P2X7 purinergic receptor engagement with extracellular ATP induces transmembrane potassium and calcium flux resulting in assembly of the NLRP3 inflammasome in LPS-primed macrophages. The role of potassium and calcium in inflammasome regulation is not well understood, largely due to limitations in existing methods for interrogating potassium in real time. The use of KS6, a novel sensor for selective and sensitive dynamic visualization of intracellular potassium flux in live cells, multiplexed with the intracellular calcium sensor Fluo-4, revealed a coordinated relationship between potassium and calcium. Interestingly, the mitochondrial potassium pool was mobilized in a P2X7 signaling, and ATP dose-dependent manner, suggesting a role for mitochondrial sensing of cytosolic ion perturbation. Through treatment with extracellular potassium we found that potassium efflux was necessary to permit sustained calcium entry, but not transient calcium flux from intracellular stores. Further, intracellular calcium chelation with BAPTA-AM indicated that P2X7-induced potassium depletion was independent of calcium mobilization. This evidence suggests that both potassium efflux and calcium influx are necessary for mitochondrial reactive oxygen generation upstream of NLRP3 inflammasome assembly and pyroptotic cell death. We propose a model wherein potassium efflux is necessary for calcium influx, resulting in mitochondrial reactive oxygen generation to trigger the NLRP3 inflammasome.

Acheron (Achn), a phylogenetically-conserved member of the Lupus antigen family of RNA binding proteins, was initially identified as a novel cell death-associated gene from the intersegmental muscles of the tobacco hawkmoth Manduca sexta. C(2)C(12) cells are a standard model for the study of myogenesis. When deprived of growth factors, these cells can be induced to: form multinucleated myotubes, arrest as quiescent satellite-like reserve cells, or undergo apoptosis. Achn expression is induced in myoblasts that form myotubes and acts upstream of the muscle specific transcription factor MyoD. Forced expression of ectopic Achn resulted in the formation of larger myotubes and massive reserve cell death relative to controls. Conversely, dominant-negative or antisense Achn blocked myotube formation following loss of growth factors, suggesting that Achn plays an essential, permissive role in myogenesis. Studies in zebrafish embryos support this hypothesis. Reduction of Achn with antisense morpholinos led to muscle fiber loss and an increase in the number of surviving cells in the somites, while ectopic Achn enhanced muscle fiber formation and reduced cell numbers. These results display a crucial evolutionarily conserved role for Achn in myogenesis and suggest that it plays key roles in the processes of differentiation and self-renewal.

Migration is a fundamental cellular behavior that plays an indispensable role in development and homeostasis, but can also contribute to pathology such as cancer metastasis. Due to its relevance to many aspects of human health, the ability to accurately measure cell migration is of broad interest, and numerous approaches have been developed. One of the most commonly employed approaches, because of its simplicity and throughput, is the exclusion zone assay in which cells are allowed to migrate into an initially cell-free region. A major drawback of this assay is that it relies on simply counting cells in the exclusion zone and therefore cannot distinguish the effects of proliferation from migration. We report here a simple modification to the exclusion zone migration assay that exclusively measures cell migration and is not affected by proliferation. This approach makes use of a lineage-tracing vital stain that is retained through cell generations and effectively reads out migration relative to the original, parental cell population. This modification is simple, robust, non-perturbing, and inexpensive. We validate the method in a panel of cell lines under conditions that inhibit or promote migration and demonstrate its use in normal and cancer cell lines as well as primary cells.

Cell adhesion to the extracellular matrix via integrins is a primary regulatory mechanism for numerous aspects of normal cellular function. However, disruption of this interaction can result in pathology. For example, one characteristic of transformed cells is loss of adhesion dependence for viability. Adhesion also is a necessary step in tumor metastasis. It has been shown previously, in HeLa cells, that cell attachment to a gelatin-coated substrate results in the release of arachidonic acid, which is metabolized by lipoxygenase. A subsequent cascade of lipid second messengers activates protein kinase C, which triggers actin polymerization leading to cell spreading. We now demonstrate by inhibitor studies and biochemical analysis, a parallel branch of arachidonic acid signaling that reorganizes the actin cytoskeleton into small bundles. This branch of the pathway is initiated by cyclooxygenase, which generates prostaglandins and causes the downstream activation of cyclic AMP-dependent protein kinase. This work elucidates a system of interacting signals in which arachidonic acid functions at a branch point in cytoskeletal signaling. The lipoxygenase branch provides polymerized actin; these actin filaments act as a substrate for the cylooxygenase branch to generate actin bundles.

Acheron (Achn) was originally identified as novel gene that is induced when insect muscles become committed to die at the end of metamorphosis. In separate studies, we have demonstrated that Achn acts upstream of MyoD and is required by mammalian myoblasts to either differentiate or undergo apoptosis following loss of growth factors. In the present study we examined the role of Achn in regulating integrin-extracellular matrix interactions that are required for myogenesis. Both control C2C12 myoblasts and those engineered to express ectopic Achn expressed the fibronectin receptor integrin alpha(5)beta(1) in the presence of growth factors and the laminin receptor alpha(7)beta(1) following growth factor withdrawal. Expression of the laminin receptor was blocked in cells expressing either Achn antisense or an Achn deletion mutant that blocks differentiation. Control cells and those expressing ectopic Achn undergo sequential and transient increases in both substrate adhesion and migration before cell fusion. Blockade of Achn expression reduced these effects on laminin but not on fibronectin. Taken together, these data suggest that Achn may influence differentiation in part via its control of cell adhesion dynamics.