Matthias M. Falk

Dynamic trafficking and delivery of connexons to the plasma membrane and accretion to gap junctions in living cells

Certain membrane channels including acetylcholine receptors, gap junction (GJ) channels, and aquaporins arrange into large clusters in the plasma membrane (PM). However, how these channels are recruited to the clusters is unknown. To address this question, we have investigated delivery of GJ channel subunits (connexons) assembled from green fluorescent protein (GFP)-tagged connexin 43 (Cx43) to the PM and GJs in living cells. Fluorescence-photobleaching of distinct areas of Cx43-GFP GJs demonstrated that newly synthesized channels were accrued to the outer margins of channel clusters. Time-lapse microscopy further revealed that connexons were delivered in vesicular carriers traveling along microtubules from the Golgi to the PM. Routing and insertion of connexons occurred predominantly into the nonjunctional PM. These PM connexons can move laterally as shown by photo-bleaching and thus, can reach the margins of channel clusters. There, the apposing PMs are close enough to allow connexons to dock into complete GJ channels. When connexon delivery to the PM was inhibited by brefeldin A, or nocodazole pretreatment, the PM pool initially enabled connexon accrual to the clusters but further accrual was inhibited upon depletion. Taken together, our results indicate that GJ channel clusters grow by accretion at their outer margins from connexon subunits that were delivered to the nonjunctional PM, and explain how connexons in the PM can function in intra-/extracellular signaling before GJ channel formation and direct cell–cell communication.

Gap junction protein connexin-43 interacts directly with microtubules

Gap junctions are specialized cell-cell junctions that mediate intercellular communication. They are composed of connexin proteins, which form transmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating by growth factors [3-5]. The Cx43 tail contains various protein interaction sites, but little is known about binding partners. To identify Cx43-interacting proteins, we performed pull-down experiments using the C-terminal tail of Cx43 fused to glutathione-S-transferase. We find that the Cx43 tail binds directly to tubulin and, like full-length Cx43, sediments with microtubules. Tubulin binding to Cx43 is specific in that it is not observed with three other connexins. We established that a 35-amino acid juxtamembrane region in the Cx43 tail, which contains a presumptive tubulin binding motif, is necessary and sufficient for microtubule binding. Immunofluorescence and immunoelectron microscopy studies reveal that microtubules extend to Cx43-based gap junctions in contacted cells. However, intact microtubules are dispensable for the regulation of Cx43 gap-junctional communication. Our findings suggest that, in addition to its well-established role as a channel-forming protein, Cx43 can anchor microtubule distal ends to gap junctions and thereby might influence the properties of microtubules in contacted cells.

Cell‐free synthesis and assembly of connexins into functional gap junction membrane channels

Several different gap junction channel subunit isotypes, known as connexins, were synthesized in a cell‐free translation system supplemented with microsomal membranes to study the mechanisms involved in gap junction channel assembly. Previous results indicated that the connexins were synthesized as membrane proteins with their relevant transmembrane topology. An integrated biochemical and biophysical analysis indicated that the connexins assembled specifically with other connexin subunits. No interactions were detected between connexin subunits and other co‐translated transmembrane proteins. The connexins that were integrated into microsomal vesicles assembled into homo‐ and hetero‐oligomeric structures with hydrodynamic properties of a 9S particle, consistent with the properties reported for hexameric gap junction connexons derived from gap junctions in vivo. Further, cell‐free assembled homo‐oligomeric connexons composed of β1 or β2 connexin were reconstituted into synthetic lipid bilayers. Single channel conductances were recorded from these bilayers that were similar to those measured for these connexons produced in vivo. Thus, this is the first direct evidence that the synthesis and assembly of a gap junction connexon can take place in microsomal membranes. Finally, the cell‐free system has been used to investigate the properties of α1, β1 and β2 connexin to assemble into hetero‐oligomers. Evidence has been obtained for a selective interaction between individual connexin isotypes and that a signal determining the potential hetero‐oligomeric combinations of connexin isotypes may be located in the N–terminal sequence of the connexins.

Expression of fluorescently tagged connexins: a novel approach to rescue function of oligomeric DsRed-tagged proteins1

A novel, brilliantly red fluorescent protein, DsRed has become available recently opening up a wide variety of experimental opportunities for double labeling and fluorescence resonance electron transfer experiments in combination with green fluorescent protein (GFP). Unlike in the case of GFP, proteins tagged with DsRed were often found to aggregate within the cell. Here we report a simple method that allows rescuing the function of an oligomeric protein tagged with DsRed. We demonstrate the feasibility of this approach on the subunit proteins of an oligomeric membrane channel, gap junction connexins. Additionally, DsRed fluorescence was easily detected 12–16 h post transfection, much earlier than previously reported, and could readily be differentiated from co‐expressed GFP. Thus, this approach can eliminate the major drawbacks of this highly attractive autofluorescent protein.

Molecular reorganization of Cx43, Zo-1 and Src complexes during the endocytosis of gap junction plaques in response to a non-genomic carcinogen.

The gap junction protein connexin 43 (Cx43) exhibits dynamic trafficking that is altered in most tumor cells and in response to carcinogen exposure. A number of connexin (Cx)-binding proteins are known to be involved in endocytic internalization of gap junctions. Here, we analyzed the discrete molecular interactions that occur between Src, ZO-1 and Cx43 during Cx43 internalization in response to the non-genomic carcinogen γ-hexachlorocyclohexane (HCH). Internalization of the Cx43 gap junction plaque was significantly accelerated in Cx43-GFP transfected 42GPA9 Sertoli cells that were exposed to the carcinogen. HCH induced the rapid recruitment of Src to the plasma membrane, activation of Src within 3 minutes and the efficient inhibition of gap junctional coupling, but had no effect in the presence of the Src inhibitor PP2. Immunoprecipitation experiments demonstrated that HCH increased Cx43-Src interaction and concomitantly decreased Cx43–ZO-1 association. ZO-1 was detected on both sides of the gap junction plaques in untreated cells, but appeared to be mainly localized on one side during HCH-induced internalization. The dissociation of ZO-1 from Cx43 appears to occur specifically on the side of the plaque to which Src was recruited. These findings provide mechanistic evidence by which internalization of the Cx43 gap junction plaque might be initiated, suggesting that Src-mediated dissociation of ZO-1 from one side of the plaque initiates endocytic internalization of gap junctions and that this process is amplified in response to exposure to HCH.

Internalized gap junctions are degraded by autophagy

Direct intercellular communication mediated by gap junctions (GJs) is a hallmark of normal cell and tissue physiology. In addition, GJs significantly contribute to physical cell-cell adhesion. Clearly, these cellular functions require precise modulation. Typically, GJs represent arrays of hundreds to thousands of densely packed channels, each one assembled from two half-channels (connexons), that dock head-on in the extracellular space to form the channel arrays that link neighboring cells together. Interestingly, docked GJ channels cannot be separated into connexons under physiological conditions, posing potential challenges to GJ channel renewal and physical cell-cell separation. We described previously that cells continuously—and effectively after treatment with natural inflammatory mediators—internalize their GJs in an endo-/exocytosis process that utilizes clathrin-mediated endocytosis components, thus enabling these critical cellular functions. GJ internalization generates characteristic cytoplasmic double-membrane vesicles, described and termed earlier annular GJs (AGJs) or connexosomes. Here, using expression of the major fluorescent-tagged GJ protein, connexin 43 (Cx43-GFP/YFP/mApple) in HeLa cells, analysis of endogenously expressed Cx43, ultrastructural analyses, confocal colocalization microscopy, pharmacological and molecular biological RNAi approaches depleting cells of key-autophagic proteins, we provide compelling evidence that GJs, following internalization, are degraded by autophagy. The ubiquitin-binding protein p62/sequestosome 1 was identified in targeting internalized GJs to autophagic degradation. While previous studies identified proteasomal and endo-/lysosomal pathways in Cx43 and GJ degradation, our study provides novel molecular and mechanistic insights into an alternative GJ degradation pathway. Its recent link to health and disease lends additional importance to this GJ degradation mechanism and to autophagy in general.

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 ( approximately 0.05-0.5 mum 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 ( approximately 0.18-0.27 mum 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.

Biosynthesis and structural composition of gap junction intercellular membrane channels

Gap junction channels assemble as dodecameric complexes, in which a hexameric connexon (hemichannel) in one plasma membrane docks end-to-end with a connexon in the membrane of a closely apposed cell to provide direct cell-to-cell communication. Synthesis, assembly, and trafficking of the gap junction channel subunit proteins referred to as connexins, largely appear to follow the general secretory pathway for membrane proteins. The connexin subunits can assemble into homo-, as well as distinct hetero-oligomeric connexons. Assembly appears to be based on specific signals located within the connexin polypeptides. Plaque formation by the clustering of gap junction channels in the plane of the membrane, as well as channel degradation are poorly understood processes that are topics of current research. Recently, we tagged connexins with the autofluorescent reporter green fluorescent protein (GFP), and its cyan (CFP), and yellow (YFP) color variants and combined this reporter technology with single, and dual-color, high resolution deconvolution microscopy, computational volume rendering, and time-lapse microscopy to examine the detailed organization, structural composition, and dynamics of gap junctions in live cells. This technology provided for the first time a realistic, three-dimensional impression of gap junctions as they appear in the plasma membranes of adjoining cells, and revealed an excitingly detailed structural organization of gap junctions never seen before in live cells. Here, I summarize recent progress in areas encompassing the synthesis, assembly and structural composition of gap junctions with a special emphasis on the recent results we obtained using cell-free translation/ membrane-protein translocation, and autofluorescent reporters in combination with live-cell deconvolution microscopy.

Specific amino-acid residues in the N-terminus and TM3 implicated in channel function and oligomerization compatibility of connexin43

To identify signals that convey connexin oligomerization compatibility, we have aligned amino-acid sequences of α and β group connexins (Cx) and compared the physico-chemical properties of each homologous amino-acid residue. Four positions were identified that consistently differed betweenα and β-type connexins; two are located in the N-terminal domain (P1 and P2, corresponding to residues 12 and 13 of the Cx43 sequence), and two in the third trans-membrane-spanning domain TM3 (P3 and P4, corresponding to residues 152 and 153 of the Cx43 sequence). Replacement of each of these residues in Cx43 (an α-type connexin) with the corresponding residues of Cx32 (a β-type connexin) resulted in the assembly of all variants into gap junctions; however, only the P4 variant was functional, as indicated by lucifer yellow dye transfer assays. The other three variants exerted a moderate to severe dose-dependent, dominant-negative effect on co-expressed wild-type (wt) Cx43 channel activity. Moreover, a significant dose-dependent, trans-dominant inhibition of channel activity was observed when either one of the N-terminal variants was co-expressed with wt Cx32. Assembly analyses indicated that dominant and trans-dominant inhibitory effects appeared to be based on the oligomerization of wt and variant connexins into mixed connexons. Interestingly, the identified N-terminal amino acids coincide with the position of naturally occurring, disease-causing missense mutations of severalβ -connexin genes (Cx26, Cx30, Cx31, Cx32). Our results demonstrate that three of the identified discriminative amino-acid residues (positions 12, 13 and 152) are crucial for Cx43 channel function and suggest that the N-terminal amino-acid residues at position 12/13 are involved in the oligomerization compatibility of α and β connexins.

Double-membrane gap junction internalization requires the clathrin-mediated endocytic machinery

Direct cell–cell communication mediated by plasma membrane‐spanning gap junction (GJ) channels is vital to all aspects of cellular life. Obviously, GJ intercellular communication (GJIC) requires precise regulation, and it is known that controlled biosynthesis and degradation, and channel opening and closing (gating) are exploited. We discovered that cells internalize GJs in response to various stimuli. Here, we report that GJ internalization is a clathrin‐mediated endocytic process that utilizes the vesicle‐coat protein clathrin, the adaptor proteins adaptor protein complex 2 and disabled 2, and the GTPase dynamin. To our knowledge, we are first to report that the endocytic clathrin machinery can internalize double‐membrane vesicles into cells.