Plasma membrane depolarization can trigger cell proliferation, but how membrane potential influences mitogenic signaling is uncertain. Here, we show that plasma membrane depolarization induces nanoscale reorganization of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate but not other anionic phospholipids. K-Ras, which is targeted to the plasma membrane by electrostatic interactions with phosphatidylserine, in turn undergoes enhanced nanoclustering. Depolarization-induced changes in phosphatidylserine and K-Ras plasma membrane organization occur in fibroblasts, excitable neuroblastoma cells, and Drosophila neurons in vivo and robustly amplify K-Ras–dependent mitogen-activated protein kinase (MAPK) signaling. Conversely, plasma membrane repolarization disrupts K-Ras nanoclustering and inhibits MAPK signaling. By responding to voltage-induced changes in phosphatidylserine spatiotemporal dynamics, K-Ras nanoclusters set up the plasma membrane as a biological field-effect transistor, allowing membrane potential to control the gain in mitogenic signaling circuits.

We explored the detection of genome-wide hypomethylation in plasma using shotgun massively parallel bisulfite sequencing as a marker for cancer. Tumor-associated copy number aberrations (CNAs) could also be observed from the bisulfite DNA sequencing data. Hypomethylation and CNAs were detected in the plasma DNA of patients with hepatocellular carcinoma, breast cancer, lung cancer, nasopharyngeal cancer, smooth muscle sarcoma, and neuroendocrine tumor. For the detection of nonmetastatic cancer cases, plasma hypomethylation gave a sensitivity and specificity of 74% and 94%, respectively, when a mean of 93 million reads per case were obtained. Reducing the sequencing depth to 10 million reads per case was found to have no adverse effect on the sensitivity and specificity for cancer detection, giving respective figures of 68% and 94%. This characteristic thus indicates that analysis of plasma hypomethylation by this sequencing-based method may be a relatively cost-effective approach for cancer detection. We also demonstrated that plasma hypomethylation had utility for monitoring hepatocellular carcinoma patients following tumor resection and for detecting residual disease. Plasma hypomethylation can be combined with plasma CNA analysis for further enhancement of the detection sensitivity or specificity using different diagnostic algorithms. Using the detection of at least one type of aberration to define an abnormality, a sensitivity of 87% could be achieved with a specificity of 88%. These developments have thus expanded the applications of plasma DNA analysis for cancer detection and monitoring.epigenomics | epigenetics | next-generation sequencing | tumor markers | global hypomethylation T here is much recent interest in the biology and diagnostic applications of cell-free DNA in the plasma of human subjects. In particular, tumor-associated DNA has been detected in the plasma of cancer patients (1) and fetus-derived DNA has been found in the plasma of pregnant women (2). The finding of these types of circulating nucleic acids has implications for the detection of cancer and noninvasive prenatal testing, respectively. There are also many similarities between the two phenomena, with the

Members of the Transient Receptor Potential-Mucolipin (TRPML) constitute a family of evolutionarily conserved cation channels that function predominantly in endolysosomal vesicles. Whereas loss-of-function mutations in human TRPML1 were first identified as being causative for the lysosomal storage disease, Mucolipidosis type IV, most mammals also express two other TRPML isoforms called TRPML2 and TRPML3. All three mammalian TRPMLs as well as TRPML related genes in other species including C. elegans and Drosophila exhibit overlapping functional and biophysical properties. The functions of TRPML proteins include roles in vesicular trafficking and biogenesis, maintenance of neuronal development, function, and viability, and regulation of intracellular and organellar ionic homeostasis. Biophysically, TRPML channels are non-selective cation channels exhibiting variable permeability to a host of cations including Na+, Ca2+, Fe2+, and Zn2+, and are activated by a phosphoinositide species, PI(3,5)P2, that is mostly found in endolysosomal membranes. Here, we review the functional and biophysical properties of these enigmatic cation channels, which represent the most ancient and archetypical TRP channels.

Summary Loss-of-function mutations in TRPML1 (Transient Receptor Potential Mucolipin 1) cause a lysosomal storage disorder called mucolipidosis type IV (MLIV). Previously, we established a Drosophila model for MLIV by knocking out the single TRPML1 homolog expressed in flies [1]. The mutant animals displayed impaired autophagy, and reduced viability during the pupal period—a phase when wild-type animals rely on autophagic sources for nutrients. However, the specific defect in autophagy has remained unclear. Here, we show that TRPML, which was localized to the membranes of late-endosomes and lysosomes in vivo, was required for fusion between late-endosomes with lysosomes. We report that loss of TRPML led to accumulation of vesicles of significantly larger volume, and loss of TRPML from the late-endosomes/lysosomes led to the accumulation of higher luminal Ca2+ within those vesicles. We also found that trpml1 mutant cells showed decreased TORC1 signaling, and a concomitant upregulation of autophagy induction. Both of these defects were reversed by activating TORC1 in the mutants genetically and by feeding the mutant larvae a high-protein diet. Feeding the larvae a high-protein diet also reduced both the pupal lethality, and the increased volume of acidic vesicles. Conversely, further inhibition of TORC1 activity by rapamycin exacerbated the mutant phenotypes. Finally, TORC1 exerted reciprocal control on TRPML function. A high amino acid diet enhanced cortical localization of TRPML, and this effect was blocked by rapamycin. Our findings delineate the interrelationship between TRPML- and TORC1-mediated nutrient sensing pathways, and also raise the intriguing possibility that amino acid supplementation might reduce the severity of the clinical manifestations associated with MLIV.

Declining insect population sizes are provoking grave concern around the world as insects play essential roles in food production and ecosystems. Environmental contamination by intense insecticide usage is consistently proposed as a significant contributor, among other threats. Many studies have demonstrated impacts of low doses of insecticides on insect behavior, but have not elucidated links to insecticidal activity at the molecular and cellular levels. Here, the histological, physiological, and behavioral impacts of imidacloprid are investigated in Drosophila melanogaster, an experimental organism exposed to insecticides in the field. We show that oxidative stress is a key factor in the mode of action of this insecticide at low doses. Imidacloprid produces an enduring flux of Ca2+ into neurons and a rapid increase in levels of reactive oxygen species (ROS) in the larval brain. It affects mitochondrial function, energy levels, the lipid environment, and transcriptomic profiles. Use of RNAi to induce ROS production in the brain recapitulates insecticide-induced phenotypes in the metabolic tissues, indicating that a signal from neurons is responsible. Chronic low level exposures in adults lead to mitochondrial dysfunction, severe damage to glial cells, and impaired vision. The potent antioxidant, N-acetylcysteine amide (NACA), reduces the severity of a number of the imidacloprid-induced phenotypes, indicating a causal role for oxidative stress. Given that other insecticides are known to generate oxidative stress, this research has wider implications. The systemic impairment of several key biological functions, including vision, reported here would reduce the resilience of insects facing other environmental challenges.