Rapid plasma membrane repair is essential to restore cellular homeostasis and improve cell survival after injury. Several mechanisms for plasma membrane repair have been proposed, including formation of an intracellular vesicle patch, reduction of plasma membrane tension, lesion removal by endocytosis, and/or shedding of the wounded membrane. Under all conditions studied to date, plasma membrane repair is strictly dependent on the entry of calcium into cells, from the extracellular medium. Calcium-dependent exocytosis of lysosomes is an important early step in the plasma membrane repair process, and defects in plasma membrane repair have been observed in cells carrying mutations responsible for serious lysosomal diseases, such as Chediak–Higashi (Huynh, Roth, Ward, Kaplan, & Andrews, 2004) and Niemann–Pick Disease type A (Tam et al., 2010). A functional role for release of the lysosomal enzyme acid sphingomyelinase, which generates ceramide on the cell surface and triggers endocytosis, has been described (Corrotte et al., 2013; Tam et al., 2010). Therefore, procedures for measuring the extent of lysosomal fusion with the plasma membrane of wounded cells are important indicators of the cellular repair response. The importance of carefully selecting the methodology for experimental plasma membrane injury, in order not to adversely impact the membrane repair machinery, is becoming increasingly apparent. Here, we describe physiologically relevant methods to induce different types of cellular wounds, and sensitive assays to measure the ability of cells to secrete lysosomes and reseal their plasma membrane.

bEntamoeba histolytica is an intestinal parasite that causes dysentery and liver abscess. Parasite cell surface receptors, such as the Gal/GalNAc lectin, facilitate attachment to host cells and extracellular matrix. The Gal/GalNAc lectin binds to galactose or N-acetylgalactosamine residues on host components and is composed of heavy (Hgl), intermediate (Igl), and light (Lgl) subunits. Although Igl is constitutively localized to lipid rafts (cholesterol-rich membrane domains), Hgl and Lgl transiently associate with this compartment in a cholesterol-dependent fashion. In this study, trophozoites were exposed to biologically relevant ligands to determine if ligand binding influences the submembrane distribution of the subunits. Exposure to human red blood cells (hRBCs) or collagen, which are bona fide Gal/GalNAc lectin ligands, was correlated with enrichment of Hgl and Lgl in rafts. This enrichment was abrogated in the presence of galactose, suggesting that direct lectin-ligand interactions are necessary to influence subunit location. Using a cell line that is able to attach to, but not phagocytose, hRBCs, it was shown that physical attachment to ligands was not sufficient to induce the enrichment of lectin subunits in rafts. Additionally, the mutant had lower levels of phosphatidylinositol (4,5)-bisphosphate (PIP 2 ); PIP 2 loading restored the ability of this mutant to respond to ligands with enrichment of subunits in rafts. Finally, intracellular calcium levels increased upon attachment to collagen; this increase was essential for the enrichment of lectin subunits in rafts. Together, these data provide evidence that ligand-induced enrichment of lectin subunits in rafts may be the first step in a signaling pathway that involves both PIP 2 and calcium signaling.

Background The sodium channel, Na v 1.5, encoded by SCN 5A , undergoes developmentally regulated splicing from inclusion of exon 6A in the fetal heart to exon 6B in adults. These mutually exclusive exons differ in 7 amino acids altering the electrophysiological properties of the Na v 1.5 channel. In myotonic dystrophy type 1, SCN 5A is mis‐spliced such that the fetal pattern of exon 6A inclusion is detected in adult hearts. Cardiac manifestations of myotonic dystrophy type 1 include conduction defects and arrhythmias and are the second‐leading cause of death. Methods and Results This work aimed to determine the impact of SCN 5A mis‐splicing on cardiac function. We used clustered regularly interspaced short palindromic repeat ( CRISPR) /CRISPR‐associated protein 9 (Cas9) to delete Scn5a exon 6B in mice, thereby redirecting splicing toward exon 6A. These mice exhibit prolonged PR and QRS intervals, slowed conduction velocity, extended action potential duration, and are highly susceptible to arrhythmias. Conclusions Our findings highlight a nonmutational pathological mechanism of arrhythmias and conduction defects as a result of mis‐splicing of the predominant cardiac sodium channel. Animals homozygous for the deleted exon express only the fetal isoform and have more‐severe phenotypes than heterozygotes that also express the adult isoform. This observation is directly relevant to myotonic dystrophy type 1, and possibly pathological arrhythmias, in which individuals differ with regard to the ratios of the isoforms expressed.

bEntamoeba histolytica is an intestinal protozoan parasite and is the causative agent of amoebiasis. During invasive infection, highly motile amoebae destroy the colonic epithelium, enter the blood circulation, and disseminate to other organs such as liver, causing liver abscess. Motility is a key factor in E. histolytica pathogenesis, and this process relies on a dynamic actomyosin cytoskeleton. In other systems, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] is known to regulate a wide variety of cellular functions, including signal transduction, actin remodeling, and cell motility. Little is known about the role of PI(4,5)P 2 in E. histolytica pathogenicity. In this study, we demonstrate that PI(4,5)P 2 is localized to cholesterol-rich microdomains, lipid rafts, and the actin-rich fractions of the E. histolytica membrane. Microscopy revealed that the trailing edge of polarized trophozoites, uroids, are highly enriched in lipid rafts and their constituent lipid, PI(4,5)P 2 . Polarization and enrichment of uroids and rafts with PI(4,5)P 2 were enhanced upon treatment of E. histolytica cells with cholesterol. Exposure to cholesterol also increased intracellular calcium, which is a downstream effector of PI(4,5)P 2 , with a concomitant increase in motility. Together, our data suggest that in E. histolytica, PI(4,5)P 2 may signal from lipid rafts and cholesterol may play a role in triggering PI(4,5)P 2 -mediated signaling to enhance the motility of this pathogen.T he intestinal parasite Entamoeba histolytica is known to cause amoebic dysentery and liver abscess. E. histolytica enters the human host via contaminated food or water as an environmentally stable cyst. Excystation leads to the release of trophozoites in the small intestine, which colonize the bowel lumen. From here, the parasite can enter two non-mutually exclusive routes of infection, noninvasive or invasive disease (reviewed in reference 1). In the noninvasive mode, the trophozoite encysts and exits the host, whereas in the invasive mode, the parasite adheres to and destroys the colonic epithelium and enters the circulatory system. This results in extraintestinal infection, of which amoebic liver abscess (ALA) is the most common manifestation. Motility is a key virulence function that enables this parasite to cause extraintestinal infection (2).Motile amoebae display a polarized morphology with a pseudopod at the leading edge and a uroid (also referred to as a uropod in other eukaryotic cells) at the trailing edge. A key feature of a polarized cell is the differential distribution of proteins and lipids, including cell surface receptors, signaling molecules, and cytoskeletal elements. For example, the pseudopod of E. histolytica is enriched in F actin, myosin IB (3, 4), and signaling molecules like phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P 3 ] (5), while the uroid is enriched in myosin II and signaling molecules, including an important heterotrimeric adhesin, the galactose/N-acetylgalactosamine (Gal/GalNAc) lectin (6), F actin, and var...

Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. E. histolytica relies on motility, phagocytosis, host cell adhesion, and proteolysis of extracellular matrix for virulence. In eukaryotic cells, these processes are mediated in part by phosphatidylinositol 3-kinase (PI3K) signaling. Thus, PI3K may be critical for virulence. We utilized a functional genomics approach to identify genes whose products may operate in the PI3K pathway in E. histolytica. We treated a population of trophozoites that were overexpressing genes from a cDNA library with a near-lethal dose of the PI3K inhibitor wortmannin. This screen was based on the rationale that survivors would be overexpressing gene products that directly or indirectly function in the PI3K pathway. We sequenced the overexpressed genes in survivors and identified a cDNA encoding a Rap GTPase, a protein previously shown to participate in the PI3K pathway. This supports the validity of our approach. Genes encoding a coactosin-like protein, EhCoactosin, and a serine-rich E. histolytica protein (SREHP) were also identified. Cells overexpressing EhCoactosin or SREHP were also less sensitive to a second PI3K inhibitor, LY294002. This corroborates the link between these proteins and PI3K. Finally, a mutant cell line with an increased level of phosphatidylinositol (3,4,5)-triphosphate, the product of PI3K activity, exhibited increased expression of SREHP and EhCoactosin. This further supports the functional connection between these proteins and PI3K in E. histolytica. To our knowledge, this is the first forward-genetics screen adapted to reveal genes participating in a signal transduction pathway in this pathogen. E ntamoeba histolytica is an enteric protozoan parasite that causes amoebiasis and amoebic liver abscess in humans (1). It is prevalent in developing countries that cannot prevent its fecaloral spread. E. histolytica enters the human host upon ingestion of water or food contaminated with environmentally stable cysts. After passing through the stomach, excystation leads to the release of trophozoites, which migrate to the bowel lumen for colonization. In 10% of infected individuals, infection can progress from a noninvasive stage to an invasive stage (2), during which the parasite binds to and destroys colonic epithelium. From here, the parasites enter the circulatory system and translocate to other organs. The most common site of extraintestinal infection is the liver, characterized by the formation of amebic liver abscess (ALA).E. histolytica relies on cell motility, phagocytosis, proteolysis of host extracellular matrix, and host cell adhesion for virulence (3). In other eukaryotic cells, these processes are mediated in part by phosphatidylinositol 3-kinase (PI3K) signaling (4). PI3Ks phosphorylate phosphatidylinositol (PI) and its derivatives to generate signaling lipids such as phosphatidylinositol 3-phosphate (PI3P), phosphatidylinositol 3,4-bisphosphate [PI(3,4)P 2 ], phosphatidylinositol 3,5-bisphosphate [PI(3,5)P 2 ]...