RUI: Elucidating Regulatory Mechanisms in the Diaphanous-related Formin Proteins using an Integrated Approach to Undergraduate Research and Education

透明相关形成蛋白（DRFs）是一个保守的Rho GT3蛋白家族，它协调肌动蛋白和微管细胞骨架网络。 因此，这些蛋白质影响细胞的形状、运动性和胞质分裂;并且它们仅在响应细胞信号时被调节和激活是至关重要的。 这些蛋白质是自抑制性的，通常保持在非活性状态。 DRF蛋白通过Rho GTP酶的结合而活化。 然而，最近对哺乳动物DRF的研究表明，单独结合Rho GTP酶是不够的，另一种信号可能参与DRF的激活。 虽然这种额外信号的性质尚不清楚，但初步研究表明，p21激活的激酶1（PAK 1）特异性磷酸化小鼠p21 mDia 2。 该项目将确定参与mDia 2-PAK 1结合相互作用的特定氨基酸残基，并通过检查mDia 2的PAK 1磷酸化对细胞定位、与Rho GTPases的相互作用以及肌动蛋白成核能力的影响来确定PAK 1是否在mDia 2调节中发挥作用。 此外，该项目将确定参与DRF蛋白，形态发生相关激活因子-1（DAAM 1）和mDia 3调控的特定分子内相互作用，以了解DAAM 1/mDia 3在细胞中的定位，功能和调控。 通过具体表征DRF家族三个特定成员的调节相互作用，这项研究将显着推进DRF蛋白对细胞骨架调节的理解。 由于DRF蛋白在黏菌、酵母、蠕虫、果蝇、小鼠和人类等多种生物体中的保守性，以及细胞蛋白中自抑制调节机制的普遍性，该项目的结果将适用于广泛的生物系统。 利用参与调节DRF蛋白的特定氨基酸残基的知识，该项目还将提供对全长DRF的细胞定位和细胞骨架效应的基本见解。 此外，细胞信号传导的相关性甚至更重要，因为PAK 1可能调节DRF，PAK 1是一种已知参与相关细胞过程的酶，如细胞死亡、细胞存活、细胞周期调节和神经元生长。 更广泛的影响这个项目将提供研究经验，本科生。 使用生物物理光谱学，生物化学，细胞和分子生物学相结合的方法以前导致了与本科合著者的出版物。 两名本科生对这项研究做出重大贡献的能力表明，这些类型的项目是尖端本科研究的理想选择。 目前的大部分研究将由本科生进行，其中一半将是第一代和/或非传统的大学生。 本科生将通过独立的研究项目进行指导，以提供科学方面的全面培训。 作为一个全职的基于假设的研究经验的结果，本科生将学习各种科学技术，提高批判性思维能力，并获得实验科学的准确理解。 这也将为医学和专业学校以及工作场所的学生提供更强的能力。 虽然大峡谷州立大学的优势之一是大量高质量的本科生，RUI项目的完成将为更多值得学习的学生提供宝贵的经验，并有助于加强大峡谷州立大学的研究环境。 RUI提案产生的结果将在同行评审的期刊上传播，并由本科生研究人员在地方，地区和国家会议上提出。

Intellectual MeritDiaphanous-related formins (DRFs) are a conserved family of Rho GTPase proteins that coordinate the actin and microtubule cytoskeletal networks. Therefore, these proteins influence cellular shape, motility, and cytokinesis; and it is critical that they are regulated and activated only in response to cellular signals. The proteins are autoinhibitory and are normally maintained in an inactive state. DRF proteins are activated by the binding of Rho GTPases. However, recent studies of mammalian DRFs have demonstrated that binding of Rho GTPases alone is insufficient and that another signal may be involved in DRF activation. Although the nature of this additional signal is unknown, preliminary studies indicate that p21-activated kinase 1 (PAK1) specifically phosphorylates the mouse formin mDia2. This project will identify the specific amino acid residues involved in the mDia2-PAK1 binding interaction and determine if PAK1 plays a role in mDia2 regulation by examining the effects of PAK1 phosphorylation of mDia2 on cellular localization, interaction with Rho GTPases, and actin nucleation ability. In addition, this project will identify specific intramolecular interactions involved in the regulation of the DRF proteins, Dishevelled-associated activator of morphogenesis-1 (DAAM1) and mDia3 to understand DAAM1/mDia3 localization, function, and regulation in cells. By specifically characterizing regulatory interactions in three specific members of the DRF family, this research will significantly advance the understanding of cytoskeletal regulation by DRF proteins. Due to the conservation of DRF proteins in organisms as diverse as slime molds, yeast, worms, fruit flies, mice, and humans, as well as the prevalence of the autoinhibitory regulatory mechanism in cellular proteins, the results of the project will be applicable to a broad array of biological systems. Using the knowledge of the specific amino acid residues involved in the regulation of formin proteins, this project will also provide fundamental insight into the cellular localization and cytoskeletal effects of full length DRFs. In addition, the cellular signaling relevance is even more substantial with the potential regulation of DRFs by PAK1, an enzyme known to be involved in a host of relevant cellular processes, such as cell death, cell survival, cell cycle regulation, and neuronal outgrowth. Broader ImpactThis project will provide research experiences to undergraduate students. The approach of using a combination of biophysical spectroscopy, biochemistry, and cell and molecular biology previously resulted in a publication with undergraduate co-authors. The ability of two undergraduate students to significantly contribute to that study indicates that projects of these types are ideal for cutting-edge undergraduate research. The majority of the current research will be conducted by undergraduates; half of whom will be first generation and/or non-traditional college students. The undergraduate students will be mentored through independent research projects in order to provide comprehensive training in the sciences. As a result of a full-time hypothesis based research experience, the undergraduate students will learn a variety of scientific techniques, increased critical thinking skills, and gain an accurate understanding of experimental science. This will also provide for stronger students in medical and professional schools, as well as in the workplace. While one of the strengths of Grand Valley State University is the significant number of high quality undergraduate students, completion of the RUI projects will provide valuable experiences for even more deserving students and serve to strengthen the research environment of Grand Valley State University. The results generated from this RUI proposal will be disseminated in peer-reviewed journals, as well as presented by undergraduate researchers at local, regional, and national meetings.