Unregulated cell response to nutrients, in the context of tumor formation or non-oncogenic cellular overgrowth, has severe impact on human health and life span. mTORC1 is a key regulator for cell growth, sensing signals from extracellular nutrients and intracellular energy levels. mTORC1 activity is inhibited by nutrient removal, but also by the immunosuppressive drug rapamycin even in the presence of full nutrient support. Using Dictyostelium, we study unique mTORC1 functions, apart from other nutrient response pathways that act in parallel, and their role in control of specific metabolic and nuclear events. We have shown that withdrawal of nutrients from or addition of rapamycin to growing cells leads to a rapid (<5min) de-phosphorylation of the mTORC1 mediated growth regulator p4EBP1. In parallel, the energy status regulator AMPK is phosphorylated/activated. Even in the presence of nutrients, rapamycin suppression of mTORC1 promotes mRNA unloading from polysomes and inhibition of protein synthesis, similar to that of nutrient depleted cells. To distinguish metabolic pathways that are separately sensitive to nutrient depletion or mTORC1 inhibition, we removed nutrients from or added rapamycin to growing cells and assayed metabolic pathways by Mass Spectrometry. We show regulation of certain metabolic pathways, including glycolysis, lipolysis, and protein synthesis and degradation, by nutrient sensor mTORC1 and relate these to the energy deficit state in control of growth/development. Chemotaxis and cell migration are fundamental, universal eukaryotic processes essential for biological functions such as embryogenesis, immunity, cell renewal, and wound healing, as well as for pathogenesis of many diseases including cancer metastasis and chronic inflammation. To identify novel chemotaxis inhibitors as probes for mechanistic studies and as leads for development of new therapeutics, we developed a unique, unbiased phenotypic chemotaxis-dependent Dictyostelium aggregation assay for high-throughput screening using rapid, laser-scanning cytometry. Under defined conditions, individual Dictyostelium secrete chemoattractants, migrate, and aggregate. Chemotaxis is quantified by laser-scanning cytometry with a GFP marker expressed only in cells after chemotaxis/multi-cell aggregation. We applied the assay to screen 1,280 known compounds in a 1536-well plate format and identified two chemotaxis inhibitors. The chemotaxis inhibitory activities of both compounds were confirmed in both Dictyostelium and in human neutrophils in a directed EZ-TAXIscan chemotaxis assay. The compounds were also shown to inhibit migration of two human cancer cell lines in monolayer scratch assays. This test screen demonstrated that the miniaturized assay is highly suitable for high-throughput screening of very large libraries of small molecules to identify novel classes of chemotaxis/migratory inhibitors for drug development and research tools for targeting chemotactic pathways universal to humans and other systems. In the wild, bacteria are one of the primary food sources to support Dictyostelium growth. Dictyostelium chemotax toward bacteria and eventually phagocytose the bacteria to fulfill nutritional requirements. However, the precision of Dictyostelium chemotaxis toward bacteria has not been well studied. We have developed quantitative analyses of Dictyostelium chemotaxis towards bacteria and characterized bacterially secreted chemoattractants. The study also analyzes the separate signaling networks required for chemotaxis by growing Dictyostelium. To this end, we have characterized the major receptors expressed by growing Dictyostelium and identified a novel receptor required for detecting and responding to bacterially-secreted folate gradients. Nucleosome placement and re-positioning can direct transcription of individual genes, however the precise interactions of these events are complex and largely unresolved at the whole genome level. The Chromodomain-Helicase-DNA binding (CHD) Type III proteins are a subfamily of SWI/SNF2 proteins that control nucleosome positioning and are associated with several complex human disorders, including CHARAGE syndrome and autism. They are found only in animals and Dictyostelium, and in both cases are required for multicellular development. Type III CHDs can mediate nucleosome translocation in vitro, but their in vivo mechanism is unknown. Here, we use genome-wide analysis of nucleosome positioning and transcription profiling to investigate the in vivo relationship between nucleosome positioning and gene expression during development of wild type (WT) Dictyostelium and mutant cells lacking ChdC, a Type III CHD protein orthologue. We demonstrate major nucleosome repositioning during development, and an association with differential gene expression. Loss of chdC causes an increase of intragenic nucleosome spacing and mis-regulation of gene expression, effecting approximately 50% of genes that are remodeled during WT development. This analysis demonstrates active nucleosome re-positioning during Dictyostelium multicellular development, establishes an in vivo function of CHD Type III chromatin remodeling proteins, and provides insight into the relationship between nucleosome positioning and gene regulation. Excessive cellular triacylglyceride (TAG) storage within intracellular neutral lipid droplets is a well-known risk factor for many metabolic disorders, including insulin resistance, cardiovascular disease, and hepatic steatosis. Intracellular lipid droplets (LDs) are unique organelles that store metabolic precursors of cellular energy, membrane biosynthesis, steroid hormone synthesis, and signaling. LD surfaces are targeted by Perilipin (Plin) family proteins with specificity to different cells. is the most highly expressed LSD protein in liver. Livers of fasted or high-fat fed WT mice have increased triacylglycerol (TAG) and Plin2-LSDs, but plin2-/- mice are protected from hepatic steatosis during fasting or when fed high-fat diets. We examined primary aspects of TAG and fatty acid (FA) metabolism in liver and isolated primary hepatocytes from chow-fed/fasted, and diet-induced obese plin2+/+ and plin2-/- mice and show that plin2-/- mice are protective to ectopic lipid storage, regardless of mode, compared to plin2+/+, as the result of significantly elevated levels of lipolysis and mitochondrial fatty acid oxidation, without an accompanying increase in VLDL secretion. The protective phenotype of plin2-/- mice derives from the more permissive associative access of lipases to their hepatic LSDs, as compared to WT livers with Plin2-coated LSDs. We also assessed broad gene expression profiling differences in liver tissue between plin2+/+ and plin2-/- mice on both chow and HF diets. In both genotypes, the transcriptome response following a shift to high-fat diet was more affected than by genotype. In essence we conclude that diet affects both plin2-/- and plin2+/+ livers in a highly similar manner. Furthermore, although the lipid content of plin2-/- livers from HFD mice are phenotypically more similar to that of plin2+/+ livers from chow-fed mice than for HFD mice, they have transcriptomes more similar to plin2+/+ livers from HFD mice. Loss of lipid droplet protein Plin2 suppresses hepatic steatosis in mice, via increased hepatic lipolysis and mitochondrial fatty acid oxidation, but with limited variation to diet-induced hepatic gene expression.

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8
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2016
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U.S. National Inst Diabetes/Digst/Kidney
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Meena, Netra P; Kimmel, Alan R (2018) Quantification of Live Bacterial Sensing for Chemotaxis and Phagocytosis and of Macropinocytosis. Front Cell Infect Microbiol 8:62
Lee, Yun Kyung; Sohn, Jee Hyung; Han, Ji Seul et al. (2018) Perilipin 3 Deficiency Stimulates Thermogenic Beige Adipocytes Through PPAR? Activation. Diabetes 67:791-804
Meena, Netra Pal; Kimmel, Alan R (2017) Chemotactic network responses to live bacteria show independence of phagocytosis from chemoreceptor sensing. Elife 6:
Liao, Xin-Hua; Kimmel, Alan R (2017) A Unique High-Throughput Assay to Identify Novel Small Molecule Inhibitors of Chemotaxis and Migration. Curr Protoc Cell Biol 74:12.11.1-12.11.13
Feng, Yuan Z; Lund, Jenny; Li, Yuchuan et al. (2017) Loss of perilipin 2 in cultured myotubes enhances lipolysis and redirects the metabolic energy balance from glucose oxidation towards fatty acid oxidation. J Lipid Res :
Andersson, Linda; Drevinge, Christina; Mardani, Ismena et al. (2017) Deficiency in perilipin 5 reduces mitochondrial function and membrane depolarization in mouse hearts. Int J Biochem Cell Biol 91:9-13
Platt, James L; Kent, Nicholas A; Kimmel, Alan R et al. (2017) Regulation of nucleosome positioning by a CHD Type III chromatin remodeler and its relationship to developmental gene expression in Dictyostelium. Genome Res 27:591-600
Liao, Xin-Hua; Meena, Netra Pal; Southall, Noel et al. (2016) A High-Throughput, Multi-Cell Phenotype Assay for the Identification of Novel Inhibitors of Chemotaxis/Migration. Sci Rep 6:22273
Kimmel, Alan R; Sztalryd, Carole (2016) The Perilipins: Major Cytosolic Lipid Droplet-Associated Proteins and Their Roles in Cellular Lipid Storage, Mobilization, and Systemic Homeostasis. Annu Rev Nutr 36:471-509
Drevinge, Christina; Dalen, Knut T; Mannila, Maria Nastase et al. (2016) Perilipin 5 is protective in the ischemic heart. Int J Cardiol 219:446-54

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