Using quantum dots to visualize clathrin associations

Our current understanding of clathrin-mediated endocytosis proposes that the process is initiated at a specialized anatomical structure called a coated pit. Electron microscopy has been required for elucidation of the morphology of coated pits and the vesicles produced therein, and the presence of a bristle coat has been taken as suggestive of clathrin surrounding these vesicles. More recently, immunocytochemical methods have confirmed that endocytic vesicles are surrounded by clathrin and its adaptor proteins, but there is a need to identify precisely and to follow the fate of the cellular organelles seen by fluorescence microscopy. We used quantum immune-electron microscopy to localize clathrin in a human adrenal cortical cell line (SW-13). Clathrin was shown to associate with a variety of vesicle types including the classic clathrin-coated vesicles and pits used in receptor internalization, pentilaminar annular gap junction vesicles, and multivesicular bodies. The images obtained with quantum dot technology allow accurate and specific localization of clathrin and the clathrin adaptor protein, AP-2, with cellular organelles and suggest that some of the structures classified as typical coated vesicles by immunocytochemical light microscopic techniques actually may be membrane bound pits.     

Molecular mechanisms regulating formation,trafficking and processing of annular gap junctions

Internalization of gap junction plaques results in the formation of annular gap junction vesicles. The factors that regulate the coordinated internalization of the gap junction plaques to form annular gap junction vesicles, and the subsequent events involved in annular gap junction processing have only relatively recently been investigated in detail. However it is becoming clear that while annular gap junction vesicles have been demonstrated to be degraded by autophagosomal and endo-lysosomal pathways, they undergo a number of additional processing events. Here, we characterize the morphology of the annular gap junction vesicle and review the current knowledge of the processes involved in their formation, fission, fusion, and degradation. In addition, we address the possibility for connexin protein recycling back to the plasma membrane to contribute to gap junction formation and intercellular communication. Information on gap junction plaque removal from the plasma membrane and the subsequent processing of annular gap junction vesicles is critical to our understanding of cell-cell communication as it relates to events regulating development, cell homeostasis, unstable proliferation of cancer cells, wound healing, changes in the ischemic heart, and many other physiological and pathological cellular phenomena.     

Molecular mechanisms regulating formation,trafficking and processing of annular gap junctions

Internalization of gap junction plaques results in the formation of annular gap junction vesicles. The factors that regulate the coordinated internalization of the gap junction plaques to form annular gap junction vesicles, and the subsequent events involved in annular gap junction processing have only relatively recently been investigated in detail. However it is becoming clear that while annular gap junction vesicles have been demonstrated to be degraded by autophagosomal and endo-lysosomal pathways, they undergo a number of additional processing events. Here, we characterize the morphology of the annular gap junction vesicle and review the current knowledge of the processes involved in their formation, fission, fusion, and degradation. In addition, we address the possibility for connexin protein recycling back to the plasma membrane to contribute to gap junction formation and intercellular communication. Information on gap junction plaque removal from the plasma membrane and the subsequent processing of annular gap junction vesicles is critical to our understanding of cell-cell communication as it relates to events regulating development, cell homeostasis, unstable proliferation of cancer cells, wound healing, changes in the ischemic heart, and many other physiological and pathological cellular phenomena.

     

Migrating cells retain gap junction plaque structure and function

Cell migration is an essential process in organ development, differentiation, and wound healing, and it has been hypothesized that gap junctions play a pivotal role in these cell processes. However, the changes in gap junctions and the capacity for cell communication as cells migrate are unclear. To monitor gap junction plaques during cell migration, adrenocortical cells were transfected with cDNA encoding for the connexin 43-green fluorescent protein. Time-lapse imaging was used to analyze cell movements and concurrent gap junction plaque dynamics. Immunocytochemistry was used to analyze gap junction morphology and distribution. Migration was initiated by wounding the cell monolayer and diffusional coupling was demonstrated by monitoring Lucifer yellow dye transfer and fluorescence recovery after photobleaching (FRAP) in cells at the wound edge and in cells located some distance from the wound edge. Gap junction plaques were retained at sites of contact while cells migrated in a "sheet-like" formation, even when cells dramatically changed their spatial relationship to one another. Consistent with this finding, cells at the leading edge retained their capacity to communicate with contacting cells. When cells detached from one another, gap junction plaques were internalized just prior to cell process detachment. Although gap junction plaque internalization clearly was a method of gap junction removal during cell separation, cells retained gap junction plaques and continued to communicate dye while migrating.     

Evidence for the participation of actin microfilaments and bristle coats in the internalization of gap junction membrane

Thin sections of rabbit granulosa, human SW-13 adrenal cortical adenocarcinoma, and mouse B-16 melanoma cells revealed an apparent single-layered basket of 4- to 7-nm filaments surrounding cytoplasmic gap junction vesicles. This interpretation was based upon apparent longitudinal, cross, and en face sections of structures surrounding the vesicle profiles in tissue treated with tannic acid-glutaraldehyde. In granulosa cells incubated with the S-1 fragment of heavy meromyosin, arrowhead-decorated filaments were observed at the periphery of the gap junction vesicles, suggesting that these filaments contained actin. In addition, we found that small gap junctional blebs and vesicles at the cell surface were coated with short electron-dense bristles similar in appearance to the cloathrin-containing coat of coated vesicles of nonjunctional membrane. The role of these and other cytoskeletal elements in the possible endocytosis of gap junction membrane is discussed.     

Are gap junction membrane plaques implicated in intercellular vesicle transfer?

Gap junction channels are concentrated in specialised plaques of plasma membrane where cells are in close apposition. In this communication evidence is provided showing that these specialised regions of membrane also provide a site for vesicular transfer between cells. Vesicle distribution in eye lenses was found to generally reflect the reported distribution of gap junction membrane plaques. In certain areas of the lens gap junction membrane plaques and vesicles could be seen to form combined, complex structures. Ultrastructure of the vesicle and gap junction membrane plaque complexes was consistent with the vesicles moving through membrane plaques from one lens fibre cell to the next. To investigate whether transport of substances was consistent with intercellular vesicle transfer, transport of various markers was investigated. Time course experiments showing the rate of uptake of various markers into the lens did not show dramatic differences for molecules smaller or larger then gap junction pores formed by connexons. While considered as a primary intercellular transport mechanism in the lens, connexon pores were not the sole agent mediating the observed transport. Other reported mechanisms of intercellular transport in the lens can only account for the movement of relatively small molecules. Vesicular transport may therefore be a major form of transport into the outer lens layers for larger molecules. Implicit in these observations is a new hypothesis for intercellular vesicle movement via gap junction membrane plaques. Intercellular vesicle movement could possibly provide a path for large molecules associated with intact vesicles to be transported into the eye lens tissue.     

Connexin 33 Impairs Gap Junction Functionality by Accelerating Connexin 43 Gap Junction Plaque Endocytosis

Connexin 33 (Cx33) is a testis-specific gap junction protein. We previously reported that Cx33 exerts dominant-negative effect on gap junction intercellular communication by sequestering Cx43 within early endosomes in Sertoli cells. However, the molecular mechanisms that drive this process are unknown. The present study analyzed: (i) the trafficking of Cx33 and Cx43 in wild-type Sertoli cells transfected with Cx33-DsRed2 and Cx43-green fluorescent protein vectors; (ii) the formation of heteromeric Cx33/Cx43 hemi-channels and their incorporation into gap junction plaques. Fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer and videomicroscopy studies demonstrated that Cx33 and Cx43 associated to form heteromeric oligomers that trafficked along microtubules to the plasma membrane. However, the plaques containing Cx33 were not functional. Immunoprecipitation experiments revealed that zonula occludens-1 (ZO-1), a scaffold protein proposed to secure Cx in gap junction plaques at the cell–cell boundary, associated with Cx33 in testis extracts. In cells expressing Cx33, Cx33 and ZO-1 specifically interacted with P₁ phosphorylated and P₀ unphosphorylated isoforms of Cx43, and the ZO-1 membranous signal level was reduced. It is suggested that alteration of Cx43/ZO-1 association by Cx33 could be one mechanism by which Cx33 exerts its dominant-negative effect on gap junction plaque.     

The role of the C-terminus in functional expression and internalization of rat connexin46 (rCx46)

The C-terminus (CT) of rCx46 consists of 186 residues (H230-I416). Recent studies showed that rCx46_28.2, truncated after H243, altered the formation of functional hemichannels when expressed in Xenopus oocytes, while rCx46_37.7, truncated after A333 formed gap junction hemichannels similarly to rCx46_wt. To analyze the role of the CT up to A333 in functional expression with cell imaging and dye-transfer techniques, different mutants were generated by C-terminal truncation between H243-A333, labeled with EGFP and expressed in HeLa cells. These rCx46 variants were characterized according to their compartmentalization in organelles, their presence in microscopic detectable vesicles and their ability to form gap junction plaques. rCx46 truncated after A311 (rCx46_35.3) was compartmentalized, was found in vesicles and formed functional gap junction plaques similarly to rCx46_wt. With a truncation after P284 (rCx46_32.6), the protein was not compartmentalized and the amount of vesicles containing the protein were reduced; however, functional gap junction plaque formation was not affected as compared to rCx46_35.3. rCx46_28.2 did not form functional gap junction plaques; it was not found in vesicles or in cellular compartments. Live-cell imaging and detection of annular junctions for rCx46_32.6 and rCx46_35.3 revealed that the truncation after P284 reduced the frequency of vesicle budding from gap junction plaques and the formation of annular junctions. These results suggest that the C-terminal region of rCx46 up to A311 (rCx46_35.3) is necessary for its correct compartmentalization and internalization in the form of annular junctions, while the H230-P284 C-terminal region (rCx46_32.6) is sufficient for the formation of dye coupled gap junction channels.     

Loss of Gap Junction Plaques and Inhibition of Intercellular Communication in Ilimaquinone-treated BICR-M1Rk and NRK Cells

To examine the mechanism(s) and pathways of gap junction formation and removal a novel and reversible inhibitor of protein secretion, ilimaquinone (IQ), was employed. IQ has been reported to cause the vesiculation of Golgi membranes, block protein transport at the cis-Golgi and depolymerize cytoplasmic microtubules. Connexin43 (Cx43) immunolabeling and dye microinjection experiments revealed that gap junction plaques were lost and intercellular communication was inhibited following IQ treatment for 1 hr in BICR-M1R_k rat mammary tumor cells and for 2 hr in normal rat kidney (NRK) cells. Gap junction plaques and intercellular communication recovered within 2 hr when IQ was removed. IQ, however, did not affect the distribution of zonula occludens-1, a protein associated with tight junctions. Western blot analysis revealed that the IQ-induced loss of gap junction plaques was accompanied by a limited reduction in the highly phosphorylated form of Cx43, previously shown to be correlated with gap junction plaques. The presence of IQ inhibited the formation of new gap junction plaques in BICR-M1R_k cells under conditions where preexisting gap junctions were downregulated by brefeldin A treatment. Treatment of BICR-M1R_k and NRK cells with other microtubule depolymerization agents did not inhibit plaque formation or promote rapid gap junction removal. These findings suggest that IQ disrupts intercellular communication by inhibiting the events that are involved in plaque formation and/or retention at the cell surface independent of its effects on microtubules. Our results also suggest that additional factors other than phosphorylation are necessary for Cx43 assembly into gap junction plaques. Received: 16 January 1996/Revised: 20 September 1996     

Accelerated internalization of junctional membrane proteins (connexin 43, N-cadherin and ZO-1) within endocytic vacuoles: an early event of DDT carcinogenicity

Stability of cell-to-cell interactions and integrity of junctional membrane proteins are essential for biological processes including cancer prevention. The present study shows that DDT, a non-genomic carcinogen used at a non-cytotoxic dose (1 microM), rapidly disrupted the cell-cell contacts and concomitantly induced the formation of cytoplasmic vacuoles close to the plasma membrane in the SerW3 Sertoli cell line. High-resolution deconvolution microscopy reveals that this vacuolization process was clathrin-dependent since a hyperosmotic media (0.2 M sucrose) blocked rhodamine-dextran endocytosis. In response to DDT, junctional proteins such as Cx43, N-Cadherin and ZO-1 were internalized and present in vacuoles. In Cx43-GFP transfected cells, time lapse videomicroscopy demonstrates that DDT rapidly enhanced fragmentation of the gap junction plaques and abolished the gap junction coupling without major modification of Cx43 phosphorylation status. Repeated exposure to DDT resulted in chronic gap junction coupling injury. The present results demonstrate that one of the early effect of DDT is to interfere with the plasma membrane and to perturb its function, specifically its ability to establish cell-cell junctions that are essential for tissue homeostasis and control of cell proliferation and differentiation. Such an alteration may play a specific role during carcinogenesis.     

The connexin43-interacting protein,CIP85, mediates the internalization of connexin43 from the plasma membrane

Abstract

CIP85 was previously identified as a connexin43 (Cx43)-interacting protein that is ubiquitously expressed in multiple mammalian tissues and cell types. The interaction between the SH3 domain of CIP85 and a proline-rich region of Cx43 has previously been associated with an increased rate of Cx43 turnover through lysosomal mechanisms. This report presents biochemical and immunofluorescence evidence that overexpression of CIP85 reduced the presence of Cx43 in gap junction plaques at the plasma membrane. Furthermore, this effect was dependent upon the interaction of CIP85 with Cx43 at the plasma membrane. These results indicate that CIP85 increases Cx43 turnover by accelerating the internalization of Cx43 from the plasma membrane. CIP85 was also observed to interact with clathrin, which suggested a role for CIP85 in the clathrin-mediated internalization of Cx43.     

Immunocytochemical localization of the gap junction 26 K protein in mouse liver plasma membranes

Specific binding sites for anti-26 K antibodies directed against the liver gap junction protein (26 K) were localized by immunoelectron microscopy in gap junction plaques purified from hepatic plasma membranes. Using immunofluorescence microscopy we found discrete fluorescent spots on plasma membranes in cross sections of liver tissues after incubation with anti-26 K antibodies. This is consistent with the notion of specific binding to gap junction plaques. Quantitative binding of anti-26 K antibodies was indirectly measured by the protein A-gold technique. We found that urea/detergent-treated, purified gap junction plaques bind 30-fold more anti-26 K antibodies than preimmune serum. Anti-26 K antibodies also bind specifically to native gap junction plaques within hepatic plasma membranes although only about one fifth as efficiently as to purified plaques. Possibly the anti-26 K antibodies raised after injection of SDS-denatured 26 K protein into rabbits recognize the cytoplasmic face of urea/detergent-treated plaques better than that of native plaques. Some, if not most, of the vesicular structures in preparations of purified plaques appear to be derived from split gap junction plaques and are probably sheets of gap junction hemichannels. In some vesicles the former cytoplasmic face of the hemichannels is turned outside, other vesicles have the former cell surface turned outside. The anti-26 K antibodies do not recognize any 26 K protein on the sheets of partially split gap junction plaques, on the heterogeneous vesicular structures, or on non-junctional areas of hepatic plasma membranes. These results suggest that the conformation of the 26 K protein in plaques must be different from that of the 26 K protein in earlier biosynthetic steps of plaque assembly.     

Dipyridamole-related enhancement of gap junction coupling in the GM-7373 aortic endothelial cells correlates with an increase in the amount of connexin 43 mRNA and protein as well as gap junction plaques

Previous data showed that dipyridamole enhanced gap junction coupling in vascular endothelial and smooth muscle cell lines by a cAMP-dependent mechanism. The present study investigates the level at which dipyridamole affects gap junction coupling. In the GM-7373 endothelial cell line, scrape loading/dye transfer experiments revealed a rapid increase in gap junction coupling induced during the first 6 h of dipyridamole treatment, followed by a slow increase induced by further incubation. Immunostaining analyses showed that the rapid enhancement of gap junction coupling correlated with an increased amount of Cx43 gap junction plaques and a reduced amount of Cx43 containing vesicles, while the amount of Cx43 mRNA or protein was not changed during this period, as found by semiquantitative RT-PCR and Western blot. Additionally, brefeldin A did not block this short-term-induced enhancement of gap junction coupling. Along with the dipyridamole-induced long-term enhancement of gap junction coupling, the amount of Cx43 mRNA and protein additionally to the amount of Cx43 gap junction plaques were increased. Furthermore, the anti-Cx43 antibody detected only two bands at 42 kDa and 44 kDa in control cells and cells treated with dipyridamole for 6 h, while long-term dipyridamole-treated cells showed a third band at 46 kDa. We propose that a dipyridamole-induced cAMP synthesis increased gap junction coupling in the GM-7373 endothelial cell line at different levels: the short-term effect is related to already oligomerised connexins beyond the Golgi apparatus and the long-term effect involves new expression and synthesis as well as posttranslational modification of Cx43.     

A proposed role for ZO-1 in targeting connexin 43 gap junctions to the endocytic pathway

Gap junctions are intercellular channels organized in plaque that directly link adjacent cells. Connexins (Cx), the constitutive proteins of gap junctions are associated with several partner proteins (cytoskeletal, anchoring) which could participate in plaque formation and degradation. Coimmunoprecipitation and indirect immunofluorescence analyses showed that ZO-1, a tight junction-associated protein, was linked to Cx43 in the testis. By using gamma-hexachlorocyclohexane (HCH), known to induce gap junction endocytosis, we demonstrated that endocytosis increased Cx43/ZO-1 association within the cytoplasm of treated Sertoli cells. In control cells, the two proteins were present, as expected, at the plasma membrane level, but poorly colocalized. The increased intracytoplasmic Cx43/ZO-1 complex was associated with a shift towards increased levels of Cx43 P1 and P2 isoforms. The HCH induced Cx43 hyperphosphorylation was abolished by the ERK inhibitor PD98059 suggesting that this effect could be mediated through activation of the ERK pathway. These data strongly support a novel role for ZO-1 in the turnover of Cx43 during gap junction plaque endocytosis.     

Ultrastructural observations of isolated intact and fragmented junctions of skeletal muscle by use of tannic acid mordanting

Tannic acid mordanting during fixation of isolated vesicles from skeletal muscle enhanced the resolution of the images. Isolated triadic junctions displayed two characteristic features not previously described: (a) a clear gap separated terminal cisternae from transverse tubules; (b) this gap was bridged by a separating array of structures which resembled the "feet" of intact muscle. When the triad was broken in a French press and subsequently reassembled by joining the two organelles, a similar gap was seen but the structure of the feet was less well defined. When the membrane of the triad was extracted by Triton X-100, the junctional region was retained and a similar gap between the two organelles could be discerned. The terminal cisternae characteristically displayed a thickening of the cytoplasmic leaflet of the membrane in select areas in which electron-dense material was apposed on the luminal leaflet. This thickened membrane was not observed in longitudinal reticulum or in terminal cisternae regions distal to the electron-dense matter. This thickened leaflet was not invariably associated with the junction, and some junctional regions did not display discernible thickening of the membrane. When the triad was treated with KCl, the electron-dense aggregate was dispersed and the thickened leaflet of the terminal cisternae dissipated, whereas the triadic junctional region with its feet remained unchanged. KCl treatment caused dissolution of three proteins of Mr = 77,000, 43,000, and 38,000. Treatment of Triton-resistant vesicles with KCl caused the loss of electron-dense aggregate but did not otherwise influence the appearance of the junction. A good degree of correlation both qualitatively and in quantitative parameters between the isolated vesicles and the intact muscle was observed.     

A potential novel mechanism involving connexin 43 gap junction for control of sertoli cell proliferation by thyroid hormones

There is strong evidence that thyroid hormones through triiodothyronine (T3) regulate Sertoli cell proliferation and differentiation in the neonatal testis. However, the mechanism(s) by which they are able to control Sertoli cell proliferation is unclear. In the present study in vivo approaches (PTU-induced neonatal hypothyroidism known to affect Sertoli cell proliferation) associated with in vitro experiments on a Sertoli cell line were developed to investigate this question. We demonstrated that the inhibitory effect of T3 on Sertoli cell growth, analyzed by evaluating DNA-incorporated [3H] thymidine, was associated with a time and dose-dependent increase in the levels of Cx43, a constitutive protein of gap junctions, known to participate in the control of cell proliferation and the most predominant Cx in the testis. These Cx43 changes were associated with increased gap junction communication measured by gap FRAP. Consistent with these results two specific inhibitors of gap junction coupling, AGA and oleamide, were able to significantly reverse the T3 inhibitory effect on Sertoli cell proliferation. The present data also revealed a nongenomic effect of T3 on Cx43 Sertoli cells that was evidenced by a rapid up-regulation of gap junction plaque number as identified in Cx43-GFP transfected cells exposed to the hormone. This process appears mediated through actin cytoskeleton since incubation of the cells with cytochalasin D totally reversed the T3 stimulatory effect on Cx43-GFP gap junction plaques. Based on these data, we propose a working hypothesis in which Cx43 could be an intermediate target for T3 inhibition of neonatal Sertoli cell proliferation.     

Dispersed and Aggregated Gap Junction Channels Identified by Immunogold Labeling of Freeze-Fractured Membranes

An indirect immunogold labeling technique was applied to replicas of freeze-fractured membranes of rapidly frozen unfixed cells. The endogenous gap junction protein Cx43 of BICR/M1R_krat mammary tumor cells was preferentially identified in quasi-crystalline gap junction plaques as were the transfected connexins Cx40, Cx43, and Cx45 in HeLa (human cervical carcinoma) cells. With this method we also detected contact areas with dispersed gap junction channels which are the only structural correlation for endogenous Cx45 in HeLa wild-type cells where no gap junction plaques exist. In double-transfected HeLa cells a colocalization of Cx40 and Cx43 was occasionally detected in quasi-crystalline gap junction plaques, whereas in contact areas with dispersed particles only one Cx type was present. Our results indicate that functional gap junction channels exist outside the quasi-crystalline plaques.     

Internalization of large double-membrane intercellular vesicles by a clathrin-dependent endocytic process

Beyond its well-documented role in vesicle endocytosis, clathrin has also been implicated in the internalization of large particles such as viruses, pathogenic bacteria, and even latex beads. We have discovered an additional clathrin-dependent endocytic process that results in the internalization of large, double-membrane vesicles at lateral membranes of cells that are coupled by gap junctions (GJs). GJ channels bridge apposing cell membranes to mediate the direct transfer of electrical currents and signaling molecules from cell to cell. Here, we report that entire GJ plaques, clusters of GJ channels, can be internalized to form large, double-membrane vesicles previously termed annular gap junctions (AGJs). These internalized AGJ vesicles subdivide into smaller vesicles that are degraded by endo/lysosomal pathways. Mechanistic analyses revealed that clathrin-dependent endocytosis machinery-components, including clathrin itself, the alternative clathrin-adaptor Dab2, dynamin, myosin-VI, and actin are involved in the internalization, inward movement, and degradation of these large, intercellular double-membrane vesicles. These findings contribute to the understanding of clathrin's numerous emerging functions.     

Prevention of organochlorine-induced inhibition of gap junctional communication by chaetoglobosin K in astrocytes

Innumerable toxic substances present in the environment inhibit gap junctions, intercellular membrane channels that play fundamental roles in coordinated function of cells and tissues. Included are persistent organochlorine compounds, which pose health risks to humans and animals owing to their widespread use, bioaccumulation, and ability to inhibit gap junction channel-mediated intercellular communication in liver, lung, skin, heart, and brain cells. In this study, the organochlorine xenobiotics dieldrin and endosulfan, at micromolar concentrations, were found to inhibit gap junction-mediated intercellular communication and induce hypophosphorylation of connexin 43 in cultured rat astrocytes, the predominant cell type in the brain coupled through gap junctions. This inhibition of gap junctional communication was substantially reduced by preincubation with chaetoglobosin K (ChK), a bioactive natural produce previously shown to have ras tumor suppressor activity. Chaetoglobosin K also prevented dieldrin and endosulfan-induced hypophosphorylation of connexin 43 and prevented dieldrin-induced connexin 43 plaque dissolution in both astrocytes and cultured liver epithelial cells. The results suggest that stabilization of the native, phosphorylated form of connexin 43 by ChK may contribute to its ability to prevent organochlorine-induced inhibition of gap junction-mediated communication and dissolution of gap junction plaques within the plasma membrane. This revised version was published online in July 2006 with corrections to the Cover Date.     

Gap Junction Turnover Is Achieved by the Internalization of Small Endocytic Double-Membrane Vesicles

Double-membrane–spanning gap junction (GJ) channels cluster into two-dimensional arrays, termed plaques, to provide direct cell-to-cell communication. GJ plaques often contain circular, channel-free domains (∼0.05–0.5 μm in diameter) identified >30 y ago and termed nonjunctional membrane (NM) domains. We show, by expressing the GJ protein connexin43 (Cx43) tagged with green fluorescent protein, or the novel photoconvertible fluorescent protein Dendra2, that NM domains appear to be remnants generated by the internalization of small GJ channel clusters that bud over time from central plaque areas. Channel clusters internalized within seconds forming endocytic double-membrane GJ vesicles (∼0.18–0.27 μm in diameter) that were degraded by lysosomal pathways. Surprisingly, NM domains were not repopulated by surrounding channels and instead remained mobile, fused with each other, and were expelled at plaque edges. Quantification of internalized, photoconverted Cx43-Dendra2 vesicles indicated a GJ half-life of 2.6 h that falls within the estimated half-life of 1–5 h reported for GJs. Together with previous publications that revealed continuous accrual of newly synthesized channels along plaque edges and simultaneous removal of channels from plaque centers, our data suggest how the known dynamic channel replenishment of functional GJ plaques can be achieved. Our observations may have implications for the process of endocytic vesicle budding in general.