Protein/nucleic Acid Interactions In Vertebrate Embryogenesis

脊椎动物胚胎发生中的蛋白质/核酸相互作用

• 批准号: 7968547
• 负责人: THOMAS D sargent
• 金额: $ 71.87万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7968547
• 关键词: Acetylation; Actins; Affect; Architecture; Binding; Cartilage; Cell Adhesion Molecules; Cell Line; Cells; Cellular Structures; Cephalic; Chromatin; Congenital Abnormality; Deacetylation; Defect; Development; Embryo; Embryonic Development; Epidermis; Face; Failure; Focal Adhesions; Gene Expression Regulation; Gene Targeting; Genes; Goals; Head; Histone Acetylation; Jaw; Journals; LIM Domain Kinase 1; Laboratories; Lead; Link; Mediating; Microfilaments; Microtubule Proteins; Microtubules; Modification; Molecular; Monomeric GTP-Binding Proteins; Motor; Mus; Names; Nature; Neural Crest; Nucleic Acids; Nuts; Peer Review; Peripheral Nervous System; Phosphorylation; Phosphotransferases; Play; Protein Family; Protein Kinase; Proteins; Publications; Publishing; Regulation; Reporting; Research; Role; Screening procedure; Shapes; Site; Skeleton; Staining method; Stains; Stress Fibers; TFAP2A gene; Testing; Tissues; Work; Xenopus; Yeasts; alpha Tubulin; base; blastomere structure; bone; cell behavior; cofilin; craniofacial; embryo cell; follow-up; insight; intercellular communication; migration; novel; p21 activated kinase; research study; rho; tool; transcription factor; yeast two hybrid system; zebrafish development

The NC plays a critical role in the developmental of the vertebrate head, face and jaws, providing the bulk of the craniofacial skeleton as well as peripheral nervous system and other cranial tissues. Normal craniofacial development depends on proper induction, migration and differentiation of NC cells and derivatives. Deficiencies at any of these steps, whether due to intrinsic defects in NC itself, or in failure of NC cells to interact properly with adjacent tissues, can lead to birth defects: up to a third of all congenital malformations are craniofacial in nature. In this project we have used a key NC control factor, TFAP2, as a molecular tool to identify target genes encoding proteins that are required for NC migration and differentiation into craniofacial tissue. The three genes that comprise the focus of this research are either themselves novel (PCNS and Inka) or have not previously been shown to function in NC (MyoX), and have an excellent potential to yield new insights into craniofacial development. In particular, our findings with Inka promise to lead to a new link between cytoskeletal regulation through small GTPases and PAK4 and regulation of gene expression through modulation of cell-cell signaling. In the previous two years we completed and published our studies on PCNS and MyoX, and now focus on focus on Inka and its interaction partners in cells and embryos. Using yeast two-hybrid screening we discovered that Inka interacts with p21-activated kinase 4 (PAK4). This kinase is part of a family of proteins that mediate cell-cell signaling through the small GTPase molecules Rac, Rho and Cdc42. We have found that stable expression of mouse Inca in NIH3T3 cells results in inhibition of PAK4 phosphorylation. PAK4 has been reported to bind to actin filaments and regulate their dynamic remodeling through LIM kinase 1 and cofilin. If Inka controls PAK4 activity, this would predict an effect on the microfilament network in cells. We have tested and confirmed this hypothesis by examining phalloidin staining of a stable Inca expressing NIH3T3 line and showing that these cells have enhanced stress fiber and focal adhesion networks, and also migrate more rapidly than controls. Rescue/reversion experiments showed that these functions depend on the kinase activity of PAK4. Several lines of evidence link the regulation of microfilaments and microtubules, suggesting the possibility that Inka might also affect microtubule dynamics, either indirectly by altering microfilaments or by a more direct mechanism. This was also investigated in the Inca expressing cell lines, and also in Xenopus embryos. We discovered that the level of alpha-tubulin acetylation is inhibited by Inka. This also appears to be mediated via PAK4, but via a mechanism that is independent of the kinase activity. The inhibition appears to be exerted at the level of acetylation, rather than the deacetylation step that has been characterized by other laboratories. Interestingly, Inka also inhibits the acetylation of histones, suggesting a possible role in the control of chromatin architecture. Such a molecular link between the regulation of gene expression and cytoskeletal dynamics would be represent a novel mechanism for coordinating cell behavior and differentiation.

NC在脊椎动物头部、面部和颌骨的发育中起着关键作用，提供了大部分颅面骨骼以及周围神经系统和其他颅组织。 正常的颅面发育依赖于NC细胞及其衍生物的适当诱导、迁移和分化。 在任何这些步骤中的缺陷，无论是由于NC本身的内在缺陷，还是由于NC细胞与邻近组织的相互作用失败，都可能导致出生缺陷：高达三分之一的先天性畸形是颅面畸形。 在这个项目中，我们使用了一个关键的NC控制因子，TFAP 2，作为一种分子工具，以确定目标基因编码的蛋白质，所需的NC迁移和分化成颅面组织。 构成这项研究重点的三个基因要么本身是新的（PCNS和Inka），要么以前没有被证明在NC中起作用（MyoX），并且具有很好的潜力，可以对颅面发育产生新的见解。 特别是，我们的研究结果与Inka的承诺，导致通过小GTP酶和PAK 4的细胞骨架调节和基因表达的调节，通过细胞-细胞信号的调制之间的新的联系。 在过去的两年中，我们完成并发表了我们对PCNS和MyoX的研究，现在专注于Inka及其在细胞和胚胎中的相互作用伙伴。 使用酵母双杂交筛选，我们发现Inka与p21激活的激酶4（PAK 4）相互作用。 这种激酶是通过小GT3分子Rac、Rho和Cdc 42介导细胞-细胞信号传导的蛋白质家族的一部分。 我们已经发现小鼠印加在NIH 3 T3细胞中的稳定表达导致PAK 4磷酸化的抑制。 PAK 4与肌动蛋白丝结合，通过LIM激酶1和cofilin调节肌动蛋白丝的动态重塑。 如果Inka控制PAK 4活性，这将预测对细胞中微丝网络的影响。 我们已经通过检查稳定表达NIH 3 T3的印加细胞系的鬼笔环肽染色，并显示这些细胞具有增强的应力纤维和粘着斑网络，并且比对照组迁移更快，来测试和证实这一假设。 拯救/回复实验表明，这些功能依赖于PAK 4的激酶活性。 有几条证据将微丝和微管的调节联系起来，表明Inka也可能通过改变微丝或更直接的机制间接影响微管动力学。 这也在表达印加的细胞系和非洲爪蟾胚胎中进行了研究。 我们发现Inka抑制了α-微管蛋白乙酰化水平。 这似乎也是通过PAK 4介导的，但通过一种不依赖于激酶活性的机制。 抑制作用似乎是在乙酰化水平上发挥作用，而不是在其他实验室表征的脱乙酰化步骤上发挥作用。 有趣的是，Inka还抑制组蛋白的乙酰化，这表明它可能在控制染色质结构中发挥作用。 基因表达调控和细胞骨架动力学之间的这种分子联系将代表协调细胞行为和分化的新机制。