SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

激酶mTORC1和AMPK作为能量传感器，控制营养反应和细胞生长。营养水平的变化会影响多种转录网络，这使得确定调节细胞生长或通过营养变化转向发育的下游途径具有挑战性。Dictyostelium的生命周期为研究mTORC1信号在生长发育中的作用提供了一个很好的模型。盘基骨柱在营养丰富的培养基中以单细胞形式生长，但在营养缺乏时，生长停止，细胞进入多细胞发育程序。虽然近一半的基因组显示在营养去除后基因表达发生变化，但我们假设并非所有这些基因都是程序发展所需的。通过单独操纵mTORC1活性，而不去除营养物质，我们专注于在生长和发育之间切换以调节细胞命运决定所需的核心基因网络。为了确定发育必需基因，我们寻求在没有营养损失的情况下促进发育的方法。我们首先检测了盘基骨菌在快速生长和饥饿诱导发育阶段的mTORC1和AMPK的活性，发现它们在各种条件下表现出相互调节的模式。使用这些作为初始读出，我们确定了富培养基条件，促进细胞快速生长，但在雷帕霉素灭活mTORC1后，导致生长/发育切换。对细胞命运转换过程中基因表达的检测表明，大多数饥饿调节基因的表达变化并不需要发育诱导。大约有1000个基因在雷帕霉素的作用下被下调，这些基因包括一个涉及核糖体生物发生、蛋白质合成和细胞周期过程的细胞生长网络。相反，通过雷帕霉素处理，500个基因的上调定义了发育诱导的必要信号通路，其中135个蛋白产物通过明确的cAMP/PKA网络相交。我们发现的许多雷帕霉素诱导基因目前尚未分类，对其中5个基因的突变分析表明，这是一种对发育调控至关重要的新基因类别。我们发现，在缺乏营养的情况下，操纵mTORC1/AMPK的活性足以在盘基骨柱中实现生长到发育的命运转换，这为识别早期发育所必需的转录网络和信号通路提供了一种方法。

Kinases mTORC1 and AMPK act as energy sensors, controlling nutrient responses and cellular growth. Changes in nutrient levels affect diverse transcriptional networks, making it challenging to identify downstream paths that regulate cellular growth or a switch to development via nutrient variation. The life cycle of Dictyostelium presents an excellent model to study the mTORC1 signaling function for growth and development. Dictyostelium grow as single cells in nutrient-rich media, but, upon nutrient withdrawal, growth ceases and cells enter a program for multi-cell development. While nearly half the genome shows gene expression changes upon nutrient removal, we hypothesized that not all of these genes are required for the switch to program development. Through manipulation of mTORC1 activity alone, without nutrient removal, we focused on a core network of genes that are required for switching between growth and development for regulation of cell fate decisions. To identify developmentally essential genes, we sought ways to promote development in the absence of nutrient loss. We first examined the activities of mTORC1 and AMPK in Dictyostelium during phases of rapid growth and starvation-induced development and showed they exhibited reciprocal patterns of regulation under various conditions. Using these as initial readouts, we identified rich media conditions that promoted rapid cell growth but, upon mTORC1 inactivation by rapamycin, led to a growth/development switch. Examination of gene expression during cell fate switching showed that changes in expression of most starvation-regulated genes were not required for developmental induction. Approximately 1000 genes which become downregulated upon rapamycin treatment comprise a cellular growth network involving ribosome biogenesis, protein synthesis, and cell cycle processes. Conversely, the upregulation of 500 genes by rapamycin treatment defines essential signaling pathways for developmental induction, and 135 of their protein products intersect through the well-defined cAMP/PKA network. Many of the rapamycin-induced genes we found are currently unclassified, and mutation analyses of 5 such genes suggest a novel gene class essential for developmental regulation. We show that manipulating activities of mTORC1/AMPK in the absence of nutrient withdrawal is sufficient for a growth-to-developmental fate switch in Dictyostelium, providing a means to identify transcriptional networks and signaling pathways essential for early development. Initial immunological defense mechanisms to pathogen invasion rely on innate pathways of chemotaxis and phagocytosis, original to ancient phagocytes. Chemotaxis and cell migration play pivotal roles in normal physiological processes such as embryogenesis, inflammation, and wound healing, as well as in pathological processes including chronic inflammatory disease and cancer metastasis. Although chemotaxis has been well-studied in mammalian and model systems using purified chemoattractants in defined conditions, directed movement toward live bacteria has been more difficult to assess. Dictyostelium discoideum is a professional phagocyte that chemotaxes toward bacteria during growth-phase in a process to locate nutrient sources. Using Dictyostelium as a model, we have developed a system that is able to quantify chemotaxis to very high sensitivity. Here, Dictyostelium can detect various chemoattractants at concentrations <1 nM. Given this exceedingly sensitive signal response, Dictyostelium will migrate directionally toward live gram positive and gram negative bacteria, in a highly quantifiable manner, and dependent upon bacterially-secreted chemoattractants. Additionally, we have developed a real-time, quantitative assay for phagocytosis of live gram positive and gram negative bacteria. To extend the analyses of endocytic functions, we further modified the system to quantify cellular uptake via macropinocytosis of smaller (<100 kDa) molecules. These various approaches provide novel means to dissect potential for identification of novel chemoattractants and mechanistic factors that are essential for chemotaxis, phagocytosis, and/or macropinocytosis and for more detailed understanding in host-pathogen interactive defenses. Cell-cell interactions and response are enhanced by increased cell density. We were interested to identify novel secreted proteins that accumulate in parallel to the collective local cell population and that can direct developmental decisions. We chose Dictyostelium, which grow as individual cells in rich nutrient sources, but initiate a multi-cell developmental program as nutrients become depleted. Cell density sufficiency is critical to multi-cell formation in Dictyostelium, and we hypothesized that novel secreted proteins may serve as density-sensing factors to promote multi-cell developmental fate decisions at specific cell-density thresholds. We have purified a novel secreted protein, DPF, that acts as a density-sensing factor for development and functions to define local collective thresholds for Dictyostelium development, to facilitate cell-cell communication and multi-cell formation. Regions of high DPF expression become centers for cell-cell signal-response, multi-cell formation, and cell-fate determination. Additionally, DPF has separate cell autonomous functions for regulation of cellular adhesion and growth. Myocardial dysfunction is commonly associated with accumulation of cardiac lipid droplets (LDs). Perilipin 2 (Plin2) is a LD protein that is involved in LD formation, stability and trafficking events within the cell. Even though Plin2 is highly expressed in the heart, little is known about its role in myocardial lipid storage. A recent report shows that cardiac overexpression of Plin2 result in massive myocardial steatosis suggesting that Plin2 stabilizes LDs. In this study, we hypothesized that deficiency in Plin2 would result in reduced myocardial lipid storage. In contrast to our hypothesis, we found increased accumulation of triglycerides in hearts, and specifically in cardiomyocytes, from Plin2-/- mice. Although Plin2-/- mice had markedly enhanced lipid levels in the heart, they had normal heart function under baseline conditions and under mild stress. However, after an induced myocardial infarction, stroke volume and cardiac output were reduced in Plin2-/- mice compared with Plin2+/+ mice. We further demonstrated that the increased triglyceride accumulation in Plin2-deficient hearts was caused by altered lipophagy. Together, our data show that Plin2 is important for proper hydrolysis of LDs. Beige adipocytes can dissipate energy as heat. Elaborate communication between metabolism and gene expression is important in the regulation of beige adipocytes. Although lipid droplet (LD) binding proteins play important roles in adipose tissue biology, it remains unknown whether perilipin 3 (Plin3) is involved in the regulation of beige adipocyte formation and thermogenic activities. In this study, we demonstrate that Plin3 ablation stimulates beige adipocytes and thermogenic gene expression in inguinal white adipose tissue (iWAT). Compared with wild-type mice, Plin3 knockout mice were cold tolerant and displayed enhanced basal and stimulated lipolysis in iWAT, inducing peroxisome proliferator-activated receptor (PPAR) activation. In adipocytes, Plin3 deficiency promoted PPAR target gene and uncoupling protein 1 expression and multilocular LD formation upon cold stimulus. Moreover, fibroblast growth factor 21 expression and secretion were upregulated, which was attributable to activated PPAR in Plin3-deficient adipocytes. These data suggest that Plin3 acts as an intrinsic protective factor preventing futile beige adipocyte formation by limiting lipid metabolism and thermogenic gene expression.