Global stimulation of Dictyostelium with different chemoattractants elicits multiple transient signaling responses, including synthesis of cAMP and cGMP, actin polymerization, activation of kinases ERK2, TORC2, and PI3K, and Ras-GTP accumulation;mechanisms that down-regulate these responses are poorly understood. Here we examine transient activation of TORC2 in response to chemically distinct chemoattractants, cAMP and folate, and suggest that TORC2 is regulated by adaptive, de-sensitizing responses to stimulatory ligands that function independently of TORC2 inactivation. Cells with acquired insensitivity to one chemoattractant remain fully responsive to TORC2 activation if stimulated with the other ligand. Thus, TORC2 responses to cAMP or folate are not cross-inhibitory. Using a series of signaling mutants, we show that folate and cAMP activate TORC2 through an identical GEF/Ras pathway, but separate receptors and G protein couplings. Since the common GEF/Ras pathway also remains fully responsive to one chemoattractant after de-sensitization to the other, GEF/Ras must act downstream and independently of adaptation to persistent ligand stimulation. When initial chemoattractant concentrations are immediately diluted, cells rapidly regain full responsiveness. We suggest that ligand adaptation functions in upstream inhibitory pathways that involve chemoattractant-specific receptor/G protein complexes and regulate multiple response pathways. The TOR protein kinase functions in two distinct complexes, TOR Complexes 1 (TORC1) and 2 (TORC2). TORC1 is required for growth in response to growth factors, nutrients, and cellular energy state;TORC2 is documented to regulate AKT signaling, which can modulate cytoskeletal polarization. In its ecological niche, Dictyostelium engulf bacteria and yeast for nutrient capture. Despite the essential role of TORC1 in control of cellular growth, we show that nutrient particle capture, phagocytosis, in Dictyostelium is independent of TORC1 mediated nutrient sensing and growth regulation. However, loss of Dictyostelium TORC2 components Rictor/Pia, SIN1/RIP3, and Lst8 promotes nutrient particle uptake;inactivation of TORC2 leads to increased efficiency and speed of phagocytosis. In contrast to phagocytosis, we show that macropinocytosis, an AKT-dependent process for cellular uptake of fluid phase nutrients, is not regulated by either TOR complex. The integrated and balanced regulations of TORC1 and TORC2 may be critical in Dictyostelium to coordinate growth and energy needs with other essential TOR-regulated processes. Chemotaxis and cell migration are fundamental and universal eukaryotic processes that are essential for embryogenesis, immunity, cell renewal, and wound healing and that impact many diseases, including cancer metastasis and chronic inflammation. Novel chemotaxis inhibitors are valuable for mechanistic studies and as a basis to develop new therapeutics for the treatment of chemotaxis-related diseases. We have developed a novel chemotaxis/aggregation assay for identification of chemotaxis inhibitors, using a rapid laser scanning cytometric plate reader and Dictyostelium in a unique phenotypic and fluorescent screen. Under defined conditions, single-celled Dictyostelium secrete chemoattractants, migrate toward each other, and form large multi-cellular aggregates. Using a GFP expression marker, which is only induced in cells that have undergone chemotaxis and aggregation, we can quantify chemotaxis with a laser scanning cytometry plate reader in a high-throughput 1536-well plate format. We applied the assay to a test screen of 1,280 known compounds and identified two putative chemotaxis inhibitors. This miniaturized chemotaxis/aggregation assay is highly suitable for high-throughput screening of very large libraries of small molecules to identify novel classes of chemotaxis inhibitors. Wnt-signaling regulates multiple pathways in metazoa. As a morphogen, Wnt functions as an effective inhibitor of GSK3, a developmental switch that regulates cell fate determination. Wnts also organize planar cell polarity and coordinate cell migration, but a dependent role for GSK3 is not clear. In many respects, extracellular cAMP in Dictyostelium is a functional Wnt analog. cAMP regulates activation/de-activation of GSK3 which, in turn, establishes developmental patterning. In addition, Dictyostelium cells polarize in response to cAMP. Previously, we had shown that ZAK1 is an activating tyrosine kinase of GSK3 and now identify ZAK2, the other tyrosine kinase in the cAMP-activation pathway for GSK3;no additional family members exist. We also show that tyrosine phosphorylation/activation of GSK3 by ZAK2 and ZAK1 are differentially required to regulate GSK3 within distinct differentiated cell populations. ZAK2/GSK3 can also act in a non-autonomous manner to regulate alternative cell fate decisions that further parallels mechanisms of non-autonomy during Wnt signaling. Finally, we show that efficient polarization of Dictyostelium toward cAMP depends on ZAK-mediated tyrosine phosphorylation of GSK3. Our results extend the complexity of GSK3 signaling during development. We suggest that combinatorial regulation of GSK3 guides cell polarity, directional cell migration, and cell fate specification in Dictyostelium and potentially other systems. Perilipin family proteins (Plins) coat the surface of intracellular neutral lipid storage droplets in various cell types. Studies across diverse species demonstrate that Plins regulate lipid storage metabolism through recruitment of lipases and other regulatory proteins to lipid droplet surfaces. Mammalian genomes encode 5 distinct Plin gene members and additional proteins forms derived from specific mRNA splice variants. However, it is not known if the different Plins have distinct functional properties. Using biochemistry, cellular imaging, and flow cytometry, we now show that within individual cells of various types, the different Plin proteins preferentially sequester to separate pools of lipid storage droplets. By examining ectopically expressed GFP fusions and all endogenous Plin protein forms, we demonstrate that different Plins sequester to lipid droplets, comprised distinctly of either triacylcerides or of cholesterol esters. Our data suggest diversity of Plin function, alter previous assumptions about shared collective actions of the Plins, and indicate that each Plin can have separate and unique functions.

Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2011
Total Cost
$1,319,304
Indirect Cost
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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
Meena, Netra Pal; Kimmel, Alan R (2017) Chemotactic network responses to live bacteria show independence of phagocytosis from chemoreceptor sensing. Elife 6:
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
Meena, Netra Pal; Kimmel, Alan R (2016) Biochemical Responses to Chemically Distinct Chemoattractants During the Growth and Development of Dictyostelium. Methods Mol Biol 1407:141-51
Kimmel, Alan R; Sztalryd, Carole (2014) Perilipin 5, a lipid droplet protein adapted to mitochondrial energy utilization. Curr Opin Lipidol 25:110-7

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