Signalling Processes in Dictyostelium

描述（由申请人提供）：趋化性，或细胞沿化学梯度的定向运动，在广泛的细胞行为中起重要作用。幼稚的，随机移动的细胞，如白细胞和Dictyosteoprotein细胞必须能够定向感测极浅的细胞外化学引诱物梯度，并通过建立一个极化的细胞骨架与F-肌动蛋白为基础的突出伪足或片足的前缘和收缩后缘。这种极化是通过在最接近化学引诱物来源的皮质部位局部激活途径，通过外部梯度的空间限制性放大来实现的。 虽然在识别控制趋化性的许多成分方面取得了重大进展，但我们还不了解细胞如何感知梯度的方向并限制对前沿的反应，新聚合的F-肌动蛋白网络如何稳定以形成伪足，或者是什么调节这些事件的时间。此外，趋化细胞能够在细胞群体内进行通信，以组织更多的全局社会反应，例如网骨藻细胞中的自分泌聚集反应，其中细胞响应并随后分泌化学引诱物cAMP，导致约105个细胞的协调聚集（信号中继）或巨噬细胞和乳腺肿瘤细胞使用的旁分泌反应系统，导致转移期间两种细胞类型的相互依赖的迁移。由于许多途径在进化上是保守的，我们将使用网骨藻来实现三个重要目标：1）我们已经证明，TORC 2通过激活PKB和PKBR 1，整合多个输入，通过控制定向传感和F-肌动蛋白聚合来调节趋化性，并通过调节腺苷酸环化酶来控制信号中继。此外，我们发现cAMP通过激活PKA，作为一个重要的反馈回路来负调节TORC 2/PKB：PKBR 1途径。我们将研究这个关键反馈回路的分子基础。 2)我们发现GSK-3是一个重要的趋化调节因子，通过比较野生型和GSK-3空细胞的磷酸化蛋白质组，已经确定了推定的GSK-3底物，包括控制趋化性的Ras/Rap 1/F-actin途径的已知组分。我们的目标是定义关键的GSK- 3底物，并了解GSK-3如何控制趋化性。 3)我们已经确定了一种新的调控途径，在形成和稳定的伪足在趋化性中发挥着核心作用。我们已经表明，Rab 1A结合，并需要激活Roco 2，一个成员的Roco家族的GTP酶的蛋白激酶。Roco 2在F-肌动蛋白交联蛋白细丝蛋白的上游起作用，并且两者都是伪足延伸所需的。我们将研究这一途径的调节机制，以及它如何控制趋化性。

DESCRIPTION (provided by applicant): Chemotaxis, or directional movement of cells up a chemical gradient, plays important roles in a wide range of cellular behaviors. Naive, randomly moving cells, such as leukocytes and Dictyostelium cells must be able to directionally sense extremely shallow extracellular chemoattractant gradients and respond by establishing a polarized cytoskeleton with an F-actin-based protruding pseudopod or lamellipod at the leading edge and a contractile trailing edge. This polarization is achieved through the spatially restricted amplification of the external gradient through the localized activation of pathways at the site of the cortex closest to the chemoattractant source. While significant advancements have been made in identifying many components that control chemotaxis, we do not yet understand how cells sense the direction of the gradient and restrict responses to the leading edge, how newly polymerized F-actin networks are stabilized to form pseudopod, or what regulates the timing of these events. Furthermore, chemotaxing cells are able to communicate, within the cell population, to organize more global social responses, such as the autocrine aggregation response in Dictyostelium cells in which cells respond to and then secrete the chemoattractant cAMP leading to the coordinate aggregation of ~105 cells (signal relay) or the paracrine response system used by macrophages and mammary tumor cells, resulting in interdependent migration of the two cell types during metastasis. As many of the pathways are evolutionarily conserved, we will use Dictyostelium to achieve three important goals: 1) We have demonstrated that TORC2, through the activation of PKB and PKBR1, integrates multiple inputs to regulate chemotaxis by controlling directional sensing and F-actin polymerization, and to control signal relay through the regulation of adenylyl cyclase. Furthermore, we have found that cAMP, through the activation of PKA, acts as an important feedback loop to negatively regulate the TORC2/PKB:PKBR1 pathway. We will investigate the molecular basis of this key feedback loop. 2) We have found that GSK-3 is an important regulator of chemotaxis and by comparing the phosphoproteomes of wild-type and GSK-3 null cells have identified putative GSK-3 substrates, including known components of the Ras/Rap1/F-actin pathways that control chemotaxis. Our goal is to define key GSK- 3 substrates and understand how GSK-3 functions to control chemotaxis. 3) We have identified a novel regulatory pathway that plays a central role in forming and stabilizing the pseudopod during chemotaxis. We have shown that Rab1A binds to and is required to activate Roco2, a member of the Roco family of GTPase-containing protein kinases. Roco2 acts upstream of the F-actin cross- linking protein filamin and both are required for pseudopod extension. We will examine the mechanism by which this pathway is regulated and how it controls chemotaxis.