The actin binding proteins Calponin 2 and 3: effectors of Wnt/PCP-mediated cell migration and neural tube closure

肌动蛋白结合蛋白 Calponin 2 和 3：Wnt/PCP 介导的细胞迁移和神经管闭合的效应子

• 批准号: 321791725
• 负责人: Professor Dr. Martin Blum
• 金额: --
• 依托单位: Fachgebiet Zoologie (190z)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/321791725?language=en
• 关键词: actin; binding; proteins; Calponin; effectors

Dynamic changes of the actin cytoskeleton drive the morphogenetic processes which shape the vertebrate embryo during development. Migration of neural crest cells (NCCs) and closure of the neural tube represent two central such processes. Both are regulated by Wnt/planar cell polarity (PCP) signaling. The calponins (Cnn) represent an evolutionarily highly conserved but poorly characterized family of three actin-binding proteins. While several studies have demonstrated the ability of Cnn proteins to modulate the dynamics of the actin cytoskeleton in vitro, their in vivo functions and regulators have remained elusive. We have recently demonstrated a novel role for Cnn2 in promoting the migration of NCCs in chick and frog embryos. Cnn2 function was found to be regulated via the activity of the Wnt/PCP mediator RhoA kinase (ROCK) at the rear end of the emigrating cell, which resulted in the polarization of Cnn2 to the cell's leading edge. Our preliminary data also support a role of Cnn3 in promoting NCC migration and differentiation in Xenopus and chick embryos. Moreover, Cnn3 function was found to be required for neural tube closure, as its loss-of-function resulted in lack of apical constriction resulting in neural tube closure defects and non-polarized morphology of the cells. A recently published but so far uncharacterized conditional Cnn3 knockout mouse displays exencephaly as well, demonstrating the conserved nature of Cnn3 function in vertebrate neural tube closure. Based on these findings, we hypothesize that Cnn2 and Cnn3 act as mediators of Wnt/PCP/ROCK signaling to promote actin-dynamics essential for NCC migration and neural tube closure. To determine the function and regulation of Cnn2 and Cnn3 we aim at (I) identifying the relevant ROCK target sequences in Cnn2; (II) studying the relevance of Cnn2 degradation/polarization in NCCs via ROCK and proteasomal pathways active in this process; (III) determining the role of Cnn3 in NCC development and differentiation; (IV) revealing the function of Wnt/PCP-mediated Cnn3 function in neural tube closure. To achieve these goals, a combination of advanced developmental, molecular and cellular approaches will be utilized in chick, frog and mouse embryos in vivo and ex vivo. Based on our extensive preliminary and published data and the experimental plan, we expect to reveal that Cnn2 and Cnn3 act as essential mediators of Wnt/PCP signaling upstream of actin dynamics during morphogenesis. The anticipated results will contribute important new insights into the understanding of cytoskeletal remodeling during morphogenetic processes in vivo.

肌动蛋白细胞骨架的动态变化驱动了脊椎动物胚胎发育过程中的形态发生过程。神经嵴细胞（NCC）的迁移和神经管的关闭代表了两个中心的过程。两者都受Wnt/平面细胞极性（PCP）信号传导调节。Calponins（Cnn）是一个进化上高度保守但特征不明显的三种肌动蛋白结合蛋白家族。虽然一些研究已经证明了Cnn蛋白在体外调节肌动蛋白细胞骨架动力学的能力，但它们在体内的功能和调节剂仍然难以捉摸。我们最近已经证明了Cnn 2在促进鸡和青蛙胚胎中NCC迁移中的新作用。发现Cnn 2功能通过在迁移细胞后端的Wnt/PCP介体RhoA激酶（ROCK）的活性来调节，这导致Cnn 2向细胞前缘的极化。我们的初步数据也支持Cnn 3在促进非洲爪蟾和鸡胚NCC迁移和分化中的作用。此外，发现Cnn 3功能是神经管闭合所需的，因为其功能丧失导致缺乏顶端收缩，从而导致神经管闭合缺陷和细胞的非极化形态。最近发表的，但到目前为止尚未表征的条件Cnn 3基因敲除小鼠显示露脑，以及，证明了保守的性质Cnn 3功能在脊椎动物神经管关闭。基于这些发现，我们假设Cnn 2和Cnn 3作为Wnt/PCP/ROCK信号传导的介质，促进NCC迁移和神经管闭合所必需的肌动蛋白动力学。 为了确定Cnn 2和Cnn 3的功能和调节，我们的目标是（I）鉴定Cnn 2中的相关ROCK靶序列;（II）研究NCC中Cnn 2降解/极化通过ROCK和在该过程中活跃的蛋白酶体途径的相关性;（III）确定Cnn 3在NCC发育和分化中的作用;（IV）揭示Wnt/PCP介导的Cnn 3功能在神经管闭合中的功能。为了实现这些目标，先进的发育，分子和细胞方法的组合将在鸡，青蛙和小鼠胚胎体内和体外使用。基于我们广泛的初步和已发表的数据和实验计划，我们希望揭示Cnn 2和Cnn 3作为Wnt/PCP信号上游肌动蛋白动力学在形态发生过程中的重要介质。预期的结果将有助于重要的新见解的理解细胞骨架重塑过程中的形态发生在体内。