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The voltage-gated sodium channel is critical for cardiomyocyte function. It consists of a protein complex comprising a pore-forming α subunit and associated β subunits. In polarized Madin-Darby canine kidney cells, we show evidence by acyl-biotin exchange that β2 is S-acylated at Cys-182. Interestingly, we found that palmitoylation increases β2 association with detergent-resistant membranes. β2 localizes exclusively to the apical surface. However, depletion of plasma membrane cholesterol, or blocking intracellular cholesterol transport, caused mislocalization of β2, as well as of the non-palmitoylable C182S mutant, to the basolateral domain. Apical β2 did not undergo endocytosis and displayed limited diffusion within the plane of the membrane; such behavior suggests that, at least in part, it is cytoskeleton anchored. https://www.selleckchem.com/products/jg98.html Upon acute cholesterol depletion, its mobility was greatly reduced, and a slight reduction was also measured as a result of lack of palmitoylation, supporting β2 association with cholesterol-rich lipid rafts. Indeed, lipid raft labeling confirmed a partial overlap with apical β2. Although β2 palmitoylation was not required to promote surface localization of the α subunit, our data suggest that it is likely implicated in lipid raft association and the polarized localization of β2.Lysosomes are acidic Ca2+ stores often mobilised in conjunction with endoplasmic reticulum (ER) Ca2+ stores. Glycyl-L-phenylalanine 2-naphthylamide (GPN) is a widely used lysosomotropic agent that evokes cytosolic Ca2+ signals in many cells. However, whether these signals are the result of a primary action on lysosomes is unclear in light of recent evidence showing that GPN mediates direct ER Ca2+ release through changes in cytosolic pH. Here, we show that GPN evoked rapid increases in cytosolic pH but slower Ca2+ signals. NH4Cl evoked comparable changes in pH but failed to affect Ca2+ The V-type ATPase inhibitor, bafilomycin A1, increased lysosomal pH over a period of hours. Acute treatment modestly affected lysosomal pH and potentiated Ca2+ signals evoked by GPN. In contrast, chronic treatment led to more profound changes in luminal pH and selectively inhibited GPN action. GPN blocked Ca2+ responses evoked by the novel nicotinic acid adenine dinucleotide phosphate-like agonist, TPC2-A1-N. Therefore, GPN-evoked Ca2+ signals were better correlated with associated pH changes in the lysosome compared to the cytosol, and were coupled to lysosomal Ca2+ release. We conclude that Ca2+ signals evoked by GPN most likely derive from acidic organelles.Phenotypic heterogeneity is increasingly acknowledged to confer several advantages to cancer progression and drug resistance. Here, we probe the collective importance of heterogeneity in cell size and deformability in breast cancer invasion. A computational model of invasion of a heterogeneous cell aggregate predicts that combined heterogeneity in cell size and deformability enhances invasiveness of the whole population, with maximum invasiveness at intermediate cell-cell adhesion. We then show that small cells of varying deformability, a subpopulation predicted to be enriched at the invasive front, exhibit considerable overlap with the biophysical properties of cancer stem cells (CSCs). In MDA-MB-231 cells, these include CD44 hi CD24- mesenchymal CSCs, which are small and soft, and CD44 hi CD24+ hybrid CSCs, which exhibit a wide range of size and deformability. We validate our predictions by tracking the pattern of cell invasion from spheroids implanted in three-dimensional collagen gels, wherein we show temporal enrichment of CD44 hi cells at the invasive front. Collectively, our results illustrate the advantages imparted by biophysical heterogeneity in enhancing cancer invasiveness.This article has an associated First Person interview with the first author of the paper.Movement of the cell nucleus typically involves the cytoskeleton and either polymerization-based pushing forces or motor-based pulling forces. In the fission yeast Schizosaccharomyces pombe, nuclear movement and positioning are thought to depend on microtubule polymerization-based pushing forces. Here, we describe a novel, microtubule-independent, form of nuclear movement in fission yeast. Microtubule-independent nuclear movement is directed towards growing cell tips, and it is strongest when the nucleus is close to a growing cell tip, and weakest when the nucleus is far from that tip. Microtubule-independent nuclear movement requires actin cables but does not depend on actin polymerization-based pushing or myosin V-based pulling forces. The vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) Scs2 and Scs22, which are critical for endoplasmic reticulum-plasma membrane contact sites in fission yeast, are also required for microtubule-independent nuclear movement. We also find that in cells in which microtubule-based pushing forces are present, disruption of actin cables leads to increased fluctuations in interphase nuclear positioning and subsequent altered septation. Our results suggest two non-exclusive mechanisms for microtubule-independent nuclear movement, which may help illuminate aspects of nuclear positioning in other cells.Escherichia coli is one of the most common etiological agents responsible for clinical bovine mastitis. Here, we report the draft genome sequences and annotations of 113 E. coli strains that were isolated from Holstein cows with intramammary infections in Canada.Here, we report the near-complete genome sequences of vesicular stomatitis virus (VSV) serotype Indiana isolates from the 2020 U.S. outbreak. The sequences were obtained from swabs collected from Kansas horses in July and August. The four genome sequences help improve our understanding of VSV outbreak dynamics in the United States.Characterizing the microbiome of spacecraft assembly cleanrooms is important for planetary protection. We report two bacterial metagenome-assembled genomes (MAGs) reconstructed from metagenomes produced from cleanroom samples from the Kennedy Space Center's Payload Hazardous Servicing Facility (KSC-PHSF) during the handling of the Phoenix spacecraft. Characterization of these MAGs will enable identification of the strategies underpinning their survival.
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