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 connexins of only 1-5 hours, 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 (GJIC). Increased Cx43 protein content in cx 43lh10 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 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. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Roberts syndrome (RBS) is a rare developmental disorder that can include craniofacial abnormalities, limb malformations, missing digits, intellectual disabilities, stillbirth, and early mortality. The genetic basis for RBS is linked to autosomal recessive loss-of-function mutation of the establishment of cohesion (ESCO) 2 acetyltransferase. ESCO2 is an essential gene that targets the DNA-binding cohesin complex. ESCO2 acetylates alternate subunits of cohesin to orchestrate vital cellular processes that include sister chromatid cohesion, chromosome condensation, transcription, and DNA repair. Although significant advances were made over the last 20 years in our understanding of ESCO2 and cohesin biology, the molecular etiology of RBS remains ambiguous. In this review, we highlight current models of RBS and reflect on data that suggests a novel role for macromolecular damage in the molecular etiology of RBS.

Yeast Eco1 (ESCO2 in humans) acetyltransferase converts chromatin-bound cohesins to a DNA tethering state, thereby establishing sister chromatid cohesion. Eco1 establishes cohesion during DNA replication, after which Eco1 is targeted for degradation by SCF E3 ubiquitin ligase. SCF E3 ligase, and sequential phosphorylations that promote Eco1 ubiquitination and degradation, remain active throughout the M phase. In this way, Eco1 protein levels are high during S phase, but remain low throughout the remaining cell cycle. In response to DNA damage during M phase, however, Eco1 activity increases—providing for a new wave of cohesion establishment (termed Damage-Induced Cohesion, or DIC) which is critical for efficient DNA repair. To date, little evidence exists as to the mechanism through which Eco1 activity increases during M phase in response to DNA damage. Possibilities include that either the kinases or E3 ligase, that target Eco1 for degradation, are inhibited in response to DNA damage. Our results reveal instead that the degradation machinery remains fully active during M phase, despite the presence of DNA damage. In testing alternate models through which Eco1 activity increases in response to DNA damage, the results reveal that DNA damage induces new transcription of ECO1 and at a rate that exceeds the rate of Eco1 turnover, providing for rapid accumulation of Eco1 protein. We further show that DNA damage induction of ECO1 transcription is in part regulated by Yap5—a stress-induced transcription factor. Given the role for mutated ESCO2 (homolog of ECO1) in human birth defects, this study highlights the complex nature through which mutation of ESCO2, and defects in ESCO2 regulation, may promote developmental abnormalities and contribute to various diseases including cancer.

Roberts Syndrome (RBS) is a multi-spectrum developmental disorder characterized by severe limb, craniofacial, and organ abnormalities and often intellectual disabilities. The genetic basis of RBS is rooted in loss-of-function mutations in the essential N-acetyltransferase ESCO2 which is conserved from yeast (Eco1/Ctf7) to humans. ESCO2/Eco1 regulate many cellular processes that impact chromatin structure, chromosome transmission, gene expression, and repair of the genome. The etiology of RBS remains contentious with current models that include transcriptional dysregulation or mitotic failure. Here, we report evidence that supports an emerging model rooted in defective DNA damage responses. First, the results reveal that redox stress is elevated in both eco1 and cohesion factor Saccharomyces cerevisiae mutant cells. Second, we provide evidence that Eco1 and cohesion factors are required for the repair of oxidative DNA damage such that ECO1 and cohesin gene mutations result in reduced cell viability and hyperactivation of DNA damage checkpoints that occur in response to oxidative stress. Moreover, we show that mutation of ECO1 is solely sufficient to induce endogenous redox stress and sensitizes mutant cells to exogenous genotoxic challenges. Remarkably, antioxidant treatment desensitizes eco1 mutant cells to a range of DNA damaging agents, raising the possibility that modulating the cellular redox state may represent an important avenue of treatment for Roberts Syndrome and tumors that bear ESCO2 mutations.

In this report, the authors describe the effects of stressed culture conditions on Vero cells, which are a form of epithelial cells derived from the African green monkey kidney. This project was designed in large part to replicate a previously published report on the effects of nutritional stress on the growth patterns of these cells. Culture conditions that include nutritional limitation and cell crowding have been shown to transform these cells into cells that differ morphologically, develop into spheroid- shaped clusters, as well as exhibit altered protein expression. It was suggested that these changes might mirror those occurring in metastatic cancer cells, and could therefore provide a useful experimental model system. In the current study, the investigators successfully cultured Vero cells in control cultures, and then compared their growth patterns and morphology to cells grown in nutritionally stressed and overcrowded cell culture conditions defined by extensive days in culture in unchanged media which did or did not contain glucose. The results confirmed that nutritionally stressed and over-crowded cultures result in cells that change morphologically, detach from the substrate, and exhibit spheroid-shaped clusters of cells. Sub-culturing these detached cells demonstrated that the results were permanent, meaning that the new growth patterns and clustering persisted. The results suggest that deprivation of nutrition and other factors essential to life may facilitate aberrant growth. KEYWORDS: Vero Cells, Nutritional Stress, Cell Morphology, Neoplastic Transformation, Malignant Cells, Spheroid Formation.