Elucidation of a Dictyostelium chalone

描述(申请人提供)：发育生物学中一个基本但知之甚少的问题是，当该细胞类型的细胞密度达到正确的点时，细胞如何停止增殖，无论是组织还是整个身体。从理论上讲，可以做到这一点的一种方法是，如果细胞分泌一种特定的扩散因子，抑制Tht细胞类型的增殖。当分泌细胞的密度增加时，当这种细胞类型的密度达到正确的水平时，这种称为抑素的细胞外浓度就会增加。 在这一点上，相应的高水平的Chone将阻止它们的扩散。尽管有证据表明查尔酮的存在，但人们对查尔酮的身份及其信号转导途径知之甚少。在简单的真核模型系统中，当细胞达到高密度时，即使有足够的营养物质，细胞也会停止增殖。这是由于抑素在细胞外的积累。我们对Chone进行了部分纯化，发现它是一种小于2 kDa的耐热和耐蛋白酶的阴离子分子。Chone信号转导通路涉及PTEN和CNRN磷酸酶以及BzpN转录因子，并通过阻止G2向 细胞周期的M期。我提出了三个特定的目标，将使用Dictyostelius模型系统的力量来阐明这种Chone及其信号转导途径。首先，我们将完成Chone的提纯和鉴定，并对Chone功能的理论模型进行验证。其次，我们将验证Chone调节PI3激酶/Akt通路以调节增殖的假设，并使用遗传筛选来确定该通路的其他组成部分。第三，我们将验证Chone通过抑制G2/M调节因子Cyclin B和CDc2的活性来阻断细胞周期的假设。这项工作将天然产物化学、物理和数学模型、遗传学、细胞生物学和生物化学结合在一个通用的模型系统中，将阐明抑素的分子机制。

DESCRIPTION (provided by applicant): A fundamental but poorly understood question in developmental biology is how cells stop proliferating when the density of cells of that cell type, n a tissue or the entire body, reaches the correct point. Theoretically, one way this can be accomplished is if the cells secrete a specific diffusible factor that inhibits proliferation of tht cell type. The extracellular concentration of such a factor, called a chalone, would increase as the density of the secreting cells increase, and when the density of that cell type reaches the correct point, the corresponding high levels of the chalone would stop their proliferation. Despite evidence for the existence of chalones, little is known about the identity of chalones and their signal transduction pathways. In the simple eukaryotic model system Dictyostelium discoideum, cells stop proliferating when they reach a high cell density even if adequate nutrients are presen+t. This is due to the extracellular accumulation of a chalone. We have partially purified the chalone and found that it is a heat- and protease-resistant anionic molecule smaller than 2 kDa. The chalone signal transduction pathway involves the PTEN and CnrN phosphatases and the BzpN transcription factor, and blocks proliferation by preventing the transition from the G2 to the M phase of the cell cycle. I propose three specific aims that will use the power of the Dictyostelium model system to elucidate this chalone and its signal transduction pathway. First, we will finish the purification and identify the chalone, and test theoretical models of chalone function. Second, we will test the hypothesis that the chalone regulates the PI3 kinase/ Akt pathway to regulate proliferation, and use genetic screens to identify additional components of the pathway. Third, we will test the hypothesis that the chalone blocks the cell cycle by inhibitin the activity of the G2/ M regulators Cyclin B and Cdc2. Together, this work combining natural products chemistry, physics and mathematical modeling, genetics, cell biology, and biochemistry in a versatile model system wil elucidate the molecular mechanism of a chalone.