SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

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Multi-protein complexes mTORC1 and mTORC2 are required for growth and development of eukaryotes. mTORC1 is a nutrient sensor that integrates metabolic signals and energy state to regulate cell growth/proliferation; mTORC2 primarily regulates developmental processes. Dictyostelium proliferate in rich growth media, but initiate development upon nutrient depletion. Both mTOR complexes play essential roles in Dictyostelium, where growth and developmental cycles independently require, respectively, mTORC1 or mTORC2. Many protein associations and regulatory pathways for mTORC1 and mTORC2 in Dictyostelium have context similarity to mammalian cells and specificity to inhibition by the immunosuppressive drug rapamycin. In Dictyostelium, mTORC1 function is inactivated upon starvation-induced development, but development is directly induced through rapamycin-mediated inhibition of mTORC1 activity, even in the absence of nutrient withdrawal. Pharmacologic inhibition of mTORC1, in the absence of nutrient loss, has allowed the identification of a class of essential up-regulated, developmentally-associated signaling genes and down-regulated, growth genes. We showed functional pathway regulations that integrate mTORC1/mTORC2 activities and emphasize complexity of small GTPase regulation of mTORC2 activity. Finally, epistases experiments indicate novel upstream pathway cross-talk in Dictyostelium that requires mTORC1 and mTORC2, but for separate and independent downstream functions. Cellular functions can be regulated by cell-cell interactions and influenced by extra-cellular, density-dependent signaling factors. Dictyostelium grow as individual cells in nutrient-rich sources. As nutrients become depleted, they initiate a multi-cell developmental program dependent upon a cell-density threshold. We hypothesized that novel secreted proteins may serve as density-sensing factors to promote multi-cell developmental fate decisions at a specific cell-density threshold, and use Dictyostelium in the identification of such a factor. We show that multi-cell developmental aggregation in Dictyostelium is lost upon minimal (2-fold) reduction in local cell density. Remarkably, developmental aggregation response at non-permissive cell densities is rescued by addition of conditioned media from high-density, developmentally competent cells. Using rescued aggregation of low-density cells as an assay, we purified a single, 150-kDa extra-cellular protein with density aggregation activity. MS/MS peptide sequence analysis identified the gene sequence, and cells that overexpress the full-length protein accumulate higher levels of a development promoting factor (DPF) activity than parental cells, allowing cells to aggregate at lower cell densities; cells deficient for this DPF gene lack density-dependent developmental aggregation activity and require higher cell density for cell aggregation compared to WT. Density aggregation activity co-purifies with tagged versions of DPF and tag-affinity-purified DPF possesses density aggregation activity. In mixed development with WT, cells that overexpress DPF preferentially localize at centers for multi-cell aggregation and define cell-fate choice during cytodifferentiation. Finally, we show that DPF is synthesized as a larger precursor, single-pass transmembrane protein, with the p150 fragment released by proteolytic cleavage and ectodomain shedding. The TM/cytoplasmic domain of DPF possesses cell-autonomous activity for cell-substratum adhesion and for cellular growth. Post-transcriptional processes mediated by mRNA binding proteins represent important control points in the regulation of gene expression. In mammals, mRNAs containing specific AU-rich motifs are regulated by proteins of the tristetraprolin (TTP) family, which bind to these motifs through a tandem zinc finger (TZF) domain. This binding leads to promotion of subsequent deadenylation and decay, partly through a conserved carboxyl-terminal CNOT1 binding domain. Four TTP family members are expressed in mice, and possible functional redundancy makes structure-function explorations difficult. We explored the physiological role of the single TTP family member (ttpA) expressed in Dictoystelium discoideum. Two null mutants exhibited slow growth and a multinuclear phenotype. A cysteine point mutant within the TZF domain, which should completely prevent mRNA binding, phenocopied the two null mutations exactly in terms of gene expression patterns. The carboxyl-terminal mutation, which removed the predicted CNOT1 binding domain, exhibited a phenotype indistinguishable from that of the null mutants, raising the possibility that this domain is primarily responsible for interactions with the CCR4/NOT mRNA decay apparatus in this species. Three transcripts which are highly upregulated in the loss-of-function mutants, are co-regulated during differentiation and possess the conserved AU-target motif and. These data suggest that ttpA controls an RNA "regulon", in which a single RNA binding protein co-regulates the expression of a gene group. Most cells store triacylglycerol (TAG) in cytoplasmic lipid droplets (LDs), but steroidogenic cells store primarily cholesteryl esters (CEs). The LD binding protein Plin2 regulates degradation of LDs containing TAG. To determine if Plin2 is also regulates CEs, we characterized adrenals of Plin2 null mice. We observed significantly enlarged adrenal glands, increased size and numbers of CE-LD, elevated cellular free cholesterol levels, and increased expression of macrophage markers and genes facilitating reverse cholesterol transport in adrenals of Plin2-/- mice as compared to Plin2+/+ mice. Mobilization of CE-LDs and secretion of corticosterone induced by ACTH stimulation or starvation were unaffected by the lack of Plin2 in young mice. There was also accumulation of ceroid-like structures containing multilamellar bodies in the adrenal cortex-medulla boundary accompanied with high levels of adrenal phosphatidylglycerols, implying that these lipids are components of the accumulated ceroid-like structures. Our findings indicate that Plin2 is important for regulation of CE-LDs and cellular cholesterol balance in adrenal cortical cells. Perilipin 5 (Plin5) is abundantly expressed in heart where it binds to cardiac lipid droplets (LDs) and facilitates physical interaction between LDs and mitochondria. We isolated cardiomyocytes from adult Plin5+/+ and Plin5-/- mice to study the role of Plin5 for tolerance to hypoxia and ischemia, fatty acid uptake, LD accumulation, and fatty acid oxidation. Cardiomyocytes from Plin5-/- mice stored less LDs than Plin5+/+ following oleic acid stimulation, but comparable levels to Plin5+/+ cardiomyocytes when adipose triglyceride lipase activity was inhibited. The ability to oxidize fatty acids into CO2 was similar between Plin5+/+ and Plin5-/- cardiomyocytes, but Plin5-/- cardiomyocytes had a transient increase in intracellular fatty acid oxidation intermediates. After pre-incubation with oleic acids, Plin5-/- cardiomyocytes retained a higher content of glycogen and showed improved tolerance to hypoxia. Deletion of Plin5 had no important effect on ventricular pressures or infarct size after ischemia in isolated, perfused hearts. Old Plin5-/- mice had reduced levels of cardiac triacylglycerides, increased heart weight and elevated Myh7 and Cd36 mRNA expression compared to Plin5+/+. Still, most other genes involved in fatty acid oxidation, glycogen metabolism and glucose utilization were essentially unchanged by age or removal of Plin5. Plin5 seems to facilitate cardiac LD storage primarily by repressing adipose triglyceride lipase activity without altering cardiac fatty acid oxidation capacity. Expression of Plin5 and cardiac LD content does not appear to be of vital importance for tolerance to acute hypoxia and ischemia.