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It is now clear that connexin-based, gap junction “hemichannels” in an undocked state are capable of opening and connecting cytoplasm to the extracellular milieu. Varied studies also suggest that such channel activity plays a vital role in diverse cell processes and abnormal hemichannel activity contributes to pathogenesis. To pursue fundamental questions in this area, investigators require methods for studying hemichannel permeability and dynamics that are quantitative, sensitive, versatile, and available to most cellular and molecular laboratories. Here we first provide a theoretical background for this work, including the role of cellular membrane potentials. We then describe in detail our computer-assisted methods for both dye uptake and leakage along with illustrative results from different cell systems. A key feature of our protocol is the inclusion of a mechanical stimulation step. We describe dye uptake, interpreted as connexin dependent, that is shown to be enhanced with reduced extracellular Ca2+, mechanically responsive, inhibited by TPA, inhibited by EL186 antibodies for Cx43 and sustained for more than 15 min following mechanical stimulation. We describe dye leakage that displays these same properties, with estimates of hemichannel numbers per cell being derived from leakage rates. We also describe dye uptake that is shown to be unaffected by a reduction in external Ca2+, insensitive to EL186 antibodies and relatively short-lived following mechanical stimulation; this uptake may occur via pannexin 1 channels expressed in the cells studied here. It is unlikely that cell damage plays a significant role in dye uptake following mechanical stimulation, given compelling results from various control experiments.

The article discusses cellular communication. Topics include the multiple modes of information exchange cells use, such as hormone signals, research of the gap junction assembly process in cells, which contain thousands of cell communication channels, by the author and his colleagues, a type of connexin protein called Cx43, and genetic evidence found in the 1990s that connexins can participate in disease, such as the developmental disease oculodentodigital dysplasia (ODDD).

Gap junction (GJ) “formation plaques” are distinct membrane domains with GJ precursors; they assemble by means of a series of defined steps. The C-terminus of Cx43 is required for normal progression of assembly, normal aggregation of 10-nm particles into small GJs, and negative regulation of assembly involving protein kinase C., Using an established gap junction (GJ) assembly system with experimentally reaggregated cells, we analyzed “formation plaques” (FPs), apparent sites of GJ assembly. Employing freeze-fracture electron microscopy methods combined with filipin labeling of sterols and immunolabeling for connexin43 (Cx43), we demonstrated that FPs constitute distinct membrane “domains” and that their characteristic 10-nm particles contain connexin43, thus representing precursors (i.e., GJ hemichannels) engaged in assembly. Analysis of FPs in new systems—HeLa and N2A cells—resolved questions surrounding several key but poorly understood steps in assembly, including matching of FP membranes in apposed cells, reduction in the separation between FP membranes during assembly, and the process of particle aggregation. Findings also indicated that “docking” of GJ hemichannels occurs within FP domains and contributes to reduction of intermembrane separation between FPs. Other experiments demonstrated that FPs develop following a major C-terminal truncation of Cx43 (M257), although assembly was delayed. Particle aggregation also occurred at lower densities, and densities of particles within developing GJ aggregates failed to achieve full-length levels. With regard to regulation, inhibition of assembly following protein kinase C activation failed to occur in the M257 truncation mutants, as measured by intercellular dye transfer. However, several C-terminal serine mutations failed to disrupt inhibition.

Early patterning of vertebrate embryos involves the generation of asymmetric signals across the left–right (L–R) axis that position and are required for the proper function of internal organs. This patterning is directed by a conserved nodal/lefty signaling cascade on the left side of the embryo, thought to be asymmetrically directed by ciliary beating that generates a leftward fluid flow in the mammalian node and in Kupffer's vesicle (KV), the related structure in zebrafish. Following morpholino knockdown of Cx43.4, asymmetric gene expression and global organ distribution are randomized, consistent with the expression of Cx43.4 in KV. Randomization is recapitulated in mosaic embryos in which Cx43.4 is depleted preferentially in KV cells, showing that Cx43.4 is specifically required in KV for proper L–R axis formation. The mechanistic basis for the laterality anomalies in Cx43.4-deficient embryos is a primary morphogenesis defect during lumen formation in KV. Additionally, the role of Cx43.4 appears to be conserved given that its ortholog, human Cx45, is able to functionally compensate for zebrafish Cx43.4 during L–R patterning. This is the first report linking connexin function in the ciliated, node-like cells of KV with normal L–R axis development.

The Monte Carlo method was used to simulate grain growth in thin Cu films. The model, based on energetic principles, was compared with the evolution of measured film structures. Surface, interface, grain boundary, and elastic strain energies were applied to determine the preferred microstructure in terms of different annealing conditions and film thicknesses. Four microstructural cases, relating to different film thicknesses, were developed in this paper. Twinning in the Cu films is simulated by arbitrary re-assignment of randomly selected crystallite lattice orientations. The observed evolution in crystallographic texture for each film thickness can be obtained from the Monte Carlo simulations.

To address the role of gap junction hemichannels in apoptosis, the cell death induced by staurosporine (ST) was evaluated in wild type HeLa cells (HeLa-WT) and transfectants expressing either full-length connexin43 (HeLa-Cx43) or a C-terminal truncation of Cx43 (HeLa-DeltaCT). Cell death was measured with fluorescence-activated cell sorting (FACS), both DNA and nuclear fragmentation methods and assays for PARP and caspase 3. The ST-mediated cell death was accelerated in HeLa-Cx43 cells compared to HeLa-WT and HeLa-DeltaCT. To determine why HeLa-Cx43 cells were more susceptible to ST, the phosphorylation state and the localization of Cx43 protein within cells were examined using specific Cx43 antibodies. The phosphorylated forms of Cx43 were sharply reduced in HeLa-Cx43 cells treated with ST. Moreover, in ST-treated HeLa-Cx43 cells, Cx43 was mainly observed at the cell surface. In contrast, the truncated form of Cx43 found in HeLa-DeltaCT cells, which lacks many of the normal phosphorylation sites, was observed in the cytosol with ST treatment. To examine the hemichannels in the plasma membranes of ST-treated HeLa-Cx43 cells, several dye uptake methods using carboxyfluorescein and propidium iodide were employed. While the number of fluorescent cells did not change in HeLa-WT and HeLa-DeltaCT cells with ST treatment, the number of fluorescent HeLa-Cx43 cells increased more than ten-fold. These results indicate that the increases in cell surface Cx43 seen with immunofluorescence and the elevated hemichannel activities detected with dye uptake could help explain the accelerated cell death observed in ST-treated HeLa-Cx43 cells.

The role of cytoskeletal elements in gap junction (GJ) assembly has been studied using Novikoff hepatoma cells treated with cytochalasin B (CB) to disrupt actin filaments or with colchicine or nocodazole to disrupt microtubules. After 60 min of cell reaggregation, freeze-fracture was used to evaluate quantitatively the "initiation," "maturation," and "growth" phases of GJ assembly. The development of junctional permeability to fluorescent dyes was also analyzed. The only effects of CB on the structure or permeability of the developing junctions involved an elongation of GJ aggregates and a small decrease in formation plaque areas. Colchicine (but not the inactive form, lumicolchicine) prevented the enhancement of GJ growth by cholesterol, but its effect on basal growth was equivocal. Nocodazole inhibited the growth of GJ, even under basal conditions, without an effect on initiation. Nocodazole also blocked the forskolin-enhanced increase in the growth of GJs and, in living MDCK cells, reduced the movement of transport intermediates containing green fluorescent protein-tagged connexin43. Thus, neither actin filaments nor microtubules appear to restrict GJ assembly by anchoring intramembrane GJ proteins, nor are they absolutely required for functional GJs to form. However, microtubules are necessary for enhanced GJ growth and likely for facilitating connexin trafficking under basal conditions.

The assembly of gap junctions (GJs) is a process coordinated by growth factors, kinases, and other signaling molecules. GJ assembly can be enhanced via the elevation of cAMP and subsequent stimulation of connexon trafficking to the plasma membrane. To study the positive regulation of GJ assembly, fibroblasts derived from connexin (Cx)43 knockout (KO) and wild-type (WT) mice were transfected with WT Cx43 (WTCx43) or mutant Cx43. GJ assembly between untransfected WT fibroblasts or stably transfected WTCx43/KO fibroblasts was increased two- to fivefold by 8Br-cAMP, and this increase could be blocked by inhibition of cAMP-dependent protein kinase (PKA) or truncation of the Cx43 COOH terminus (CT). Although serine 364 (S364) of the Cx43 CT was determined to be a major site of phosphorylation, the molar ratio of Cx43 phosphorylation was not increased by 8Br-cAMP. Importantly, GJ assembly between either S364ECx43/KO or S364ECx43/WT fibroblasts was stimulated by 8Br-cAMP, but that between S364ACx43/KO or S364PCx43/KO fibroblasts was not stimulated, indicating that phosphorylation or a negative charge at S364 is required for enhancement of GJ assembly by cAMP. Furthermore, GJ assembly between S364ACx43/WT fibroblasts could be stimulated by 8Br-cAMP, but could not be between S364PCx43/WT fibroblasts. Thus, S364PCx43 interferes with enhanced GJ assembly when coexpressed with WTCx43.

Phorbol esters (e.g., TPA) activate protein kinase C (PKC), increase connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. We asked whether PKC directly phosphorylates and regulates Cx43. Rat epithelial T51B cells metabolically labeled with (32)P(i) yielded two-dimensional phosphotryptic maps of Cx43 with several phosphopeptides that increased in intensity upon TPA treatment. One of these peptides comigrated with the major phosphopeptide observed after PKC phosphorylation of immunoaffinity-purified Cx43. Purification of this comigrating peptide and subsequent sequencing indicated that the phosphorylated serine was residue 368. To pursue the functional importance of phosphorylation at this site, fibroblasts from Cx43(-/-) mice were transfected with either wild-type (Cx43wt) or mutant Cx43 (Cx43-S368A). Intercellular dye transfer studies revealed different responses to TPA and were followed by single channel analyses. TPA stimulation of T51B cells or Cx43wt-transfected fibroblasts caused a large increase in the relative frequency of approximately 50-pS channel events and a concomitant loss of approximately 100-pS channel events. This change to approximately 50-pS events was absent when cells transfected with Cx43-S368A were treated with TPA. These data strongly suggest that PKC directly phosphorylates Cx43 on S368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication.

Gap junctional communication (GJC) is regulated in the early Xenopus embryo and quantitative differences in junctional communication correlate with the specification of the dorsal-ventral axis. To address the mechanism that is responsible for regulating this differential communication, we investigated the function of beta-catenin during the formation of the dorsal-ventral axis in Xenopus embryos by blocking its synthesis with antisense oligodeoxynucleotides. This method has previously been shown to reduce the level of beta-catenin in the early embryo, prior to zygotic transcription, and to inhibit the formation of the dorsal axis (Heasman et al., 1994, Cell 79, 791-803). We show here that antisense inhibition of beta-catenin synthesis also reduces GJC among cells in the dorsal hemisphere of 32-cell embryos to levels similar to those observed among ventral cells. Full-length beta-catenin mRNA can restore elevated levels of dorsal GJC when injected into beta-catenin-deficient oocytes, demonstrating the specificity of the beta-catenin depletion with the antisense oligonucleotides. Thus, endogenous beta-catenin is required for the observed differential GJC. This regulation of GJC is the earliest known action of the dorsal regulator, beta-catenin, in Xenopus development. Two lines of evidence, presented here, indicate that beta-catenin acts within the cytoplasm to regulate GJC, rather than through an effect on cell adhesion. First, when EP-cadherin is overexpressed and increased adhesion is observed, embryos display both a ventralized phenotype and reduced dye transfer. Second, a truncated form of beta-catenin (i.e., the ARM region), that lacks the cadherin-binding domain, restores dorsal GJC to beta-catenin-depleted embryos. Thus, beta-catenin appears to regulate GJC independent of its role in cell-cell adhesion, by acting within the cytoplasm through a signaling mechanism.

Epithelial cells in primary ovine lens cultures express the gap junction proteins connexin43 (Cx43) and connexin49 (Cx49; a.k.a. MP70), a homologue of mouse connexin50. In contrast, lens cultures of differentiated, fiber-like cells (termed lentoid cells) express Cx49 and connexin46 (Cx46), but not Cx43. To investigate the regulation of lens cell gap junctions by protein kinase C (PKC), differentiating lens cultures were treated with the PKC activator 12-O-tetradecanoylphorbol-13-acetate (beta-TPA). Within 10 min, beta-TPA significantly inhibited the transfer of Lucifer Yellow dye between epithelial, but not lentoid, cells. This inhibition was correlated with the phosphorylation of Cx43 and was followed by the gradual disappearance of Cx43 from cell interfaces. The protein kinase inhibitor staurosporine prevented Cx43 phosphorylation and the loss of Cx43 from intercellular junctions. Following treatment of cultures with beta-TPA for 2-6 hr, Cx49 disappeared from epithelial cell interfaces, and by 24 hr of beta-TPA treatment, levels of Cx49 detected on immunoblots of purified epithelial membrane fractions had also diminished significantly. The beta-TPA-induced loss of Cx49 both from regions of epithelial cell contact and from isolated membranes was correlated with the disappearance of Cx49 mRNA. In contrast to the epithelial connexins, the lentoid connexins Cx49 and Cx46 were unaffected by even extended beta-TPA treatment. In spite of lentoid dye transfer being refractory to beta-TPA, significant levels of PKC-alpha (a beta-TPA-sensitive isoform) were detected in the lentoid cell. The response of lens gap junctions to beta-TPA depends upon the stage of differentiation and the complement of connexins expressed. The contrasting effects of beta-TPA on Cx43 and Cx49 in lens epithelial cells indicate a fundamental difference in the regulation of these connexin proteins in the developing mammalian lens.

Communication via gap junctions provides a mechanism for the cell-cell transfer and coordination of developmental signals. The spatial restriction of gap junctions may also serve to organize cells into domains of coordinated behavior. To investigate the role of gap junctions during embryogenesis, we have characterized the expression of a member of the gap junction gene family, zebrafish connexin43.4, a homolog of connexin45 in chicken and mammals. Expression of connexin43.4 was induced in the early gastrula, coincident with the first definitive assignments of axial cell fate and the onset of the cell movements comprising convergence and extension in zebrafish. In situ hybridization and immunohistochemistry revealed that during gastrulation connexin43.4 mRNA and protein were progressively enriched in the germ ring and in the notochord primordia on the dorsal side of the embryo. Later in development connexin43.4 expression was detected in the notochord, the paraxial mesoderm, and the tail bud but was not observed after the differentiation of these tissues. In no tail mutant embryos which are defective in tail formation and proper morphogenesis of the notochord, connexin43.4 expression was absent during gastrulation from the caudal embryonic shield and notochord primordia. During somite stages in no tail embryos, connexin43.4 expression remained absent in the notochordal precursor cells and was lost in the tail bud. Thus, the no tail gene product, a transcription factor, was required for the expression of connexin43.4 in both the notochord and tail bud during morphogenesis. By microinjection of mRNA coding for a connexin43.4/green fluorescent protein fusion in the 1-cell zebrafish embryo, we showed that connexin43.4 is capable of assembling into structures reminiscent of gap junctions. The progressively restricted, developmental expression of the zebrafish connexin43.4 gene suggests that this gap junctional protein participates in the coordination of gastrulation and the formation of the notochord and tail.

During the assembly of gap junctions, a hemichannel in the plasma membrane of one cell is thought to align and dock with another in an apposed membrane to form a cell-to-cell channel. We report here on the existence and properties of nonjunctional, plasma membrane connexin43 (Cx43) hemichannels. The opening of the hemichannels was demonstrated by the cellular uptake of 5(6)-carboxyfluorescein from the culture medium when extracellular calcium levels were reduced. Dye uptake exhibited properties similar to those of gap junction channels. For example, using different dyes, the levels of uptake were correlated with molecular size: 5(6)-carboxyfluorescein (approximately 32%), 7-hydroxycoumarin-3-carboxylic acid (approximately 24%), fura-2 (approximately 11%), and fluorescein-dextran (approximately 0.4%). Octanol and heptanol also reduced dye uptake by approximately 50%. Detailed analysis of one clone of Novikoff cells transfected with a Cx43 antisense expression vector revealed a reduction in dye uptake levels according to uptake assays and a corresponding decrease in intercellular dye transfer rates in microinjection experiments. In addition, a more limited decrease in membrane resistance upon reduction of extracellular calcium was detected in electrophysiological studies of antisense transfectants, in contrast to control cells. Studies of dye uptake in HeLa cells also demonstrated a large increase following transfection with Cx43. Together these observations indicate that Cx43 is responsible for the hemichannel function in these cultured cells. Similar dye uptake results were obtained with normal rat kidney (NRK) cells, which express Cx43. Dye uptake can be dramatically inhibited by 12-O-tetradeconylphorbol-13-acetate-activated protein kinase C in these cell systems and by a temperature-sensitive tyrosine protein kinase, pp60v-src in LA25-NRK cells. We conclude that Cx43 hemichannels are found in the plasma membrane, where they are regulated by multiple signaling pathways, and likely represent an important stage in gap junction assembly.

We investigated whether the growth state of NRK cells (proliferating or quiescent by serum deprivation) affected the ability of oncogenic Ki-ras p21 and the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), to alter gap junctional communication. We evaluated gap junctional permeance by rate analysis of the transfer of a fluorescent dye, Lucifer Yellow, between cell pairs. We found that while the gap junctions of proliferating NRK cells were unresponsive to both TPA and to Ki-ras p21, junctional communication in quiescent cells was significantly inhibited by brief exposures to 100 ng/ml TPA. Furthermore, activity of Ki-ras p2l 2 h prior TPA exposure enhanced the inhibitory effect of TPA in quiescent cells. Junctional sensitivity to TPA was transient, with inhibition of junctional communication detected at 10 min and refractory after 60 min of continuous exposure. The suppression of junctional communication by TPA was completely prevented if the oncogenic p21 had been active for a longer period of time (48 h). The application of a phorbol ester derivative (4α-PDD), which does not activate protein kinase C, did not affect the ability of quiescent cells to communicate. From these results we conclude that there is a cell-state dependence of junctional sensitivity to TPA in NRK cells and that ras p21 activity potentiates the junctional response to TPA. One interesting possibility is that this involved a cell-cycle effect.

We examined the roles of the extracellular domains of a gap junction protein and a cell adhesion molecule in gap junction and adherens junction formation by altering cell interactions with antibody Fab fragments. Using immunoblotting and immunocytochemistry we demonstrated that Novikoff cells contained the gap junction protein, connexin43 (Cx43), and the cell adhesion molecule, A-CAM (N-cadherin). Cells were dissociated in EDTA, allowed to recover, and reaggregated for 60 min in media containing Fab fragments prepared from a number of antibodies. We observed no cell-cell dye transfer 4 min after microinjection in 90% of the cell pairs treated with Fab fragments of antibodies for the first or second extracellular domain of Cx43, the second extracellular domain of connexin32 (Cx32) or A-CAM. Cell-cell dye transfer was detected within 30 s in cell pairs treated with control Fab fragments (pre-immune serum, antibodies to the rat major histocompatibility complex or the amino or carboxyl termii of Cx43). We observed no gap junctions by freeze-fracture EM and no adherens junctions by thin section EM between cells treated with the Fab fragments that blocked cell-cell dye transfer. Gap junctions were found on approximately 50% of the cells in control samples using freeze-fracture EM. We demonstrated with reaggregated Novikoff cells that: (a) functional interactions of the extracellular domains of the connexins were necessary for the formation of gap junction channels; (b) cell interactions mediated by A-CAM were required for gap junction assembly; and (c) Fab fragments of antibodies for A-CAM or connexin extracellular domains blocked adherens junction formation.

Currently little is known about the regulation of gap junction communication in the lens. We report here on the effects of the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), on cultured bovine lens cells which appeared to be epithelial in nature. Dramatically reduced intercellular transfer of the fluorescent dye Lucifer yellow was observed when the cultured lens cells were treated with octanol, a known inhibitor of gap junction communication. TPA (4 beta isomer) was also shown to reduce intercellular permeability within these cultures. In contrast, an inactive form of TPA, 4 alpha-TPA, did not decrease dye transfer. Permeability was evaluated in terms of both the number of cells receiving dye and the rate of decrease in fluorescence intensity in the injected cell. The maximum decreases in dye transfer occurred at 2 h of TPA treatment and dye transfer gradually increased to control levels over a time course of many hours. Incubation of cultures with 32Pi and immunoprecipitation using antibodies to the N- and C-terminal regions of connexin43 demonstrated a gap junction phosphoprotein of 43,000 Da. Phosphorylation of connexin43 increased during the first 2 h of TPA treatment. These results suggest that protein kinase C has a direct or indirect effect on gap junction communication in cultured lens cells.

Gap junctions are plasma membrane specializations involved in direct cell-cell communication. Intercellular communication is dependent upon the assembly of gap junction structures and would be influenced by agents which alter the assembly process. We investigated the effects of low density lipoprotein (LDL) on gap junction assembly between cultured Novikoff cells using quantitative dye transfer and freeze-fracture electron microscopic methods. We observed a concentration-dependent increase in dye transfer (maximum effect at 2.5 μg protein/ml) and a sixfold increase in the number of aggregated gap junction particles per cell. Immunoblots of Novikoff cells probed with anti-connexin43 antibody revealed no detectable increase in gap junction protein (connexin) levels. The influence of the different components of LDL on junction formation was also examined. First, we treated cells with cholesterol (0–150 μ M ) in serum-free BSA media and observed a decrease in junction assembly. Second, we added apolipoprotein-B (apo-B) in phosphatidyl choline vesicles to the cells and observed a concentration-dependent increase in dye transfer (maximum effect at 2.5 μg protein/ml) and a fivefold increase in the number of aggregated gap junction particles per cell. The addition of phosphatidyl choline vesicles without apo-B had no effect on gap junction formation. Thus, we demonstrated that gap junction assembly can be modulated by LDL and apo-B treatments.

We have characterized the membrane protein of apparent molecular weight 26 kD from bovine lenses (MP26 or MIP) with respect to six different electrophoretic and chromatographic procedures. These include one- and two-dimensional gel electrophoretic procedures, as well as SDS-hydroxylapatite chromatography. The two-dimensional gels include isoelectric focusing with both conventional ampholytes and buffer focusing methods. With buffer focusing, the membranes are solubilized without the use of SDS and the isoelectric focusing is performed in the absence of SDS. As specific probes for MP26, a monoclonal antibody and an anti-MP26 rabbit serum were used, the latter prepared against electrophoretically purified MP26. These separation techniques were adapted to MP26 in order to permit a more detailed characterization of this protein and to search for any heterogeneity in this size range, specifically other junctional proteins or protein fragments. We have found evidence for charge heterogeneity in MP26, but no evidence for multiple membrane proteins of M r 26 000 in urea-treated membranes. The charge heterogeneity appears to be related to a phosphorylation of MP26. The results reported here aid the interpretation of a variety of data, especially findings on the reconstitution of MP26 in artificial membranes and results from work with polyclonal MP26 antibodies. These investigations are all designed to evaluate the proposed role of MP26 as a protein of cell-to-cell channels in the lens fiber cell.

Gap junctions (GJ) are important regulators of cellular function. They provide channels for the direct movement of small molecules between cells and thus control cell-to-cell transfer of metabolites and the transmission of various stimuli. Gap junctions have been shown to be involved in a multitude of cellular processes ranging from cell synchronization and neuronal function to cell differentiation and carcinogenesis. Much knowledge has been gained in recent years concerning the structure and molecular organization of GJ proteins; yet, the mechanisms that control and modulate gap junction assembly and function are still not well understood. Although it is quite apparent that the GJ proteins assemble in the lipid milieu of the plasma membrane, and that the cluster of proteins assembled in the junction do function in a lipid environment, there is a general paucity of information on the role of lipids in the gap junction assembly process and in the function of gap junctions. The present review is a comprehensive account of current knowledge on gap junction lipids. We also discuss what is known to date on the involvement of lipids in gap junction formation. Special emphasis is being placed on the potential role of membrane cholesterol in gap junction assembly and function.

Novikoff hepatoma cells provide an excellent model system for the study of gap junction assembly, a process that could be influenced by lipids and other factors at numerous points. Since it is possible to alter the cellular levels of cholesterol in these cells, it was added to the cells in serum-supplemented medium and changes in gap junction assembly were evaluated. Cells were dissociated and reaggregated following exposure to a range of cholesterol concentrations for 24 h. A five- to sixfold increase in the number of aggregated gap junction particles and a 50% increase in cellular cholesterol content were observed with 20 microM added cholesterol. A 1-h exposure to added cholesterol, during cell reaggregation, resulted in a fourfold increase in the number of aggregated gap junction particles, demonstrating that the effect was rapid. The number of aggregated gap junction particles and formation plaque areas were used as measures of junction assembly and assayed by quantitative freeze-fracture and electron microscopy. Junctional permeabilities were evaluated by means of dye transfer times following the intracellular microinjection of Lucifer Yellow. Increased dye transfer was observed between cholesterol-treated cells, which suggested that the increase in assembly was accompanied by an increase in junction permeability. Cells were treated with cycloheximide (100 micrograms ml-1) and actinomycin D (10 micrograms ml-1) to determine whether protein and RNA syntheses were involved in the enhanced gap junction assembly. Cycloheximide but not actinomycin D blocked the increased junction assembly observed with added cholesterol. These results suggested that protein synthesis, but not RNA synthesis, is necessary for the increased gap junction formation observed.(ABSTRACT TRUNCATED AT 250 WORDS)

We cloned and sequenced the zebrafish (Danio rerio) connexin43 (Cx43α1) gene. The predicted protein sequence shows a high degree of sequence conservation. Transcript analyses revealed multiple transcription start sites and a potential alternative transcript encoding a N-terminally truncated Cx43α1 protein. Maternal Cx43α1 transcripts were detected, with zygotic expression initiated before gastrulation. In situ hybridization revealed many Cx43α1 expression domains, including the notochord and brain, heart and vasculature, many resembling patterns seen in mammalian embryos. Of interest, a reporter construct under control of the mouse Cx43α1 promoter was observed to drive green fluorescent protein expression in zebrafish embryos in domains mimicking the native Cx43α1 expression pattern in fish and mice. Sequence comparison between the mouse and zebrafish Cx43α1 promoter sequences showed the conservation of several transcription factor motifs, which otherwise shared little overall sequence homology. The conservation of protein sequence and developmental gene regulation would suggest that Cx43α1 gap junctions are likely to have conserved roles in vertebrate embryonic development. Developmental Dynamics 233:890–906, 2005. © 2005 Wiley-Liss, Inc.

Cells expressing connexin43 are able to upregulate gap junction (GJ) communication by enhancing the assembly of new GJs, apparently through increased connexin trafficking. Because G proteins are known to regulate different aspects of protein trafficking, we examined the effects of pertussis toxin (PTX; a specific inhibitor of certain G proteins) on GJ assembly. Dissociated Novikoff hepatoma cells were reaggregated for 60 min to form nascent junctions. PTX inhibited GJ assembly, as indicated by a reduction in dye transfer. Electron microscopy also revealed a 60% decrease in the number of GJ channels per cell interface. Importantly, PTX blocked the twofold enhancement in GJ assembly found in the presence of low-density lipoprotein. Two Giαproteins (Giα2and Giα3), which have been implicated in the control of membrane trafficking, reacted with PTX in ADP-ribosylation studies. PTX and/or the trafficking inhibitors, brefeldin A and monensin, inhibited GJ assembly to comparable degrees. In addition, assays for GJ hemichannels demonstrated reduced plasma membrane levels of connexin43 following PTX treatment. These results suggest that PTX-sensitive G proteins regulate connexin43 trafficking, and, as a result of inhibition with PTX, the number of plasma membrane hemichannels available for GJ assembly is reduced.

Given the rapid turnover of connexin proteins, gap junction (GJ) assembly represents an important means of regulating the extent of GJ communication between cells. This report describes an increase in the level of GJ assembly within one hour following treatment with cAMP-elevating reagents or low density lipoprotein (LDL). Dye transfer methods and freeze-fracture with electron microscopy were used to assay junctional permeability and structure, respectively, subsequent to the dissociation, recovery and reaggregation of Novikoff hepatoma cells. Reaggregating cells in the presence of agents that increase cAMP levels (8-Br-cAMP, forskolin and IBMX) enhanced both dye transfer rates between cells and the extent of GJ formation 2- to 3-fold. These data and studies with the protein kinase A inhibitor, H-89, indicate that cAMP signaling plays a key role in enhanced assembly. The response to LDL parallels that to cAMP and relies on the activity of both adenylyl cyclase and protein kinase A. Immunoblot analysis revealed no change in the level of connexin43 (Cx43) or its phosphorylation states over a period of 2.5 hours. However, three agents (brefeldin A, monensin and nocodazole), that inhibit intracellular membrane trafficking by different mechanisms, all blocked the enhanced assembly of GJs when triggered by either elevated cAMP or exposure to LDL. Related studies, which employed trafficking inhibitors at different stages in GJ assembly, suggested that Cx43 trafficking during enhanced assembly is regulated, in part, by cell contact. Intracellular sources of Cx43 were characterized by colabeling for several markers of cytoplasmic membrane systems. We conclude that an increase in GJ assembly: (i) occurs rapidly in the presence of elevated cAMP or LDL, (ii) does not require an increase in Cx43 levels or major changes in Cx43 phosphorylation and (iii) is dependent upon the trafficking of Cx43 from intracellular storage sites.

Nuclear receptors play key roles in anterior/posterior (A/P) axis formation during vertebrate embryogenesis. Within this gene family, retinoic acid receptors and retinoic acid itself have profound influences on the establishment of the A/P axis. Thyroid hormone receptors are expressed during early periods of development, long before the establishment of the thyroid gland, and are able to interact with retinoic acid receptors. Here we examined the ability of the thyroid hormone receptor alpha 1 to affect early embryonic development by mRNA injection of either repressor or activator forms of the thyroid hormone receptor. Overexpression of either the thyroid hormone receptor alpha 1 or a constitutive repressor form, v-erbA, caused a swelling in the rostral hindbrain. These defects were associated with disorganization and loss of rhombomere borders as well as an increase in the number of acetylcholine esterase positive cells. This phenotype correlated with a reduction in hoxa1 expression during gastrulation. Furthermore, injection of either thyroid hormone receptor alpha 1 or v-erbA mRNA repressed a reporter gene that contained a retinoic acid response element, demonstrating the ability of the thyroid hormone receptor alpha 1 to repress retinoic acid signaling during gastrulation. In contrast, embryos treated with retinoic acid alone or embryos injected with thyroid hormone receptor alpha 1 and treated with the thyroid hormone analog TRIAC displayed a similar set of defects, including loss of the midbrain-hindbrain border and severe disruption of the rostral hindbrain. These studies support the involvement of retinoic acid and its receptors in the direct control of Hox gene expression and the early patterning of the zebrafish central nervous system.

Intercellular communication mediated by gap junctions is important for tissue homeostasis in the avascular lens, and extensive areas of gap junctions form between fiber cells during fiber cell differentiation and lens development. We examined the role of the calcium-dependent cell adhesion molecule, N-cadherin, in the process of gap junction formation between fiber cells. Lentoids, multicellular structures with characteristics of differentiated fiber cells, were isolated from embryonic chick lens cultures and subsequently paired to provide an in vitro model of fiber cell interactions. Gap junction formation between cells of paired lentoids was monitored by observing the lentoid-to-lentoid transfer of fluorescent dyes, either calcein or Lucifer yellow, over a time course of up to 48 hr. Dye transfer between lentoids was inhibited upon the addition to the medium of Fab fragments (100-622 microgram/ml) of a monoclonal antibody specific for N-cadherin, and also by the reduction of extracellular calcium in the incubation medium. However, the addition of Fab fragments (100-1500 microgram/ml) of an antibody to a fiber-cell-specific integral membrane protein, MIP, did not change the time course nor extent of dye transfer between lentoids. Our results, using cultured embryonic cells, extend those from previous studies with cell lines and transfected cells. We conclude that cadherin interactions facilitate the formation of gap junctions between embryonic lens fiber cells, by the stabilization of membrane appositions and/or by the generation of an intracellular signal(s).

The present study was undertaken to address the question whether lipid differentiation occurs in junctional domains which could imply a functional requirement for specific lipids in junctional structures. Junction enriched membranes were isolated from bovine lens fiber cells using Tris and urea treatment, and the presence of junctional structures was ascertained by electron microscopy. Enrichment in major intrinsic protein (MIP, MP26) was monitored by SDS polyacrylamide gel electrophoresis. Junctional lipids were extracted by a modified Folch procedure, to quantitatively recover cholesterol, and lipid classes were analyzed. While 99.5% of total lens protein was solubilized in the course of junction isolation, 43.9% of cell phospholipids (PL) and 64.1% of cell cholesterol (Chol) were conserved. Cholesterol was by far the predominant lipid in the junction enriched lens fiber cell membranes (833 nmol/mg protein) and was more abundant than all phospholipids combined (682 nmol/mg protein). In isolating the junctional membranes, cholesterol levels increased 144-fold, and average phospholipid levels increased 99-fold, which resulted in an increase in Chol/PL ratio from 0.84 to 1.22. Different phospholipids showed substantially different degrees of enrichment with highest enrichments seen for the phosphatidylethanolamine fraction (152-fold) and sphingomyelin (101-fold). Thus, the phospholipids of the junction enriched membranes consisted mainly of ethanolamine glycerophospholipids (37.3%) and sphingomyelin (28.6%), with lesser amounts of choline glycerophospholipids (23.5%) and phosphatidylserine (9.2%) present. Our data suggest that the MP26 junction enriched membranes of bovine lens fiber cells contain differentiated lipid domains, and that cholesterol, ethanolamine glycerophospholipids and sphingomyelin are the prevalent boundary lipids of the major intrinsic protein in these domains.

A number of previous observations have indicated that the cadherins play an important role in the regulation of gap junction communication. In the experiments reported here, the process of gap junction assembly was specifically monitored between Novikoff hepatoma cells in culture. In the presence of reduced levels of extracellular calcium, gap junction assembly was substantially inhibited, although not completely blocked. The mechanism of this inhibition was explored with an analysis of connexin43 phosphorylation, determinations of cytoplasmic calcium, and immunofluorescence methods for cadherins and connexins. Two models are considered - one for simple adhesion and one for signalling.

Gap junction structures were assembled in vitro from octyl-beta-D-glucopyranoside-solubilized components of lens fiber cell membranes. Individual pore structures (connexons), short double-membrane structures, and other amorphous material were evident in the solubilized mixture. Following the removal of the detergent by dialysis, these connexons associated to form single- and double-layered, two-dimensional hexagonal arrays (unit cell size a = b = 8.5 nm). The formation of larger arrays was dependent on the lipid-to-protein ratio and the presence of Mg2+ ions. Crystallographic analysis of electron micrographs revealed that lens junctional connexons consisted of six subunits surrounding a stain-filled channel. Upon further detergent treatment, in vitro assembled gap junctions were insoluble and formed three-dimensional stacks while other components were solubilized. SDS-PAGE and mass data from scanning transmission electron microscopy strongly suggest that a 38-kDa polypeptide, which is a processed form of the lens specific gap junction protein MP70, is a major component of the arrays. The in vitro assembly of gap junctions opens new avenues for the structural analysis of gap junctions and for the study of the intermolecular interactions of connexons during junctional assembly.

The main intrinsic membrane protein of the lens fiber cell, MIP, has been previously shown to be phosphorylated in preparations of lens fragments. Phosphorylation occurred on serine residues near the cytoplasmic C-terminus of the molecule. Since MIP is thought to function as a channel protein in lens plasma membranes, possibly as a cell-to-cell channel protein, phosphorylation could regulate the assembly or gating of these channels. We sought to identify the specific serines which are phosphorylated in order to help identify the kinases involved in regulating MIP function. To this end we purified a peptide fragment from native membranes that had not been subjected to any exogenous kinases or kinase activators. Any phosphorylation detected in these fragments must be due to cellular phosphorylation and thus is termed in vivo phosphorylation. Purified membranes were also phosphorylated with cAMP-dependent protein kinase to determine the mobility of phosphorylated and unphosphorylated MIP-derived peptides on different HPLC columns and to determine possible cAMP-dependent protein kinase phosphorylation sites. Lens membranes, which contain 50-60% of the protein as MIP, were digested with lysylendopeptidase C. Peptides were released from the C-terminal region of MIP and a major product of 21-22 kDa remained membrane-associated. Separation of the lysylendopeptidase-C-released peptides on C8 reversed-phase HPLC demonstrated that one of these fragments, corresponding to residues 239-259 in MIP, was partially phosphorylated. The phosphorylated and nonphosphorylated forms of this peptide were separated on QAE HPLC. In vivo phosphorylation sites were found at residues 243 and 245 through phosphoserine modification via ethanethiol and sequence analysis. Phosphorylation was never detected on serine 240. The phosphorylation level of serine 243 could be increased by incubation of membranes with cAMP-dependent protein kinase under standard assay conditions. Other kinases that phosphorylate serines found near acidic amino acids must be responsible for the in vivo phosphorylation demonstrated at serine 245.

Embryonic chicken lenses, which had been disrupted by trypsin, were grown in culture. These cultures mimic lens development as it occurred in vivo , forming lens-like structures known as lentoids. Using a variety of techniques including electron microscopic analysis, autoradiography, immunofluorescence, and polyacrylamide gel electrophoresis, it was shown that the lentoid cells had many characteristics in common with the differentiated cells of the intact lens, the elongated fiber cells. These characteristics included a shut off of DNA synthesis, a loss of cell organelles, an increase in cell volume, an increase in δ-crystallin protein, and the development of extensive intercellular junctions. The cultures began as a simple epithelial monolayer but then underwent extensive morphogenesis as they differentiated. This morphogenesis involved three distinctive morphological types which appeared in sequence as an epithelial monolayer of polygonal shaped cells with pavement packing, elongated cells oriented end to end, and the multilayered, multicellular lentoids. These distinct morphological stages of differentiation in culture mimic morphogenesis as it occurs in the lens.

Electrophysiological studies of low-resistance junctions between Novikoff hepatoma cells grown in suspension cultures were carried out and correlated with gap-junctional areas per inferface determined by freeze-fracture. The mean coupling coefficient between isolated cell pairs was 0.773 +/- 0.025 (SEM) in 67G medium and 0.653 +/- 0.028 in M67 medium; the respective means for the central pairs of four-cell chains were 0.714 +/- 0.034 and 0.595 +/- 0.026. Mean estimates of nonjunctional resistances for cell pairs were 3.0 +/- 0.32 x 10(7) ohm (67G) and 2.01 +/- 0.01 x 10(7) ohm (M67), and the respective estimates for specific nonjunctional resistances were 158.6 +/- 8.1 ohm-cm2 (67G) and 133.0 +/- 812 ohm-cm2 (M67). Mean estimates of junctional conductances were 0.409 +/- 0.058 x 10(-6) mho (67G) and 0.211 +/- 0.018 x 10(-6) mho (M67) for pairs and 0.291 +/- 0.063 x 10(-6) mho (67G) and 0.212 +/- 0.04 mho (M67) for four-cell chains. The mean area of gap junction per interface for separate cell populations was 0.187 +/- 0.049 micron 2 and 0.269 +/- 0.054 micron 2 for cells fixed in loose pellets and in suspension, respectively. When compared with the mean junctional conductance, these values gave specific junctional conductance estimates of 1.13 x 10(2) mho/cm2 and 0.78 x 10(2) mho/cm2, respectively. These values are higher than most previous estimates, but are consistent with the hypothesis that gap-junctional particles contain central hydrophilic channels, about 2 nm in diameter, which have cytoplasmic resistivity.

Direct transfer of [3H]uridine-derived metabolites between reaggregated Novikoff hepatoma cells was monitored autoradiographically over the first 4 h following initial cell contact. The percentage of significantly labeled primary recipients increased progressively over the reaggregation period, reaching a level of more than 90% by 4 h. When reaggregation was promoted by centrifuging the cells on to a polylysine-coated slide, over 50% of the potential recipients became labeled within 15 min. Transfer was contact-dependent, required RNA synthesis in the recipients for detection, was not reduced by excess cold uridine in the medium, and did not occur when L-67 cells were used or recipients. Similar transfer of [3H]hypoxanthine-derived metabolites occurred between HGPRT+ and HGPRT− Novikoff cells reaggregated up to 4 h. By that time, 1:1 cocultures of the two cell types incorporated 29% more hypoxanthine than expected from the number of HGPRT+ cells present, implying a cooperative effect on nucleotide metabolism. The development of metabolite transfer had a time course similar to that reported elsewhere for the formation of gap junctions and for the appearance of junctional permeability to other small molecules between Novikoff cells. We conclude that the labeled metabolites were transferred via newly formed gap junctions.

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

Investigators with interests in cell junctions, and gap junctions in particular, have been intrigued over the last 5-10 years by the extensive junctional specializations found in the lens. Attention has focused on the suggested role of MP26 as the major protein component. The fundamental question has been whether the abundant junctions seen with thin-section or freeze-fracture analyses correspond to lens “gap junctions.” That is, do these cell surface specializations between lens fiber cells provide for the direct cell-to-cell movement of small molecules? Unfortunately, we still lack a definitive answer to the question. For some investigators, enthusiasm waned when protein analysis uncovered no homology between MP26 and a major liver gap junction protein. However, we have much to learn about the nature and role of the lens junction.

Using thin-section and freeze-fracture techniques, the fates of formation plaques with and without gap junctions [designated FP(+), FP(−)] have been studied following the dissociation of Novikoff hepatoma cells with EDTA. Quantitative analysis shows dissociated cells to possess less numerous FP(+)'s and FP(−)'s than undissociated suspension cultures. Both EM techniques showed gap junctions remaining on cell surfaces, after one junctional membrane had been ripped from its cell and had apparently resealed forming a bleb. These structures were only infrequently observed to invaginate from the cell surface, suggesting that endocytosis is rare. Following the 30-min dissociation, some cells were placed in growth medium and incubated on a gyratory shaker (200 rpm) for 5–90 min. Under these conditions, the cells “recovered” from the dissociation process, but failed to reaggregate. FP(−)'s, dramatically reduced in dissociated preparations, completely disappeared by 30 min of recovery. FP(+)'s were reduced by dissociation, continued to fall over the first 15 min of recovery, and declined slowly to a low level by 90 min of recovery. Substantial numbers of the larger gap junctions were surrounded by numerous smaller junctional aggregates, an unusual configuration for undissociated cells. From these data, we suggest that: (a) a fraction of the FP(+)'s persists on cell surfaces for some time after dissociation; (b) FP(−)'s are more labile and are apparently not produced during breakdown; (c) FP(−)'s do not develop in the absence of prolonged cell contact; and (d) irregular junctions with adjacent, smaller junctions may represent junctional breakdown.

Freshwater planaria ( Dugesia ) were found to have three distinct forms of gap junctions. The three forms differ from one another in morphology and in the kinds of cells that they are found between. Utilizing freeze-fracture and lanthanum tracer preparations, the three types were recognized by differences in the spacing and pattern of their particles or subunits and in the membrane faces to which the particles adhere. One of the three types is associated with gastrodermal, parenchymal, and excretory cells; another is found on muscles and nerves; and the third is localized apparently on secretory cells. The possibility of the three junctional forms being correlated with functional differences is discussed. Muscle and nerve cells also have a nonjunctional type of specialization consisting of intramembranous particles arranged in a rhombic pattern.

Several polypeptides have been described in the past as components of gap junction fractions. Of these, a peptide of Mr 26,000 is found in gap junctions isolated from livers of different species under conditions that minimize proteolysis. Tryptic digestion of purified "intact" junctions causes the rapid disappearance of this peptide with a concomitant appearance of a band of Mr 10,000, which has previously been found to be characteristic of junctional fractions isolated with the aid of proteolytic treatment. Both the peptides of Mr 26,000 and 10,000 are missing from gap junction preparations after partial hepatectomy, when gap junctions are absent from the surface of the hepatocytes. They have reappeared in fractions from the livers of animals killed 3 days postoperatively, when gap junctions are again present in vivo.

MP26, a protein thought to form gap junctional channels in the lens, and other lens proteins were phosphorylated under conditions that activate protein kinase C. Phosphorylation was detected both in lens fiber cell fragments in an "in vivo" labeling procedure with 32P-phosphate and in cell homogenates with 32P-ATP. In these experiments, both calcium and 12-O-tetradecanoylphorbol 13-acetate (TPA) were necessary for maximal phosphorylation of MP26. Calcium stimulated the phosphorylation of MP26 approximately fourfold and TPA with calcium led to a sevenfold increase. If TPA was present, 1 microM calcium was sufficient for maximal labeling. Phosphoamino acid analysis demonstrated approximately 85% phosphoserine, 15% phosphothreonine, and no phosphotyrosine when MP26 was phosphorylated in lens homogenates in the presence of TPA and calcium and then electrophoretically purified. Phosphorylation occurred near the cytoplasmic, C-terminal of MP26. The possible involvement of other kinases was also examined. The Walsh inhibitor, which affects cAMP-dependent protein kinases, had no influence on the TPA-mediated increase in phosphorylation. In studies with isolated membranes and added kinases, MP26 was also found to not be a substrate for calcium/calmodulin-dependent protein kinase II. Thus, protein kinase C may have phosphorylated MP26 in a direct manner.

Specific antibodies are versatile tools for analyzing cell surface proteins. This study involves the characterization of monoclonal antibodies which are specific for the junctional protein found in the lens fiber cell. This protein can be expected to include regions on the external membrane surface for junction formation, others on the cytoplasmic surface for regulation of junctional properties and, if cell-cell channels are indeed involved, transmembrane domains forming the hydrophilic connection between adjacent cytoplasms. Antibodies to these various regions would provide for an experimental analysis of the junctional protein, e.g., the identification of "active sites" for junction formation. Three monoclonal antibodies specific for the lens junctional protein in the chicken are described here. The first, termed B2, also recognizes the bovine junctional protein, MP26 (5). We have characterized the submolecular specificity of B2 and have found that it binds approximately ten amino acid residues from the C-terminus of MP26. In isolated lens junction preparations, B2 binds to the cytoplasmic surfaces of the lens junctions (both 12 nm and 16 nm thick forms). Thus, we consider MP26 a component of the lens junction. Monoclonal A4, the second antibody considered in detail here, was produced by immunization with lens membranes after treatment with low pH. We have found that lens junctional membranes are separated, or "split," by treatment at pH 2.5-3.0. It appears that A4 binds to the external surface of the junctional membrane; EM studies to confirm this are in progress. In order to map the A4 binding site within the chicken junctional protein and to explore the arrangement of this protein within the membrane, a number of procedures were used to generate fragments of MP26. These included reactions with N-chlorosuccinimide and proteases after acid treatment. Antibody binding to fragments was evaluated with immunotransfer ("Western") procedures. These studies mapped the A4 binding site to the center of the molecule and suggested that MP26 projected externally from the membrane at two different points. These results are consistent with a recent model, based on sequence data (6), for the arrangement of MP26 within the bovine lens membrane.

Two intrinsic proteins of bovine lens membranes with apparent relative molecular masses (Mr, app) of 26,000 and 18,000 were phosphorylated in intact membranes by protein kinase C prepared from either bovine brain or lens. The kinase preparations exhibited histone H1 phosphorylation dependent on calcium and phospholipid but not on cAMP. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the lens membranes showed a major band at Mr, app = 26,000 (identified as MP26, the main intrinsic protein of lens fiber cells), an intermediate band at Mr, app = 18,000 and several minor bands. Autoradiography of complete assay mixture containing protein kinase C, calcium, magnesium and [gamma-32P]ATP showed major bands at Mr, app = 18,000 and 26,000. Several lines of evidence indicated that the label at Mr, app = 26,000 was associated with MP26, a protein which has been found in lens junctions and which may form cell-cell channels. Treatment of the phosphorylated membranes with chymotrypsin and V8 protease cleaved the major band at Mr, app = 26,000 to fragments of Mr, app .= 22,000 and 24,000. Label was not detected in the resulting Mr, app = 22,000 peptide, but the Mr, app = 24,000 peptide was found to be labeled. Phosphoamino acid analysis of MP26 indicated that approximately 75% of the label was on phosphoserine and 25% was on phosphothreonine. No label was found on phosphotyrosine. These results differ from those reported for cAMP-dependent phosphorylation of lens proteins. Phosphorylation by protein kinase C may account for some of the labeling of MP26 detected in vivo.

Gap junctions between three different cell combinations in Limulus are studied with thin-section, lanthanum-tracer, and freeze-fracture techniques. Midgut epithelial (E) cells form E—E gap junctions. Reserve (R) cells adjacent to the outer surface of the midgut form R—R gap junctions. E—R junctions are formed when R-cell processes extend through a basement membrane and contact E-cells. All junctions possess parallel membranes with 30 A gaps, polygonal substructure when treated with lanthanum and sectioned en face , and electron-dense 25 A dots in subunit centers. In freeze-fracture replicas only E—E and R—R junctions can be identified. All junctions possess aggregates of 125 A particles in each membrane. Particles are observed only on outer fracture faces and, like the complementary pits, are not highly patterned. In both particle centers and pits, 30 A spots of platinum are seen. The substructure of gap junctions between cells of the same type appears indistinguishable from that between different cell types.

Cell junctions between Novikoff hepatoma cells (N1S1-67) growing as small clumps or chains in suspension culture have been studied with ultrastructural, electrophysiological, and dye-injection techniques. Cells within clumps are commonly electrically coupled and can exchange dyes with a molecular weight of 332 to 500. Gap junctions and intermediate junctions are present, whereas true tight junctions and desmosomes are absent or very rare. This system should provide a useful model for studying the properties of "communicating" junctions.

We have combined freeze-fracture and electrophysiological methods in a study of gap junction formation between reaggregated Novikoff hepatoma cells. Cell clumps are dissociated with EDTA, and the resulting single cells are allowed to reaggregate (5-180 min) in loose pellets in the presence of calcium at 37°. The earliest electron microscopic evidence for the genesis of new junctions is the appearance of flattened regions of the plasma membrane with a relative paucity of small intramembranous particles. These regions contain instead loosely organized groupings of 9- to 11-nm intramembranous particles, which are seen on the A face of the fractured plasma membrane, while corresponding pits occur on the membrane B face. We have termed the specialized membrane regions “formation plaques.” They are seen as early as 5 min after reaggregation and are quite numerous by 30 min. Larger plaques are observed at later times. Plaques seen at 30 min are consistently matched with other plaques on apposed cells, although the extracellular space separating these structures still exceeds 10 nm. By 60 min, some matched plaques display a reduced extracellular space, resembling that of normal gap junctions. Between 30 and 60 min, aggregates of closely packed particles on A faces and hexagonally arranged pits on B faces frequently appear in the formation plaques. The aggregates, which are indistinguishable from small gap junctions, appear to enlarge over the subsequent 2-hr period as the number of unaggregated 9- to 11-nm particles declines. Microelectrode studies demonstrate progressive increases in the percent of interfaces containing lowresistance junctions and in the degree of elctrical coupling in preparations incubated up to 2 hr. Coupling is first detected at about the same time as particle aggregates (or formation plaques with reduced extracellular spaces), and increases as aggregate sizes increase.

cAMP-dependent protein kinase, derived from either calf lens or bovine heart, promotes the phosphorylation of three lens plasma membrane proteins of molecular mass 28 kDa, 26 kDa and 18 kDa. Correlation of the maximal level of phosphorylation of these components with the Coomassie blue staining intensity of fractionated lens membranes suggests that the phosphorylation of the 28 kDa and 18 kDa components may be approximately stoichiometric. The protein kinase substrates could be dephosphorylated by a cardiac sarcoplasmic-reticulum-bound protein phosphatase activity. The 26 k Da component comigrated with MP26, the major lens membrane component that has been localized to the lens fiber cell junction. Treatment of phosphorylated lens membranes with chymotrypsin did not suggest that any of the three major phosphorylated components was derived from the partial proteolysis of a larger phosphoprotein. After electrophoretic separation of phosphorylated proteins, treatment with N-chlorosuccinimide confirmed that there was little similarity in the structure of the three phosphoproteins. Chymotrypsin did, however, reveal a cryptic phosphorylation site in a 22 kDa fragment that appeared to be derived from MP26. Treatment of phosphorylated membranes with reducing agents resulted in the disappearance of the 28 kDa phosphorylated component and the appearance of a new phosphorylated component of 18 kDa; neither MP26 nor the original 18 kDa component was affected by such treatment. It is not clear whether the original 18 kDa phosphoprotein, present in unreduced samples, is the same as that generated with reducing agents from the 28 kDa phosphorylated lens membrane component.

Mitochondrial movements in Saccharomyces cerevisiae (Sc) zygotes were monitored with phase-contrast microscopy and compared to known mitochondrial inheritance systems. The mitochondria of Sc were convincingly identified by integrated use of phase-contrast, cytochemical and electron microscopic observations. Mitochondria in Sc appear to move by saltatory jumps, which appear to be oriented towards movement of mitochondria into developing buds. Tracking of mitochondria of different genotypes was made possible by positive identification of each mitochondrial population before zygosis, and by the low degree of mixing (less than 10%) of mitochondrial populations before first bud septation. A grande by grande cross demonstrated equal numbers of mitochondria from each haploid moving into the first zygotic bud. A grande by neutral petite cross gave a 2:1 ratio of grande to petite mitochondria. However, a grande by suppressive petite cross gave equal numbers of grande and petite mitochondria. Using drug resistance systems, a comparison was made of highly biased (97%) and moderately biased (71%) chloramphenicol resistant inheritance patterns. In both cases, the ratios of drug resistant to sensitive mitochondria were 1:1. When numbers of mitochondria moving into an individual bud were compared to the phenotypic content of the clone of that bud, no model could be constructed which could predict the latter from the former. The data indicate (with the exception of the neutral petite by grande cross) that the numbers of each mitochondrial type "inserted" into the first zygotic bud are equal, regardless of the degree of asymmetry of inheritance of mitochondrial markers.

Lens membranes, purified from calf lenses, have been labeled by covalent cross-linking to membrane-bound 125I-calmodulin with dithiobis(succinimidyl propionate). Electrophoretic analysis in sodium dodecyl sulfate demonstrated two major 125I-containing products of Mr = 49 000 and 36 000. That the formation of these two components was specifically inhibited by unlabeled calmodulin, or calmodulin antagonists, would indicate that the formation of these components was calmodulin-specific. The size of these two 125I-labeled components was unchanged over a range of 125I-calmodulin or dithiobis(succinimidyl propionate) concentrations indicating that they represent 1:1 complexes between 125I-calmodulin (Mr = 17 000) and Mr-32 000 and Mr-19 000 lens membrane components respectively. Although formation of both cross-linked components exhibited an absolute dependence on Mg2+, the autoradiographic intensity of these components was enhanced when Ca2+ was included with Mg2+ during the cross-linking reaction. Labeling was maximal in 10 mM MgCl2 and approximately 1 microM Ca2+. Treatment of lens membranes with chymotrypsin resulted in the cleavage of MP26 (the major lens membrane protein), with the appearance of a major proteolytic fragment of Mr = 22 000. This proteolysis was not associated with any significant change in either the size or amount of the 125I-calmodulin-labeled membrane components. These results suggest that calmodulin interacts with two membrane proteins, but not significantly with MP26, in the intact lens cell membrane. Our results indicate the need to maintain caution in interpreting direct calcium plus calmodulin effects on MP26 and lens cell junctions.