Membrane Localization of Atypical Protein Kinase C during Neuroblast Polarization

项目总结 细胞膜的极性是将质膜组织成不连续的区域。在动物细胞中，极性是 尤其重要的是它们高度有序的多细胞组织。为了满足这些需求 在极性上组织了多细胞的地方，动物进化出了特殊的机械，称为PAR 复杂，它控制着多种细胞类型的极化。在发育过程中，干细胞必须极化 在它们分裂以产生细胞多样性之前。PAR复合体的工作是将偏振引导到 有丝分裂中的干细胞。PAR复合体由适配蛋白PAR-6和非典型蛋白激酶C组成 (APKC)。正是aPKC的催化活性导致了下游组分的极化。 为了在有丝分裂开始时履行其催化职责，必须将a PKC招募到细胞膜上， 在它极化的膜上发现了命运的决定因素。这项建议旨在阐明aPKC如何 与脂类的相互作用有助于其催化活性的适当定位和调节。在 APKC我们已经确定了一个称为C1域的脂结合结构域。我们假设，对 APKC的C1域调控着aPKC的膜募集。我们建议通过以下方式来检验这一假设 在分子和细胞水平上表征了aPKC的C1域的调控。具体目标：(1) 确定C1域被自动抑制的分子内机制。(2)确定c1如何 域已激活。研究设计：为了表征aPKC的C1域的自抑制机制，我们 将使用突变策略来剖析抑制C1域的分子内相互作用。C1域 活性将通过aPKC对脂质体的亲和力在体外进行测量。激酶活性分析将使我们能够 揭示脂质结合如何影响aPKC的催化活性。我们将使用果蝇神经母细胞(神经干细胞 细胞)，以跟踪荧光标记的aPKC在体内的定位。我们将在高时间(每个 20秒)和使用活细胞共聚焦显微镜的空间分辨率。根据这些数据，我们将组成3- 三维重建以可视化aPKC的体内定位动力学。除了评估c1 抑制，我们将探索C1的激活模式。我们将分析aPKC绑定伙伴如何做出贡献 用脂质体结合试验检测对aPKC的C1域的激活。此外，我们还将探讨 通过检测aPKC与不同脂类脂质体的亲和力，C1域优先结合特定的脂类 作文。最后，我们将追踪肌醇磷脂在极化神经母细胞中的定位，以便 如果aPKC定位于肌醇磷脂在体内的浓缩部位。学习如何调节C1值 域控制的本地化将为解决以下更大的问题提供所需的背景 细胞如何启动极化。

PROJECT SUMMARY Cell polarity is the organization of the plasma membrane into discrete domains. In animal cells, polarity is particularly important because of their highly ordered, multicellular organization. In order to meet the demands that organized multicellularity places on polarity, animals have evolved specialized machinery, known as the Par complex, that controls polarization in a broad range of cell types. During development, stem cells must polarize before they divide in order to generate cellular diversity. It is the job of the Par complex to direct polarization in stem cells during mitosis. The Par complex consists of the adapter protein Par-6 and atypical Protein Kinase C (aPKC). It is the catalytic activity of aPKC which is responsible for the polarization of downstream components. In order to perform its catalytic duties at the start of mitosis, aPKC must get recruited to the cell membrane, where it polarizes membrane found fate determinants. This proposal is directed at elucidating how aPKC's interactions with lipids contribute to its proper localization and regulation of its catalytic activity. Within aPKC we have identified a lipid binding domain called the C1 domain. We hypothesize that regulation of the aPKC C1 domain governs the membrane recruitment of aPKC. We propose to test this hypothesis by characterizing the regulation of aPKC's C1 domain both at the molecular and cellular levels. Specific Aims: (1) Determine the intramolecular mechanism by which the C1 domain is autoinhibited. (2) Determine how the C1 domain is activated. Research design: To characterize the autoinhibition mechanism of aPKC's C1 domain, we will use mutagenesis strategies to dissect the intramolecular interactions that repress the C1 domain. C1 domain activity will be measured through aPKC's affinity for liposomes in vitro. Kinase activity assays will allow us to uncover how lipid binding affects the catalytic activity of aPKC. We will use Drosophila neuroblasts (neural stem cells) to track the localization of fluorescently-tagged aPKC in vivo. We will image aPKC at high temporal (every 20 seconds) and spatial resolution using live-cell confocal microscopy. From this data, we will compose 3- dimensional reconstructions to visualize the in vivo localization dynamics of aPKC. In addition to evaluating C1 repression, we will explore the modes of C1 activation. We will analyze how aPKC binding partners contribute to the activation of aPKC's C1 domain using liposome binding assays. Additionally, we will explore whether the C1 domain preferentially binds specific lipids by testing aPKC's affinity for liposomes with different lipid compositions. Finally, we will track the localization of phosphoinositide lipids in polarizing neuroblasts in order to address if aPKC localizes to sites of phosphoinositide enrichment in vivo. Learning how the regulation of the C1 domain controls aPKC's localization will provide the required context for addressing larger questions concerning how cells initiate polarization.