Membrane Localization of Atypical Protein Kinase C during Neuroblast Polarization

神经母细胞极化过程中非典型蛋白激酶 C 的膜定位

• 批准号: 10001981
• 负责人: Bryce LaFoya
• 金额: $ 6.53万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10001981
• 关键词: 3-Dimensional; Adaptor Signaling Protein; Address; Affect; Affinity; Anaphase; Animals; Apical; Automobile Driving; Back; Binding; Biochemical; Biological Assay; Biological Models; Brain; Cell Lineage; Cell Polarity; Cell membrane; Cells; Complement component C1s; Complex; Confocal Microscopy; Cytoplasm; DAG/PE-Binding Domain; Data; Development; Drosophila genus; Ensure; Foundations; Image; In Vitro; Interphase; Learning; Length; Ligands; Lipid Binding; Lipids; Liposomes; Measures; Mediating; Membrane; Metaphase; Mitosis; Molecular; Mutagenesis; Nature; Occupations; PAR-6 protein; PARD6A gene; Pattern; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipids; Phosphotransferases; Process; Proteins; Regulation; Repression; Research Design; Resolution; Site; Testing; apical membrane; atypical protein kinase C; cell type; daughter cell; in vivo; live cell imaging; nerve stem cell; neuroblast; protein complex; protein kinase C kinase; reconstruction; recruit; stem cells; temporal measurement

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.

项目摘要 细胞极性是质膜组织成离散域。在动物细胞中，极性 因为它们高度有序的多细胞组织而显得尤为重要。为了满足 有组织的多细胞生物在极性上的作用，动物已经进化出专门的机器，称为Par 复杂的，控制极化在广泛的细胞类型。在发育过程中，干细胞必须 在它们分裂之前产生细胞多样性。Par复合体的工作是引导极化， 有丝分裂过程中的干细胞。Par复合物由衔接蛋白Par-6和非典型蛋白激酶C组成 （aPKC）。正是aPKC的催化活性负责下游组分的极化。 为了在有丝分裂开始时履行其催化职责，aPKC必须被募集到细胞膜上， 在它极化膜的地方发现了命运决定因素。该建议旨在阐明aPKC如何 与脂质的相互作用有助于其适当的定位和调节其催化活性。内 我们已经鉴定了一个称为C1结构域的脂质结合结构域。我们假设， aPKC C1结构域控制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aPKC的C1结构域的调节。具体目标：（1） 确定C1结构域自我抑制的分子内机制。(2)确定C1 域被激活。研究设计：为了表征aPKC C1结构域的自抑制机制，我们 将使用诱变策略来剖析抑制C1结构域的分子内相互作用。C1结构域 活性将通过体外aPKC对脂质体的亲和力来测量。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）来追踪荧光标记的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。从这些数据中，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 我们将探索C1激活的模式。我们将分析aPKC结合伙伴如何发挥作用， 使用脂质体结合试验检测aPKC的C1结构域的激活。此外，我们还将探讨 通过检测aPKC对不同脂质体的亲和力，发现C1结构域优先结合特定的脂质 组合物的最后，我们将跟踪磷酸肌醇脂质在极化成神经细胞中的定位， 说明aPKC是否定位于体内磷酸肌醇富集位点。了解C1的调节 域控制aPKC的本地化将为解决更大的问题提供所需的背景， 细胞如何启动极化。