Protein/nucleic Acid Interactions In Vertebrate Embryogenesis

NC在脊椎动物头部、面部和颌骨的发育中起着至关重要的作用，它提供了颅面骨骼的大部分以及周围神经系统和其他颅骨组织。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。这些步骤中的任何一个步骤的缺陷，无论是由于NC本身的固有缺陷，还是由于NC细胞无法与邻近组织正确地相互作用，都可能导致出生缺陷：多达三分之一的先天性畸形本质上是颅面畸形。在这个项目中，我们使用了一个关键的NC控制因子TFAP2作为分子工具来识别编码NC迁移和分化到头面部组织所需的蛋白质的靶基因。构成这项研究重点的三个基因本身都是新的(PCNS和INKA)，或者以前没有被证明在NC(MyoX)中起作用，并且有很好的潜力对颅面发育产生新的见解。特别是，我们对INKA的发现有望在通过小GTP酶和PAK4调节细胞骨架和通过调节细胞-细胞信号调节基因表达之间建立新的联系。在前两年，我们完成并发表了我们对PCNS和MyoX的研究，现在重点关注Inka及其在细胞和胚胎中的相互作用伙伴。 通过酵母双杂交筛选，我们发现INKA与p21激活的蛋白4(PAK4)相互作用。该激酶是通过小GTP酶分子Rac、Rho和CDc42介导细胞-细胞信号转导的蛋白质家族的一部分。我们发现，小鼠INCA在NIH3T3细胞中稳定表达会抑制PAK4的磷酸化。据报道，PAK4与肌动蛋白细丝结合，并通过LIM激酶1和粘连蛋白调节其动态重构。如果INKA控制了PAK4的活性，这将预示着对细胞内微丝网络的影响。我们通过检测稳定表达NIH3T3的印加细胞系的鬼臼蛋白染色来验证并证实了这一假设，并表明这些细胞增强了应力纤维和焦点黏附网络，而且迁移速度也比对照组更快。救援/逆转实验表明，这些功能依赖于PAK4的激酶活性。 有几条证据将微丝和微管的调节联系在一起，这表明INKA也可能通过改变微丝或通过更直接的机制间接影响微管动力学。这一点也在印加表达细胞系中进行了研究，在非洲爪哇胚胎中也进行了研究。我们发现INKA对α-微管蛋白乙酰化水平有抑制作用。这似乎也是通过PAK4介导的，但通过一种独立于激酶活性的机制。这种抑制似乎是在乙酰化水平上施加的，而不是其他实验室已经确定的脱乙酰化步骤。有趣的是，Inka还抑制组蛋白的乙酰化，这表明它可能在染色质结构的控制中发挥作用。这种基因表达调控和细胞骨架动力学之间的分子联系将代表着一种协调细胞行为和分化的新机制。

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.