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.

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.

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.

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.

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.

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.

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.

New bioactive glass scaffolds with exceptional qualities for bone tissue regeneration: response of osteoblasts and osteoclasts.

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.

Phosphorylation regulates connexin43/ZO-1 binding and release, an important step in gap junction turnover.

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.

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.

Connexin43 phosphorylation by PKC and MAPK signals VEGF-mediated gap junction internalization.

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.

Degradation of connexins and gap junctions.

Connexin proteins are short-lived within the cell, whether present in the secretory pathway or in gap junction plaques. Their levels can be modulated by their rate of degradation. Connexins, at different stages of assembly, are degraded through the proteasomal, endo-/lysosomal, and phago-/lysosomal pathways. In this review, we summarize the current knowledge about connexin and gap junction degradation including the signals and protein-protein interactions that participate in their targeting for degradation.

EGF induces efficient Cx43 gap junction endocytosis in mouse embryonic stem cell colonies via phosphorylation of Ser262, Ser279/282, and Ser368.

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.

Two tyrosine-based sorting signals in the Cx43 C-terminus cooperate to mediate gap junction endocytosis.

Gap junction (GJ) channels that electrically and chemically couple neighboring cells are formed when two hemichannels (connexons) of apposed cells dock head-on in the extracellular space. Remarkably, docked connexons are inseparable under physiological conditions, and we and others have shown that GJs are internalized in whole, utilizing the endocytic clathrin machinery. Endocytosis generates double-membrane vesicles (annular GJs or connexosomes) in the cytoplasm of one of the apposed cells that are degraded by autophagosomal and, potentially, endo/lysosomal pathways. In this study, we investigated the structural motifs that mediate Cx43 GJ endocytosis. We identified three canonical tyrosine-based sorting signals of the type "YXXΦ" in the Cx43 C-terminus, two of which function cooperatively as AP-2 binding sites. We generated a set of green fluorescent protein-tagged and untagged Cx43 mutants that targeted these two sites either individually or together. Mutating both sites completely abolished Cx43-AP-2/Dab2/clathrin interaction and resulted in increased GJ plaque size, longer Cx43 protein half-lives, and impaired GJ internalization. Interestingly, Dab2, an accessory clathrin adaptor found earlier to be important for GJ endocytosis, interacts indirectly with Cx43 via AP-2, permitting the recruitment of up to four clathrin complexes per Cx43 protein. Our analyses provide a mechanistic model for clathrin's efficient internalization of large plasma membrane structures, such as GJs.

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.

Nanoporosity significantly enhances the biological performance of engineered glass tissue scaffolds.

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.