The Stk1/Stp1 and GraSR signal-transduction pathways are two distinct pathways in Staphylococcus aureus that rely on a reversible phosphorylation process in transducing external stimuli intracellularly. Stk1/Stp1 is an eukaryote-like Ser/Thr kinase phosphatase pair involved in purine biosynthesis, cell-wall metabolism, and autolysis. GraSR is a two-component system involved in resistance to cationic antimicrobial peptides. Both systems are implicated in S. aureus virulence and resistance to cell-wall inhibitors. Our study shows that the response regulator protein GraR undergoes phosphorylation by Stk1 at three threonine residues in the DNA-binding domain. Phosphorylation by Stk1 depends on the structural integrity of GraR as well as the amino acid sequences flanking the phosphorylation sites. Its homologue in Bacillus subtilis , BceR, which harbors two of the three phosphorylation sites in GraR, does not undergo Stk1-dependent phosphorylation. GraR is involved in regulation of the dltABCD operon, the gene products of which add the d-Ala on wall teichoic acid (WTA). Investigation of WTA isolated from the S. aureus RN6390 ΔgraR strain by NMR spectroscopy showed a clear negative effect that graR deletion has on the d-Ala content of WTA. Moreover, complementation of ΔgraR mutant with graR lacking the Stk1 phosphorylation sites mirrors this effect. These findings provide evidence that GraR is a target of Stk1 in vivo and suggest that modification of WTA by d-Ala is modulated by Stk1. The crosstalk between these two otherwise independent signaling pathways may facilitate S. aureus interaction with its environment to modulate processes such as cell growth and division and virulence.

Background: Alterations in hERG-encoded K ϩ channel current can cause fatal cardiac electrical disturbances.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a myocardial disease characterized by fibro-fatty replacement of myocardium in the right ventricular free wall and frequently results in life-threatening ventricular arrhythmias and sudden cardiac death. A heterozygous missense mutation in the transmembrane protein 43 (TMEM43) gene, p.S358L, has been genetically identified to cause autosomal dominant ARVC type 5 in a founder population from the island of Newfoundland, Canada. Little is known about the function of the TMEM43 protein or how it leads to the pathogenesis of ARVC. We sought to determine the distribution of TMEM43 and the effect of the p.S358L mutation on the expression and distribution of various intercalated (IC) disc proteins as well as functional effects on IC disc gap junction dye transfer and conduction velocity in cell culture. Through Western blot analysis, transmission electron microscopy (TEM), immunofluorescence (IF), and electrophysiological analysis, our results showed that the stable expression of p.S358L mutation in the HL-1 cardiac cell line resulted in decreased Zonula Occludens (ZO-1) expression and the loss of ZO-1 localization to cell-cell junctions. Junctional Plakoglobin (JUP) and α-catenin proteins were redistributed to the cytoplasm with decreased localization to cell-cell junctions. Connexin-43 (Cx43) phosphorylation was altered, and there was reduced gap junction dye transfer and conduction velocity in mutant TMEM43-transfected cells. These observations suggest that expression of the p.S358L mutant of TMEM43 found in ARVC type 5 may affect localization of proteins involved in conduction, alter gap junction function and reduce conduction velocity in cardiac tissue.

Background: A reduction in either I Kr or I Ks can cause long QT syndrome. Results: Enhanced endocytic degradation of I Kr decreases the expression of both I Kr and I Ks in the plasma membrane. Conclusion: I Kr and I Ks form a macrocomplex at the plasma membrane. Significance: Elucidation of I Kr -I Ks interaction is important for understanding the pathology of cardiac arrhythmias and designing anti-arrhythmic strategies.

Compared to robotic injection of suspended cells (e.g., embryos and oocytes), fewer attempts were made to automate the injection of adherent cells (e.g., cancer cells and cardiomyocytes) due to their smaller size, highly irregular morphology, small thickness (a few micrometers thick), and large variations in thickness across cells. This paper presents a robotic system for automated microinjection of adherent cells. The system is embedded with several new capabilities: automatically locating micropipette tips; robustly detecting the contact of micropipette tip with cell culturing surface and directly with cell membrane; and precisely compensating for accumulative positioning errors. These new capabilities make it practical to perform adherent cell microinjection truly via computer mouse clicking in front of a computer monitor, on hundreds and thousands of cells per experiment (versus a few to tens of cells as state of the art). System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4000 cells. This paper also reports the use of the new robotic system to perform cell-cell communication studies using large sample sizes. The gap junction function in a cardiac muscle cell line (HL-1 cells), for the first time, was quantified with the system.