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Cohesins are ATPase complexes that play central roles in cellular processes such as chromosome division, DNA repair, and gene expression. Cohesinopathies arise from mutations in cohesin proteins or cohesin complex regulators and encompass a family of related developmental disorders that present with a range of severe birth defects, affect many different physiological systems, and often lead to embryonic fatality. Treatments for cohesinopathies are limited, in large part due to the lack of understanding of cohesin biology. Thus, characterizing the signaling networks that lie upstream and downstream of cohesin-dependent pathways remains clinically relevant. Here, we highlight alterations in cohesins and cohesin regulators that result in cohesinopathies, with a focus on cardiac defects. In addition, we suggest a novel and more unifying view regarding the mechanisms through which cohesinopathy-based heart defects may arise.

The nanostructure of engineered bioscaffolds has a profound impact on cell response, yet its understanding remains incomplete as cells interact with a highly complex interfacial layer rather than the material itself. For bioactive glass scaffolds, this layer comprises of silica gel, hydroxyapatite (HA)/carbonated hydroxyapatite (CHA), and absorbed proteins—all in varying micro/nano structure, composition, and concentration. Here, we examined the response of MC3T3-E1 pre-osteoblast cells to 30 mol% CaO–70 mol% SiO2 porous bioactive glass monoliths that differed only in nanopore size (6–44 nm) yet resulted in the formation of HA/CHA layers with significantly different microstructures. We report that cell response, as quantified by cell attachment and morphology, does not correlate with nanopore size, nor HA/CHO layer micro/nano morphology, or absorbed protein amount (bovine serum albumin, BSA), but with BSA’s secondary conformation as indicated by its β-sheet/α-helix ratio. Our results suggest that the β-sheet structure in BSA interacts electrostatically with the HA/CHA interfacial layer and activates the RGD sequence of absorbed adhesion proteins, such as fibronectin and vitronectin, thus significantly enhancing the attachment of cells. These findings provide new insight into the interaction of cells with the scaffolds’ interfacial layer, which is vital for the continued development of engineered tissue scaffolds.

Michael Levin,1 Robert C Spiro,2 Himanshu Jain,3 Matthias M Falk4 1Department of Bioengineering, P.C. Rossin College of Engineering & Applied Science, Lehigh University, Bethlehem, PA, 18015, USA; 2Research and Development, Aesculap Implant Systems, LLC, Breinigsville, PA, 18031, USA; 3Department of Materials Science & Engineering, P.C. Rossin College of Engineering & Applied Science, Lehigh University, Bethlehem, PA, 18015, USA; 4Department of Biological Sciences, Lehigh University, Bethlehem, PA, 18015, USACorrespondence: Himanshu Jain; Matthias M Falk, Email H.Jain@lehigh.edu; MFalk@lehigh.eduPurpose: Titanium is commonly used for implants because of its corrosion resistance and osseointegration capability. It is well known that surface topology affects the response of bone tissue towards implants. In vivo studies have shown that in weeks or months, bone tissue bonds more efficiently to titanium implants with rough surfaces compared to smooth surfaces. In addition, stimulating early endosseous integration increases the long-term stability of bone-implants and hence their clinical outcome. Here, we evaluated the response of human MG-63 osteoblast-like cells to flat and solid, compared to rough and porous surface topologies in vitro 1– 6 days post seeding. We compared the morphology, proliferation, and attachment of cells onto three smooth surfaces: tissue culture (TC) plastic or microscope cover glasses, machined polyether-ether-ketone (PEEK), and machined solid titanium, to cells on a highly porous (average Ra 22.94 μm) plasma-sprayed titanium surface (composite Ti-PEEK spine implants).Methods: We used immuno-fluorescence (IF) and scanning electron microscopy (SEM), as well as Live/Dead and WST-1 cell proliferation assays.Results: SEM analyses confirmed the rough topology of the titanium implant surface, compared to the smooth surface of PEEK, solid titanium, TC plastic and cover glasses. In addition, SEM analyses revealed that MG-63 cells seeded onto smooth surfaces (solid titanium, PEEK) adopted a flat, planar morphology, while cells on the rough titanium surface adopted an elongated morphology with numerous filopodial and lamellipodial extensions interacting with the substrate. Finally, IF analyses of focal adhesions (vinculin, focal adhesion kinase), as well as proliferation assays indicate that MG-63 cells adhere less and proliferate at a slower rate on the rough than on a smooth titanium surface.Conclusion: These observations suggest that bone-forming osteoblasts adhere less strongly and proliferate slower on rough compared to smooth titanium surfaces, likely promoting cell differentiation, which is in agreement with other porous implant materials.Keywords: cell adhesion and proliferation, implant surface topology, MG-63 cells, polyether-ether-ketone, PEEK, porous titanium implants, Ti-PEEK

Gap junction proteins, termed connexins (Cx), mediate direct cell-to-cell communication by forming channels that physically couple cells, thereby linking their cytoplasm, permitting exchange of molecules, ions, and electrical impulses. The most ubiquitously expressed gap junction protein, connexin43 (Cx43) has been implicated in cardiovascular diseases including arrhythmias, cardiomyopathies, hypertension and diabetes. The Cx43 C-terminal (CT) domain serves as the regulatory hub of the protein affecting all aspects of gap junction function. Here, deletion within the Cx43 CT (amino acids 256-289), a region known to encode key residues regulating gap junction turnover is employed to examine the effects of dysregulated Cx43 gap junction endocytosis using cultured cells (Cx43Δ256-289) and zebrafish model (cx43lh10). We report that this CT deletion causes defective gap junction endocytosis as well as increased gap junction intercellular communication (GJIC). Increased Cx43 protein content in cx43lh10zebrafish, specifically in the cardiac tissue, larger gap junction plaques and longer Cx43 protein half-lives coincide with severely impaired cardiovascular development. These findings suggest that normal, unimpaired Cx43 gap junction endocytosis and turnover is an essential aspect of gap junction function as demonstrated here for cardiovascular development that when impaired can give rise to arrhythmias, heart malformations and aberrant vasculature structure and function.

The gap junction protein Connexin43 (Cx43) contributes to cell fate decisions that determine the location of fin ray joints during regeneration. Here, we provide insights into how Cx43, expressed medially, influences changes in gene expression in lateral skeletal precursor cells. Using the Gap27 peptide inhibitor specific to Cx43, we show that Cx43-gap junctional intercellular communication (GJIC) influences Cx43-dependent skeletal phenotypes, including segment length. We also demonstrate that Cx43-GJIC influences expression of the Smp/β-catenin pathway in the lateral skeletal precursor cells, and does not influence the Sema3d pathway. Moreover, we show that the cx43 lh10 allele, which has increased Cx43 protein levels, exhibits increased regenerate length and segment length. These phenotypes are rescued by Gap27, suggesting that increased Cx43 is responsible for the observed Cx43 phenotypes. Finally, our findings suggest that inhibition of Cx43 hemichannel activity does not influence Cx43-dependent skeletal phenotypes. These data provide evidence that Cx43-GJIC is responsible for regulating cell fate decisions associated with appropriate joint formation in the regenerating fin.

Introduction/Objective The tailored amorphous multi-porous (TAMP) material fabrication technology has led to a new class of bioactive materials possessing versatile characteristics. It has not been tested for dental applications. Thus, we aimed to assess its biocompatibility and ability to regenerate dental mineral tissue. Methods 30CaO-70SiO2 model TAMP discs were fabricated by a sol-gel method followed by in vitro biocompatibility testing with isolated human or mini-swine dental pulp stem cells (DPSCs). TAMP scaffolds were tested in vivo as a pulp exposure (pin-point, 1 mm, 2 mm, and entire pulp chamber roof) capping material in the molar teeth of mini-swine. Results The in vitro assays showed that DPSCs attached well onto the TAMP discs with comparable viability to those attached to culture plates. Pulp capping tests on mini-swine showed that after 4.5 months TAMP material was still present at the capping site, and mineral tissue (dentin bridge) had formed in all sizes of pulp exposure underneath the TAMP material. Conclusions TAMP calcium silicate is biocompatible with both human and swine DPSCs in vitro and with pulp in vivo, it may help regenerate the dentin bridge after pulp exposure.

A series of hierarchical phosphorylation/dephosphorylation events at serines 373, 365, and 368 (known as the Akt, PKA, and PKC sites) regulate—through ZO-1 binding and its release—connexon docking, channel accrual, and transitioning of peripheral, functional into permanently closed, centrally located GJ channels., To investigate whether connexin phosphorylation regulates the known role of zonula occludens-1 protein (ZO-1) in gap junction (GJ) function, we generated and analyzed a series of phosphomimetic and phosphorylation-dead mutants by mutating known conserved regulatory serine (S) residues 255, 279/282, 365, 368, and 373 located in the C-terminal domain of connexin43 (Cx43) into glutamic acid (E) or alanine (A) residues. All connexin mutants were translated into stable, full-length proteins and assembled into GJs when expressed in HeLa or Madin–Darby canine kidney epithelial cells. However, mutants with S residues exchanged at positions 365, 368, and 373 exhibited a significantly altered ZO-1 interaction profile, while mutants with S residues exchanged at 255 and 279/282 did not. Unlike wild-type Cx43, in which ZO-1 binding is restricted to the periphery of GJ plaques, S365A, S365E, S368A, S368E, and S373A mutants bound ZO-1 throughout the GJ plaques, while the S373E mutant did not bind ZO-1 at all. Inability to disengage from ZO-1 correlated with increased GJ plaque size and increased connexin protein half-life, while maintaining GJ channels in an open, functional state. Quantitative clathrin-binding analyses revealed no significant alterations in clathrin-binding efficiency, suggesting that the inability to disengage from ZO-1 prevented maturation of functional into nonfunctional/endocytic channels, rather than ZO-1 interfering with GJ endocytosis directly. Collectively, our results indicate that ZO-1 binding regulates channel accrual, while disengagement from ZO-1 is critical for GJ channel closure and transitioning GJ channels for endocytosis. Intriguingly, these transitional ZO-1 binding/release and channel-aging steps are mediated by a series of hierarchical phosphorylation/dephosphorylation events at S373, S365, and S368, well-known Cx43 Akt, protein kinase A, and protein kinase C phosphorylation sites located in the vicinity of the ZO-1 binding site.

For many years now, researchers have known of a sensory appendage on the surface of most differentiated cell types called primary cilium. Primary cilia are both chemo- and mechano-sensory in function and have an obvious role in cell cycle control. Because of this, it has been thought that primary cilia are not found on rapidly proliferating cells, for example, cancer cells. Here we report using immunofluorescent staining for the ciliary protein Arl13b that primary cilia are frequently found on HeLa (human epithelial adenocarcinoma) and other cancer cell lines such as MG63 (human osteosarcoma) commonly used for cell culture studies and that the ciliated population is significantly higher (ave. 28.6% and 46.5%, respectively in starved and 15.7-18.6% in un-starved cells) than previously anticipated. Our finding impacts the current perception of primary cilia formed in highly proliferative cells.

Phosphorylation on well-recognized, regulatory connexin43 amino acid residues by a series of kinases serves as an early molecular signal that triggers not only inhibition of gap junction (GJ)–mediated cell-to-cell communication but also GJ internalization. The findings contribute to the newly evolving dynamic picture of GJs., Gap junctions (GJs) exhibit a complex modus of assembly and degradation to maintain balanced intercellular communication (GJIC). Several growth factors, including vascular endothelial growth factor (VEGF), have been reported to disrupt cell–cell junctions and abolish GJIC. VEGF directly stimulates VEGF-receptor tyrosine kinases on endothelial cell surfaces. Exposing primary porcine pulmonary artery endothelial cells (PAECs) to VEGF for 15 min resulted in a rapid and almost complete loss of connexin43 (Cx43) GJs at cell–cell appositions and a concomitant increase in cytoplasmic, vesicular Cx43. After prolonged incubation periods (60 min), Cx43 GJs reformed and intracellular Cx43 were restored to levels observed before treatment. GJ internalization correlated with efficient inhibition of GJIC, up to 2.8-fold increased phosphorylation of Cx43 serine residues 255, 262, 279/282, and 368, and appeared to be clathrin driven. Phosphorylation of serines 255, 262, and 279/282 was mediated by MAPK, whereas serine 368 phosphorylation was mediated by PKC. Pharmacological inhibition of both signaling pathways significantly reduced Cx43 phosphorylation and GJ internalization. Together, our results indicate that growth factors such as VEGF activate a hierarchical kinase program—including PKC and MAPK—that induces GJ internalization via phosphorylation of well-known regulatory amino acid residues located in the Cx43 C-terminal tail.

For hard tissue regeneration, the bioactivity of a material is measured by its ability to induce the formation of hydroxyapatite (HA) under physiological conditions. It depends on the dissolution behavior of the glass, which itself is determined by the composition and structure of glass. The enhanced HA growth on nanoporous than on nonporous glass has been attributed by some to greater specific surface area (SSA), but to nanopore size distribution by others. To decouple the influence of nanopore size and SSA on HA formation, we have successfully fabricated homogeneous 30CaO-70SiO2 (30C70S) model bioactive glass monoliths with different nanopore sizes, yet similar SSA via a combination of sol-gel, solvent exchange, and sintering processes. After incubation in PBS, HA, and Type-B carbonated HA (HA/B-CHA) form on nanoporous monoliths. The XPS, FTIR, and SEM analyses provide the first unambiguous demonstration of the influence of nanopore size alone on the formation pathway, growth rate, and microstructure of HA/CHA. Due to pore-size limited diffusion of PO4 3- , two HA/CHA formation pathways are observed: HA/CHA surface deposition and/or HA/CHA incorporation into nanopores. HA/CHA growth rate on the surface of a nanoporous glass monolith is dominated by the pore-size limited transport of Ca2+ ions dissolved from nanoporous glass substrates. Furthermore, with increasing nanopore size, HA/CHA microstructures evolve from needle-like, plate-like, to flower-like appearance. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 886-899, 2019.

Gap junctions (GJs) assembled from connexin (Cx) proteins play a pivotal role in cell-to-cell communication by forming channels that connect the cytosols of adjacent cells. Connexin 43, the best-studied Cx, is ubiquitously expressed in vertebrates. While phosphorylation is known to regulate multiple aspects of GJ function, much less is known about the role ubiquitination plays in these processes. Here we show by using ubiquitination-type specific antibodies and Cx43 lysine (K) to arginine (R) mutants that a portion of Cx43 in GJs can become K63-polyubiquitinated on K264 and K303. Relevant Cx43 K/R mutants assembled significantly larger GJ plaques, exhibited much longer protein half-lives and were internalization impaired. Interestingly, ubiquitin-deficient Cx43 mutants accumulated as hyper-phosphorylated polypeptides in the plasma membrane, suggesting that K63-polyubiquitination may be triggered by phosphorylation. Phospho-specific Cx43 antibodies revealed that upregulated phosphorylation affected serines 368, 279/282, and 255, well-known regulatory PKC and MAPK phosphorylation sites. Together, these novel findings suggest that upon internalization, some Cx43 in GJs becomes K63-polyubiquitinated, ubiquitination is critical for GJ internalization, and that K63-polyubiquitination may be induced by Cx phosphorylation.Summary StatementHere we show that connexin 43 in gap junctions becomes K63-poly ubiquitinated on lysines 264 and 303 and its requirement for gap junction endocytosis. These novel findings significantly contribute to our understanding of GJ turnover and patho-/physiology.Abbreviations usedAGJannular gap junctionAMSHassociated molecule with the SH3 domain of STAMCMEclathrin-mediated endocytosisCxConnexinCx43Connexin 43DUBdeubiquitinaseGJgap junctionMonoUbmonoubiquitinNedd4-1neural precursor cell expressed developmentally down-regulated protein 4-1PMplasma membranePolyUbpolyubiquitinTPA12-O-Tetradecanoylphorbol 13-AcetateTX-100Triton X-100RTroom temperatureUbubiquitin

We analyzed the biological performance of spinodally and droplet-type phase-separated 45S5 Bioglass® generated by quenching the melt from different equilibrium temperatures. MC3T3-E1 pre-osteoblast cells attached more efficiently to 45S5 Bioglass® with spinodal than to the one with droplet morphology, providing the first demonstration of the role of micro-/nano-scale on the bioactivity of Bioglass®. Upon exposure to biological solutions, phosphate buffered saline (PBS) and cell culture medium (α-MEM), a layer of hydroxyapatite (HA) formed on both glass morphologies. Although both Bioglass® varieties were incubated under identical conditions, and physico-chemical characteristics of the HA layers were similar, the adsorption magnitude of a model protein, bovine serum albumin (BSA, an abundant blood serum component) and its β-sheet/β-turn ratio and α-helix content were significantly higher on spinodal than droplet type Bioglass®. These results indicate that: (i) a protein layer quickly adsorbs on the surface of 45S5 Bioglass® varieties (with or without HA layer), (ii) the amount and the conformation of adsorbed proteins are guided by the glass micro-/nano-structure, and (iii) cell attachment and proliferation are influenced by the concentration and the conformation of attached proteins with a significantly better cell adhesion to spinodal type 45S5 Bioglass® substrate. Taken together, our results indicate that the biological performance of 45S5 Bioglass® can be improved further with a relatively simple, inexpensive fabrication procedure that provides a superior glass micro-/nano-structure. A simple modification to the fabrication procedure of classic 45S5 Bioglass® generates spinodal (A(a)) and droplet (A(b)) varieties and has a significant impact on protein adsorption (B) and cell adhesion (C).

To facilitate hard tissue regeneration there is a pressing need to develop biocompatible, macroporous (pore size ∼100's of µm) scaffolds that are tailored ideally to suit individual patient needs. A critical requirement of this goal is that the scaffolds degrade in the body at a rate that is comparable to that of the tissue re-growth. Among various potential materials, calcium silicate glasses are the most promising model bioactive candidates that also stimulate bone formation, but they degrade too slowly. A successful strategy to accelerate degradation is to increase the specific surface area by superimposing nanoporosity (≈ 10 nm) on macroporosity. In such a bioscaffold, nano- and macroporosities are optimized to meet the needs of a specific patient, so the desired product is a tailored amorphous multi-porous (TAMP) silicate structure. As an added benefit, nanoporosity allows improved diffusion of nutrients and waste products in and out of the scaffolds, further improving its biocompatibility. Here we present an overview of its fabrication process, biological performance and future prospects for use in patients. The TAMP bioscaffolds are fabricated by a modified sol–gel process, which is based on spinodal phase separation induced by a water-soluble polymer. Under quasi-dynamic conditions the model 30CaO–70SiO2 composition degrades in simulated body fluid within weeks depending on nanoporosity. Additionally, in vitro cell tests indicate that nanoscale topology significantly affects the biological performance of TAMP scaffolds. In vivo tests in animals demonstrate superior regeneration of hard, as well as soft, tissue depending on TAMP implant site, and preliminary results of a clinical trial provide a promising outlook for this class of bioscaffold.

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.

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.

Recently, nano-macro dual-porous, three-dimensional (3D) glass structures were developed for use as bioscaffolds for hard tissue regeneration, but there have been concerns regarding the interconnectivity and homogeneity of nanopores in the scaffolds, as well as the cytotoxicity of the environment deep inside due to limited fluid access. Therefore, mercury porosimetry, nitrogen absorption, and TEM have been used to characterize nanopore network of the scaffolds. In parallel, viability of MG 63 human osteosarcoma cells seeded on scaffold surface was investigated by fluorescence, confocal and electron microscopy methods. The results show that cells attach, migrate and penetrate inside the glass scaffold with high proliferation and viability rate. Additionally, scaffolds were implanted under the skin of a male New Zealand rabbit for in vivo animal test. Initial observations show the formation of new tissue with blood vessels and collagen fibers deep inside the implanted scaffolds with no obvious inflammatory reaction. Thus, the new nano-macro dual-porous glass structure could be a promising bioscaffold for use in regenerative medicine and tissue engineering for bone regeneration.

E-cadherin and N-cadherin affect the assembly of connexin43 into gap junctions differentially. N-cadherin disrupts assembly by triggering endocytosis of connexin43, whereas E-cadherin facilitates the assembly., Cadherins have been thought to facilitate the assembly of connexins (Cxs) into gap junctions (GJs) by enhancing cell–cell contact, however the molecular mechanisms involved in this process have remained unexplored. We examined the assembly of GJs composed of Cx43 in isogenic clones derived from immortalized and nontransformed rat liver epithelial cells that expressed either epithelial cadherin (E-Cad), which curbs the malignant behavior of tumor cells, or neuronal cadherin (N-Cad), which augments the invasive and motile behavior of tumor cells. We found that N-cad expression attenuated the assembly of Cx43 into GJs, whereas E-Cad expression facilitated the assembly. The expression of N-Cad inhibited GJ assembly by causing endocytosis of Cx43 via a nonclathrin-dependent pathway. Knock down of N-Cad by ShRNA restored GJ assembly. When both cadherins were simultaneously expressed in the same cell type, GJ assembly and disassembly occurred concurrently. Our findings demonstrate that E-Cad and N-Cad have opposite effects on the assembly of Cx43 into GJs in rat liver epithelial cells. These findings imply that GJ assembly and disassembly are the down-stream targets of the signaling initiated by E-Cad and N-Cad, respectively, and may provide one possible explanation for the disparate role played by these cadherins in regulating cell motility and invasion during tumor progression and invasion.

We report on the preparation of a bioactive CaO–SiO2 monolithic scaffold with interconnected bimodal nanomacro porosity, which simulates the morphology of a natural trabecular bone, by a newly developed modified sol-gel process. This method inherently creates nanopores, whose average diameter can be tailored to approximately 5–20 nm by solvent exchange. To achieve interconnected macroporosity (pores ∼5–300 μm in size), a polymer [poly(ethylene oxide)] is added, which causes phase separation simultaneously with the sol-gel transition. High-resolution scanning electron microscopy and mercury intrusion porosimetry demonstrate a high degree of three-dimensional interconnectivity and sharp distributions of pore size. In vitro bioactivity tests in simulated body fluid (SBF) show bioactivity of the material after soaking for approximately 5 h, as verified by the formation of a hydroxyapatite layer deep into the scaffold structure. Analysis of the SBF after the reaction indicates the dissolution of the samples, another desired feature of temporary scaffolds for bone regeneration. MG63 osteoblast-like cells seeded on our sol-gel glass samples responded better to samples with nanopores enlarged by a solvent exchange process than to the one with normal nanopores. Thus, the benefits of the high surface area achieved by sol-gel and solvent exchange procedures are most clearly demonstrated for the first time.

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.

Gap junctions, composed of connexin proteins in chordates, are the most ubiquitous form of intercellular communication. Complete connexin gene families have been identified from human (20) and mouse (19), revealing significant diversity in gap junction channels. We searched current databases and identified 37 putative zebrafish connexin genes, almost twice the number found in mammals. Phylogenetic comparison of entire connexin gene families from human, mouse, and zebrafish revealed 23 zebrafish relatives of 16 mammalian connexins, and 14 connexins apparently unique to zebrafish. We found evidence for duplication events in all genomes, as well as evidence for recent tandem duplication events in the zebrafish, indicating that the complexity of the connexin family is growing. The identification of a third complete connexin gene family provides novel insight into the evolution of connexins, and sheds light into the phenotypic evolution of intercellular communication via gap junctions.

Gap junctions (GJs) are the only known cellular structures that allow a direct transfer of signaling molecules from cell-to-cell by forming hydrophilic channels that bridge the opposing membranes of neighboring cells. The crucial role of GJ-mediated intercellular communication (GJIC) for coordination of development, tissue function, and cell homeostasis is now well documented. In addition, recent findings have fueled the novel concepts that connexins, although redundant, have unique and specific functions, that GJIC may play a significant role in unstable, transient cell–cell contacts, and that GJ hemi-channels by themselves may function in intra-/extracellular signaling. Assembly of these channels is a complicated, highly regulated process that includes biosynthesis of the connexin subunit proteins on endoplasmic reticulum membranes, oligomerization of compatible subunits into hexameric hemi-channels (connexons), delivery of the connexons to the plasma membrane, head-on docking of compatible connexons in the extracellular space at distinct locations, arrangement of channels into dynamic, spatially and temporally organized GJ channel aggregates (so-called plaques), and coordinated removal of channels into the cytoplasm followed by their degradation. Here we review the current knowledge of the processes that lead to GJ biosynthesis and degradation, draw comparisons to other membrane proteins, highlight novel findings, point out contradictory observations, and provide some provocative suggestive solutions.

To identify signals that convey connexin oligomerization compatibility, we have aligned amino-acid sequences of alpha and beta group connexins (Cx) and compared the physico-chemical properties of each homologous amino-acid residue. Four positions were identified that consistently differed between alpha and beta-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 alpha-type connexin) with the corresponding residues of Cx32 (a beta-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 several beta-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 alpha and beta connexins.

Tissue regeneration is a significantly improved alternative to tissue replacement by implants. It requires porous bioscaffolds for the restoration of natural tissue rather than relying on bio-inactive, often metallic implants. Recently, we developed technology for fabricating novel, nano-macroporous bioactive 'tailored amorphous multi-porous (TAMP)' hard tissue scaffolds using a 70 mol% SiO2-30 mol% CaO model composition. The TAMP silicate scaffolds, fabricated by a modified sol-gel process, have shown excellent biocompatibility via the rapid formation of hydroxyapatite in biological fluids as well as in early tests with bone forming cells. Here we report an in depth investigation of the response of MC3T3-E1 pre-osteoblast cells and bone marrow derived (BMD) osteoclasts to these TAMP scaffolds. Light and electron microscopic imaging, gene and protein expression, and enzyme activity analyses demonstrate that MC3T3-E1 pre-osteoblasts adhere, proliferate, colonize, and differentiate on and inside the bioactive TAMP scaffolds. Additionally, BMD precursor cells mature into active osteoclasts and remodel the scaffold, highlighting the exceptional qualities of this novel scaffold material for bone tissue regeneration.

Four principal categories of cell-cell junctions connect cells in vertebrates and form the basis for shaping distinct tissues and organs. Gap junctions (GJs), one of the four junction types, provide direct cell-to-cell communication by mediating passive diffusion of small hydrophilic signaling molecules between neighboring cells. Gap junction mediated intercellular communication (GJIC) has been shown to play a crucial role for all aspects of multicellular life, including embryonic development, tissue function, and cellular homeostasis; and mutations in the GJ forming proteins, connexins (Cxs), have been linked to severe human diseases that include inherited and sporadic nonsyndromic hearing loss, neuropathies, eye lens cataracts, cardiac diseases, craniofacial malformations, and a number of acute skin disorders. Clearly, biosynthesis and degradation significantly contribute to GJ function and need to be controlled precisely. We have previously shown that GJs are removed from the plasma membrane via the internalization of entire GJ plaques (or portions thereof) in a cellular process that resembles clathrin-mediated endocytosis. GJ endocytosis results in the formation of double-membrane vesicles (termed annular gap junctions [AGJs] or connexosomes) in the cytoplasm of one of the coupled cells. A set of recent independent studies consistent with earlier ultrastructural analyses demonstrate the degradation of endocytosed AGJs by autophagy. Some other reports, however, describe AGJ degradation by endo-/lysosomal pathways in cells that were treated with TPA. Here, I summarize evidence that supports the concept that autophagy serves as the principal cellular degradation pathway for internalized GJs under physiological and pathological conditions.

The availability of green fluorescent protein (GFP) as a tracer for observing proteins in living cells has revolutionized cell biology and spurred an intensive search for GFP variants with novel characteristics, additional autofluorescent proteins and alternative techniques of protein labelling. Two recent studies – one on tagging with tetracysteine motifs and labelling with biarsenic fluorophores of different colours, and the other on GFP tagging and fluorescence recovery after photobleaching (FRAP) – show how membrane channels are added and removed from gap junctions by using different fluorescent tags to distinguish between newly synthesized and older protein populations.

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.

High-resolution, fluorescence deconvolution (DV) microscopy was implemented to obtain a detailed view of the organization and structural composition of gap junctions assembled from one or two different connexin isotypes in live and fixed cells. To visualize gap junctions, the structural protein components of gap junction channels, the connexin polypeptides alpha1(Cx43), beta1(Cx32), and beta2(Cx26), were tagged on their C-termini with the autofluorescent tracers green fluorescent protein (GFP), and its cyan (CFP), and yellow (YFP) color variants. Tagged connexins were expressed in transiently transfected HeLa cells. Comprehensive analysis including dye-transfer analysis demonstrated that the tagged connexins trafficked, assembled, and packed normally into functional gap junction channel plaques. Such gap junction plaques were examined by single, dual, and triple-color DV microscopy. High-resolution images and three-dimensional volume reconstructions of gap junction plaques were obtained by this technique, which revealed several new aspects of gap junction structure. Specifically, the studies demonstrated that the mode of channel distribution strictly depends on the connexin isotypes. Here we present such images, and volume reconstructions in context with images obtained by other light, and electron microscopic techniques, such as laser scanning confocal, conventional wide-field fluorescence, thin section, and freeze-fracture electron microscopy. In addition, we give a simple description of the principal mechanisms of DV microscopy, name advantages and disadvantages, and discuss issues such as dual-color imaging using CFP and YFP, spatial resolution, colocalization, and avoiding imaging artifacts.

To study the organization of gap junctions in living cells, the connexin isotypes alpha(1)(Cx43), beta(1)(Cx32) and beta(2)(Cx26) were tagged with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. The cellular fate of the tagged connexins was followed by high-resolution fluorescence deconvolution microscopy and time-lapse imaging. Comprehensive analyses demonstrated that the tagged channels were functional as monitored by dye transfer, even under conditions where the channels were assembled solely from tagged connexins. High-resolution images revealed a detailed structural organization, and volume reconstructions provided a three-dimensional view of entire gap junction plaques. Specifically, deconvolved dual-color images of gap junction plaques assembled from CFP- and YFP-tagged connexins revealed that different connexin isotypes gathered within the same plaques. Connexins either codistributed homogeneously throughout the plaque, or each connexin isotype segregated into well-separated domains. The studies demonstrate that the mode of channel distribution strictly depends on the connexin isotypes. Based on previous studies on the synthesis and assembly of connexins I suggest that channel distribution is regulated by intrinsic connexin isotype specific signals.

Summary 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.

For gap junction channels to function, their subunit proteins, referred to as connexins, have to be synthesized and inserted into the cell membrane in their native configuration. Like other transmembrane proteins, connexins are synthesized and inserted cotranslationally into the endoplasmic reticulum membrane. Membrane insertion is followed by their assembly and transport to the plasma membrane. Finally, the end-to-end pairing of two half-channels, referred to as connexons, each provided by one of two neighboring cells, and clustering of the channels into larger plaques complete the gap junction channel formation. Gap junction channel formation is further complicated by the potential assembly of homo- as well as heterooligomeric connexons, and the pairing of identical or different connexons into homo- and heterotypic gap junction channels. In this article, I describe the cell-free synthesis approach that we have used to study the biosynthesis of connexins and gap junction channels. Special emphasis is placed on the synthesis of full-length, membrane-integrated connexins, assembly into gap junction connexons, homo- as well as heterooligomerization, and characterization of connexin-specific assembly signals.

Gap junctions (GJs) traverse apposing membranes of neighboring cells to mediate intercellular communication by passive diffusion of signaling molecules. We have shown previously that cells endocytose GJs utilizing the clathrin machinery. Endocytosis generates cytoplasmic double-membrane vesicles termed annular gap junctions or connexosomes. However, the signaling pathways and protein modifications that trigger GJ endocytosis are largely unknown. Treating mouse embryonic stem cell colonies – endogenously expressing the GJ protein connexin43 (Cx43) – with epidermal growth factor (EGF) inhibited intercellular communication by 64% and activated both, MAPK and PKC signaling cascades to phosphorylate Cx43 on serines 262, 279/282, and 368. Upon EGF treatment Cx43 phosphorylation transiently increased up to 4-fold and induced efficient (66.4%) GJ endocytosis as evidenced by a 5.9-fold increase in Cx43/clathrin co-precipitation.

Gap junctions (GJs) are the only known cellular structures that allow a direct cell-to-cell transfer of signaling molecules by forming densely packed arrays or “plaques” of hydrophilic channels that bridge the apposing membranes of neighboring cells. The crucial role of GJ-mediated intercellular communication (GJIC) for all aspects of multicellular life, including coordination of development, tissue function, and cell homeostasis, has been well documented. Assembly and degradation of these membrane channels is a complex process that includes biosynthesis of the connexin (Cx) subunit proteins (innexins in invertebrates) on endoplasmic reticulum (ER) membranes, oligomerization of compatible subunits into hexameric hemichannels (connexons), delivery of the connexons to the plasma membrane (PM), head-on docking of compatible connexons in the extracellular space at distinct locations, arrangement of channels into dynamic spatially and temporally organized GJ channel plaques, as well as internalization of GJs into the cytoplasm followed by their degradation. Clearly, precise modulation of GJIC, biosynthesis, and degradation are crucial for accurate function, and much research currently addresses how these fundamental processes are regulated. Here, we review posttranslational protein modifications (e.g., phosphorylation and ubiquitination) and the binding of protein partners (e.g., the scaffolding protein ZO-1) known to regulate GJ biosynthesis, internalization, and degradation. We also look closely at the atomic resolution structure of a GJ channel, since the structure harbors vital cues relevant to GJ biosynthesis and turnover.

Nanoporosity is known to impact the performance of implants and scaffolds such as bioactive glass (BG) scaffolds, either by providing a higher concentration of bioactive chemical species from enhanced surface area, or due to inherent nanoscale topology, or both. To delineate the role of these two characteristics, BG scaffolds have been fabricated with nearly identical surface area (81 and 83±2 m(2)/g) but significantly different pore size (av. 3.7 and 17.7 nm) by varying both the sintering temperature and the ammonia concentration during the solvent exchange phase of the sol-gel fabrication process. In vitro tests performed with MC3T3-E1 preosteoblast cells on such scaffolds show that initial cell attachment is increased on samples with the smaller nanopore size, providing the first direct evidence of the influence of nanopore topography on cell response to a bioactive structure. Furthermore, in vivo animal tests in New Zealand rabbits (subcutaneous implantation) indicate that nanopores promote colonization and cell penetration into these scaffolds, further demonstrating the favorable effects of nanopores in tissue-engineering-relevant BG scaffolds.

Gap junctions (GJs) are composed of tens to many thousands of double-membrane spanning GJ channels that cluster together to form densely packed channel arrays (termed GJ plaques) in apposing plasma membranes of neighboring cells. In addition to providing direct intercellular communication (GJIC, their hallmark function), GJs, based on their characteristic double-membrane-spanning configuration, likely also significantly contribute to physical cell-to-cell adhesion. Clearly, modulation (up-/down-regulation) of GJIC and of physical cell-to-cell adhesion is as vitally important as the basic ability of GJ formation itself. Others and we have previously described that GJs can be removed from the plasma membrane via the internalization of entire GJ plaques (or portions thereof) in a cellular process that resembles clathrin-mediated endocytosis. GJ endocytosis results in the formation of double-membrane vesicles [termed annular gap junctions (AGJs) or connexosomes] in the cytoplasm of one of the coupled cells. Four recent independent studies, consistent with earlier ultrastructural analyses, demonstrate the degradation of endocytosed AGJ vesicles via autophagy. However, in TPA-treated cells others report degradation of AGJs via the endo-/lysosomal degradation pathway. Here we summarize evidence that supports the concept that autophagy serves as the cellular default pathway for the degradation of internalized GJs. Furthermore, we highlight and discuss structural criteria that seem required for an alternate degradation via the endo-/lysosomal pathway.

One of the four principal categories of cell-cell junctions that hold together and shape distinct tissues and organs in vertebrates, adherens junctions (AJs) form cell-cell contacts that connect transmembrane proteins with cytoskeletal actin filaments to provide architectural strength, aid in morphogenesis, and help to maintain proper tissue homeostasis. The classical organization of AJs, consisting of transmembrane cadherins and cytoplasmically attached β-catenins and α-catenins assembled together into a multiprotein complex, was once thought obligatory to craft a robust and stable connection to actin-based cytoskeletal elements, but this architecture has since been challenged and questioned to exist. In a stimulating paper published in a recent issue of BMC Biology, Millán et al. provide convincing evidence that in confluent vascular endothelial cells a novel dynamic vascular endothelial (VE)-cadherin-based AJ type exists that interacts with and physically connects prominent bundles of tension-mediating actin filaments, stress fibers, between neighboring cells. Stress fibers were known previously to link to integrin-based focal adhesion complexes but not to cell-cell adhesion mediating AJs. These new findings, together with previous results support the concept that different AJ subtypes, sharing the same transmembrane cadherin types, can assemble in various configurations to either increase barrier function and promote physical cell-cell adhesion, or to lessen cell-cell adhesion and promote cell separation and migration.

Sol–gel-derived SiO2–CaO–P2O5 porous glass monoliths with a dual hierarchical pore structure including both macropores of ∼20–200 micrometers and mesopores ∼5–20 nanometers in size are prepared in the presence of the drying control chemical additive formamide, for a possible application as scaffolds in bone tissue regeneration. While the mesopores are intrinsic to the sol–gel processing, the interconnected macropores are achieved through a polymer-induced phase separation together with the sol–gel transition, by adding a water-soluble polymer, poly(ethylene oxide), to the precursor sol. The textural nanopore structure is controlled through solvent exchange procedures and the addition of urea. The overall pore size distribution obtained by mercury intrusion porosimetry was found to shift to larger pore sizes when formamide is added. In vitro tests are used to evaluate the bioactivity. The cell-support function of the resultant scaffolds is also assessed in vitro using osteoblast-like cells cultured for 2 days. The results show that the scaffold has a significant bioactivity and a good ability to support the attachment of MC3T3 preosteoblast cells.

Background Green fluorescent protein (GFP) and other FP fusions have been extensively utilized to track protein dynamics in living cells. Recently, development of photoactivatable, photoswitchable and photoconvertible fluorescent proteins (PAFPs) has made it possible to investigate the fate of discrete subpopulations of tagged proteins. Initial limitations to their use (due to their tetrameric nature) were overcome when monomeric variants, such as Dendra, mEos, and mKikGR were cloned/engineered. Results Here, we report that by closing the field diaphragm, selective, precise and irreversible green-to-red photoconversion (330-380 nm illumination) of discrete subcellular protein pools was achieved on a wide-field fluorescence microscope equipped with standard DAPI, Fluorescein, and Rhodamine filter sets and mercury arc illumination within 5-10 seconds. Use of a DAPI-filter cube with long-pass emission filter (LP420) allowed the observation and control of the photoconversion process in real time. Following photoconversion, living cells were imaged for up to 5 hours often without detectable phototoxicity or photobleaching. Conclusions We demonstrate the practicability of this technique using Dendra2 and mEos2 as monomeric, photoconvertible PAFP representatives fused to proteins with low (histone H2B), medium (gap junction channel protein connexin 43), and high (α-tubulin; clathrin light chain) dynamic cellular mobility as examples. Comparable efficient, irreversible green-to-red photoconversion of selected portions of cell nuclei, gap junctions, microtubules and clathrin-coated vesicles was achieved. Tracking over time allowed elucidation of the dynamic live-cycle of these subcellular structures. The advantage of this technique is that it can be performed on a standard, relatively inexpensive wide-field fluorescence microscope with mercury arc illumination. Together with previously described laser scanning confocal microscope-based photoconversion methods, this technique promises to further increase the general usability of photoconvertible PAFPs to track the dynamic movement of cells and proteins over time.

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) identified30 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.

Bioglass 45S is a promising bone implant material with superior biocompatibility. Past research showed that adhesion of bone cells to titanium is strongly affected by its surface architecture. However, little is known about the role of surface topology of glass on its use as an implant. Thus, we systematically investigated the effect of surface roughness (Ra ∼ 0.01 – 1.2 μm) on cell adhesion and proliferation on 45S Bioglass in vitro. MG63 osteosarcoma and MC3T3 osteoblast precursor cells were seeded on the glass samples, and incubated for up to 6 days. The attachment, morphology and proliferation of cells were investigated using fluorescence microscopy. Our results show that cell attachment (as indicated by cell spreading and number of focal adhesion sites), and proliferation rate decrease with increasing roughness of bioactive glass surface. These findings provide important insight for improving surface characteristics of bioactive glass bone implants.

In foot-and-mouth disease virus (FMDV)-infected cells, the disappearance of nuclear protein histone H3 and the simultaneous appearance of a new chromatin-associated protein termed Pi can be observed (P. R. Grigera and S. G. Tisminetzky, Virology 136:10-19, 1984). We sequenced the amino terminus of protein Pi and showed that Pi derives from histone H3 by proteolytic cleavage. The 20 N-terminal amino acid residues of histone H3 are specifically cleaved off early during infection. Truncated histone H3 remains chromatin associated. In addition, we showed that the histone H3-Pi transition is catalyzed by the FMDV 3C protease. The only known function of the viral 3C protease was, until now, the processing of the viral polyprotein. The viral 3C protease is the only FMDV protein required to induce the histone H3-Pi transition, as well as being the only viral protein capable of cleaving histone H3. No viral precursor fusion protein is needed for this specific cleavage as was reported for the processing of the poliovirus P1 precursor polyprotein by 3C/D protease. As the deleted part of the histone H3 corresponds to the presumed regulatory domain involved in the regulation of transcriptionally active chromatin in eucaryotes, it seems possible that this specific cleavage of histone H3 is related to the host cell transcription shutoff reported for several picornaviruses.

Gap junction channels may be comprised of either connexin or pannexin proteins (innexins and pannexins). Membrane topologies of both families are similar, but sequence similarity is lacking. Recently, connexin-like sequences have been identified in mammalian and zebrafish genomes that have only four conserved cysteines in the extracellular domains (Cx23), a feature of the pannexins. Phylogenetic analyses of the non-canonical “C4” connexins reveal that these sequences are indeed connexins. Functional assays reveal that the Cx23 gap junctions are capable of sharing neurobiotin, and further, that Cx23 connexins form hemichannels in vitro.