possible that modified δ-crystallin peptides may be present. The level of cytoplasmic contamination of the membrane fraction was estimated by preparing lens membranes in the presence of added δ-[³⁵S]crystallin. The results indicated that cytoplasmic contamination contributes significantly to the presence of δ-crystallin in lens membrane preparations.     

Molecular cloning and developmental expression of two chick embryo gap junction proteins

The connexins are a family of related gap junction proteins which contain conserved transmembrane and extracellular domains but unique cytoplasmic regions. To identify connexins with potential roles in development, a chick embryo cDNA library was screened by hybridization at low stringency with a cDNA for rat connexin-43. cDNA clones for two previously undescribed connexins were isolated. Chick connexin-45 has a predicted molecular mass of 45,376 daltons; connexin-42 has a predicted molecular mass of 41,748 daltons. Both of these predicted connexin proteins share the homologous regions noted in other members of this family, and each has its own unique regions. Southern blots of chicken genomic DNA suggest that each connexin is encoded by a distinct single copy gene. RNA blots demonstrate that while chick connexin-43, -42, and -45 are each expressed in a number of chick organs, they each have a unique tissue distribution. Each connexin mRNA is present in heart. Blots of total RNA isolated from hearts of chick embryos of different ages demonstrate that the abundance of connexin-42 and -43 mRNAs varies no more than 2-fold between the embryo and the adult. However, connexin-45 mRNA shows a dramatic change, falling 10-fold from the 6-day embryonic heart to the adult. These multiple connexins are likely to have different physiological properties and may account for the multiple physiologically distinct gap junction channels which have been observed in cardiac myocytes. They may provide a mechanism for the formation of communication compartments in the developing myocardium.     

Functional Effects of Casein Kinase I-Catalyzed Phosphorylation on Lens Cell-to-Cell Coupling

The functional consequence of the casein kinase I-catalyzed phosphorylation of the lens gap junctional protein connexin49 was investigated using a sheep primary lens cell culture system. To determine whether the phosphorylation of connexin49 catalyzed by endogenous casein kinase I results in an altered junctional communication between lens cells, the effect of the casein kinase I-specific inhibitor CKI-7 on Lucifer Yellow dye transfer between cells in the lens culture was examined. Dye transfer was analyzed in cultures of different ages because we have demonstrated previously that the expression of connexin49 increases as the cultures age while that of connexin43, which is likely not a substrate for casein kinase I, has been shown to decrease [Yang & Louis (1999) Invest. Ophthalmol. Vis. Sci. 41: 2568–2564]. In 9-day old lens cultures, in which gap junctions are composed primarily of connexin43, CKI-7 had little effect on the rate of dye transfer between lens cells. In contrast, treatment of 15-day and 28-day old cultures with CKI-7 resulted in a significant increase in the rate of dye transfer. Thus, the extent of this CKI-7-dependent increase in cell-to-cell communication was positively correlated with the level of expression of connexin49, the major casein kinase I substrate in lens plasma membranes. These results suggest that the casein kinase I-catalyzed phosphorylation of connexin49 decreases cell communication between connexin49-containing gap junctions in the lens. Received: 31 July 2000/Revised: 12 January 2001     

Distinct behavior of connexin56 and connexin46 gap junctional channels can be predicted from the behavior of their hemi-gap-junctional channels.

The gap-junctional protein rat connexin46 (Cx46) has the unusual ability to form voltage-gated channels in the nonjunctional plasma membrane of Xenopus oocytes (Paul et al., 1991; Ebihara and Steiner, 1993). These have been suggested to be gap-junctional hemichannels or connexons. The Xenopus oocyte system was used to characterize the functional properties of a closely related lens gap-junctional protein, chicken connexin56 (Cx56) (Rup et al., 1993) and to contrast them to those of rat Cx46. Single oocytes injected with either Cx56 or Cx46 cRNA developed time-dependent, outward currents that activated on depolarization. The currents induced by Cx56 and Cx46 showed differences in steady-state voltage dependence and in their degree of rectification. Furthermore, the voltage-dependent properties of the nonjunctional channels induced by the connexin cRNAs in external solutions containing low concentrations of calcium ions could account remarkably well for the behavior of the intercellular channels formed by Cx56 and Cx46 in paired oocytes. These results suggest that many of the voltage-dependent properties of the hemi-gap-junctional channels are retained by the intercellular channels.