Signalling Processes in Dictyostelium

描述（由申请人提供）：趋化性，或细胞在化学梯度上的定向运动，在广泛的细胞行为中起着重要作用。幼稚的、随机移动的细胞，如白细胞和盘基骨柱细胞，必须能够定向地感知极浅的细胞外化学引诱剂梯度，并通过建立极化的细胞骨架来做出反应，细胞骨架的前缘是基于f -肌动蛋白的突起假足或片足，后缘是收缩的。这种极化是通过外部梯度的空间受限放大，通过最接近化学引诱源的皮质区域的局部通路激活来实现的。虽然在确定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形成假足，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内进行交流，以组织更多的全局社会反应，例如Dictyostelium细胞的自分泌聚集反应，细胞响应并分泌趋化剂cAMP，导致约105个细胞的协调聚集（信号接力），或巨噬细胞和乳腺肿瘤细胞使用的旁分泌反应系统，导致两种细胞类型在转移过程中相互迁移。由于许多途径是进化保守的，我们将利用Dictyostelium实现三个重要目标：1)我们已经证明，TORC2通过激活PKB和PKBR1，整合多种输入，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传递。此外，我们发现cAMP通过激活PKA，作为负向调节TORC2/PKB:PKBR1通路的重要反馈回路。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是趋化性的重要调节因子，通过比较野生型和GSK-3空白细胞的磷酸化蛋白质组，我们鉴定出了可能的GSK-3底物，包括控制趋化性的Ras/Rap1/F-actin通路的已知组分。我们的目标是确定关键的GSK-3底物，并了解GSK-3如何控制趋化性。3)我们发现了一种新的调控途径，它在趋化过程中对假足的形成和稳定起着核心作用。我们已经证明Rab1A结合并激活Roco2是必需的，Roco2是含gtpase蛋白激酶Roco家族的成员。Roco2作用于f -肌动蛋白交联蛋白丝蛋白的上游，两者都是假足延伸所必需的。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. PUBLIC HEALTH RELEVANCE: The research will focus on understanding the basic mechanisms by which cells are able to directionally move in response to a chemical gradient or chemotaxis, a process that is used by cells during wound repair, bacterial infection, and metastasis of cancer cells. The findings illuminated by the proposed work will shed light on the molecular basis of human disease and may help, in the future, identify new targets for treatment.