Connexin 43 gap junctional intercellular communication inhibits evx1 expression and joint formation in regenerating fins.

The gap junction protein Connexin 43 (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 the expression of the Smp/ß-catenin pathway in the lateral skeletal precursor cells, and does not influence the Sema3d pathway. Moreover, we show that the cx43lh10 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.

Endocytosis and Endocytic Motifs across the Connexin Gene Family.

Proteins fated to be internalized by clathrin-mediated endocytosis require an endocytic motif, where AP-2 or another adaptor protein can bind and recruit clathrin. Tyrosine and di-leucine-based sorting signals are such canonical motifs. Connexin 43 (Cx43) has three canonical tyrosine-based endocytic motifs, two of which have been previously shown to recruit clathrin and mediate its endocytosis. In addition, di-leucine-based motifs have been characterized in the Cx32 C-terminal domain and shown to mediate its endocytosis. Here, we examined the amino acid sequences of all 21 human connexins to identify endocytic motifs across the connexin gene family. We find that although there is limited conservation of endocytic motifs between connexins, 14 of the 21 human connexins contain one or more canonical tyrosine or di-leucine-based endocytic motif in their C-terminal or intracellular loop domain. Three connexins contain non-canonical (modified) di-leucine motifs. However, four connexins (Cx25, Cx26, Cx31, and Cx40.1) do not harbor any recognizable endocytic motif. Interestingly, live cell time-lapse imaging of different GFP-tagged connexins that either contain or do not contain recognizable endocytic motifs readily undergo endocytosis, forming clearly identifiable annular gap junctions when expressed in HeLa cells. How connexins without defined endocytic motifs are endocytosed is currently not known. Our results demonstrate that an array of endocytic motifs exists in the connexin gene family. Further analysis will establish whether the sites we identified in this in silico analysis are legitimate endocytic motifs.

Connexin-43 K63-polyubiquitylation on lysines 264 and 303 regulates gap junction internalization.

Gap junctions (GJs) assembled from connexin (Cx) proteins allow direct cell-cell communication. While phosphorylation is known to regulate multiple GJ functions, much less is known about the role of ubiquitin in these processes. Using ubiquitylation-type-specific antibodies and Cx43 lysine-to-arginine mutants we show that ∼8% of a GJ, localized in central plaque domains, is K63-polyubiquitylated on K264 and K303. Levels and localization of ubiquitylation correlated well with: (1) the short turnover rate of Cxs and GJs; (2) removal of older channels from the plaque center; and (3) the fact that not all Cxs in an internalizing GJ channel need to be ubiquitylated. Connexins mutated at these two sites assembled significantly larger GJs, exhibited much longer protein half-lives and were internalization impaired. Interestingly, these ubiquitin-deficient Cx43 mutants accumulated as hyper-phosphorylated polypeptides in the plasma membrane, suggesting that K63-polyubiquitylation is triggered by phosphorylation. Phospho-specific anti-Cx43 antibodies revealed that upregulated phosphorylation affected serines 368, 279/282 and 255, which are well-known regulatory PKC and MAPK sites. Together, these novel findings suggest that the internalizing portion of channels in a GJ is K63-polyubiquitylated, ubiquitylation is critical for GJ internalization and that phosphorylation induces Cx K63-polyubiquitylation.This article has an associated First Person interview with the first author of the paper.

Role of phase separation on the biological performance of 45S5 Bioglass®.

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 ß-sheet/ß-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).

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.

Primary cilia found on HeLa and other cancer cells.

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.

Proteins and mechanisms regulating gap-junction assembly, internalization, and degradation.

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.

Cohesin: an emerging master regulator at the heart of cardiac development.

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.

Degradation of endocytosed gap junctions by autophagosomal and endo-/lysosomal pathways: a perspective.

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.

Effects of Titanium Implant Surface Topology on Bone Cell Attachment and Proliferation in vitro.

Purpose: 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.

E-cadherin differentially regulates the assembly of Connexin43 and Connexin32 into gap junctions in human squamous carcinoma cells.

It is as yet unknown how the assembly of connexins (Cx) into gap junctions (GJ) is initiated upon cell-cell contact. We investigated whether the trafficking and assembly of Cx43 and Cx32 into GJs were contingent upon cell-cell adhesion mediated by E-cadherin. We also examined the role of the carboxyl termini of these Cxs in initiating the formation of GJs. Using cadherin and Cx-null cells, and by introducing Cx43 and Cx32, either alone or in combination with E-cadherin, our studies demonstrated that E-cadherin-mediated cell-cell adhesion was neither essential nor sufficient to initiate GJ assembly de novo in A431D human squamous carcinoma cells. However, E-cadherin facilitated the growth and assembly of preformed GJs composed of Cx43, although the growth of cells on Transwell filters was required to initiate the assembly of Cx32. Our results also documented that the carboxyl termini of both Cxs were required in this cell type to initiate the formation of GJs de novo. Our findings also showed that GJ puncta composed of Cx43 co-localized extensively with ZO-1 and actin fibers at cell peripheries and that ZO-1 knockdown attenuated Cx43 assembly. These findings suggest that the assembly of Cx43 and Cx32 into GJs is differentially modulated by E-cadherin-mediated cell-cell adhesion and that direct or indirect cross-talk between carboxyl tails of Cxs and actin cytoskeleton via ZO-1 may regulate GJ assembly and growth.

Adherens junctions remain dynamic.

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 beta-catenins and alpha-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.

Nanostructure of bioactive glass affects bone cell attachment via protein restructuring upon adsorption.

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 ß-sheet/α-helix ratio. Our results suggest that the ß-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.

Impaired Cx43 gap junction endocytosis causes morphological and functional defects in zebrafish.

Gap junctions mediate direct cell-to-cell communication by forming channels that physically couple cells, thereby linking their cytoplasm, permitting the exchange of molecules, ions, and electrical impulses. Gap junctions are assembled from connexin (Cx) proteins, with connexin 43 (Cx43) being the most ubiquitously expressed and best studied. While the molecular events that dictate the Cx43 life cycle have largely been characterized, the unusually short half-life of Cxs of only 1-5 h, resulting in constant endocytosis and biosynthetic replacement of gap junction channels, has remained puzzling. 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 (Cx43∆256-289) and a zebrafish model (cx43lh10). We report that this CT deletion causes defective gap junction endocytosis as well as increased gap junction intercellular communication. Increased Cx43 protein content in cx43lh10 zebrafish, specifically in the cardiac tissue, larger gap junction plaques, and longer Cx43 protein half-lives coincide with severely impaired development. Our findings demonstrate for the first time that continuous Cx43 gap junction endocytosis is an essential aspect of gap junction function and, when impaired, gives rise to significant physiological problems as revealed here for cardiovascular development and function.

Potential of tailored amorphous multiporous calcium silicate glass for pulp capping regenerative endodontics-A preliminary assessment.

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.

Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes.

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 (alpha-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.

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.

Cx23, a connexin with only four extracellular-loop cysteines, forms functional gap junction channels and hemichannels.

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.

Acute internalization of gap junctions in vascular endothelial cells in response to inflammatory mediator-induced G-protein coupled receptor activation.

During the inflammatory response, activation of G-protein coupled receptors (GPCRs) by inflammatory mediators rapidly leads to inhibition of gap junction intercellular communication (GJIC); however, the steps that lead to this inhibition are not known. Combining high-resolution fluorescence microscopy and functional assays, we found that activation of the GPCRs PAR-1 and ET(A/B) by their natural inflammatory mediator agonists, thrombin and endothelin-1, resulted in rapid and acute internalization of gap junctions (GJs) that coincided with the inhibition of GJIC followed by increased vascular permeability. The endocytosis protein clathrin and the scaffold protein ZO-1 appeared to be involved in GJ internalization, and ZO-1 was partially displaced from GJs during the internalization process. These findings demonstrate that GJ internalization is an efficient mechanism for modulating GJIC in inflammatory response.