CAREER:Regulation of Membrane-Cytoskeletal Dynamics during Cytokinesis

这是一个为期五年的职业奖。智力优势：细胞质分裂是通过在分裂后期形成分裂沟，将分离的染色体、细胞质和细胞器之间的有丝分裂纺锤体一分为二而实现的。这项研究活动的目的是深入了解建立卵裂沟所需的机制，确定调控沟形成所需的因素，并确定一种新的Dynamin/Dyn-1相互作用蛋白DIP-1如何协调这一过程。潜在的假设是，卵裂沟是通过来自纺锤体的未知信号(S)聚集脂筏蛋白而建立的。这一信号触发DIP-1，在细胞的中期-后期转变过程中，调节已知的RAFT组分Dynamin/Dyn-1在赤道膜上的靶向和组装。Dynamin/Dyn-1随后指导肌动蛋白细丝的形成，肌动蛋白微丝组装成acto-myosin环，进而促进卵裂沟内陷和完成。这一假说基于以下观察结果：1)胞质分裂信号的靶点是赤道皮质；2)Dyn-1定位于赤道膜和新形成的分裂沟；3)Dynamin是已知的脂筏成分；4)Dynamin可以影响肌动蛋白的组装；5)DIP-1和Dyn-1一样，调节分裂沟的形成和完成。基于这些观察，本项目的实验重点是以线虫线虫为模型，研究DIP-1在动物细胞胞质分裂过程中建立分裂沟和调节Dyn-1功能的作用。对DIP-1(RNAi)胚胎的初步研究表明：(I)纺锤体中区微管组装正常，但在后期(60%)发生弯曲和消失；(Ii)形成细胞质的球形微管：：观察到GFP聚集体(40%)；(Iii)肌动蛋白皮质紊乱，细胞质分裂期间几乎没有膜褶皱；以及(Iv)DIP-1包含BRCT基序，通常用于调节与其他含有BRCT基序的蛋白质的相互作用，如BRCA2和ECT2，已知的细胞质分裂蛋白。本研究的策略是：1)确定DIP-1的细胞功能；2)表征DIP-1在胞质分裂过程中耗尽对膜细胞骨架的影响；3)确定DIP-1是否直接与Dyn-1结合；以及3)确定DIP-1：：GFP的细胞周期定位，并确定在有丝分裂过程中调节DIP-1：：GFP定位的蛋白质基序和其他因素。随着对DIP-1在这一过程中的具体作用的理解，这些研究应该为细胞质分裂过程中发生的膜-细胞骨架事件的调节和功能提供新的见解。更广泛的影响：这个职业项目还包括一个重要的、综合的教育组成部分，旨在使直接参与工作的学生和公众受益，特别是对当地的美洲原住民社区。参与或介绍这项研究的高中生、本科生和研究生将获得将活体显微镜技术与遗传学、生物化学和分子生物学相结合的实践经验。由于线虫胚胎细胞分裂的现场电影非常令人兴奋，而且很容易理解，斯科普博士的工作尤其受到所有教育背景的欢迎。由于“系统生物学”将在实验室教授和操作，这些技术的介绍将是基因组学、蛋白质组学和生物学领域非常受欢迎的技能。关于斯科普博士的工作和她的《系统生物学》课程(遗传学875)的信息将通过与她的实验室和课程相关的在线资源传播给学术界和高中社区。

This is a five-year CAREER award.Intellectual merit: Cytokinesis is achieved by the formation of a cleavage furrow in anaphase that bisects the mitotic spindle between the separated chromosomes, cytoplasm and organelles. The goal of this research activity is to gain insight into the mechanisms required to establish cleavage furrows, identify factors required to regulate furrow formation and to determine how DIP-1, a novel dynamin/DYN-1 interacting protein, coordinates this process. The underlying hypothesis is that the cleavage furrow is established by clustering of lipid raft proteins by an unknown signal(s) from the spindle. This signal triggers DIP-1 to regulate the targeting and assembly of dynamin/DYN-1, a known raft component, at the equatorial membrane of the cell during the metaphase-anaphase transition. Dynamin/DYN-1 subsequently directs actin filament formation, which assemble into the acto-myosin ring and in turn, promotes cleavage furrow invagination and completion. This hypothesis is based on the observations that: 1) The target of the signal for cytokinesis is the equatorial cortex; 2) DYN-1 localizes to equatorial membranes and newly formed cleavage furrows; 3) Dynamin is a known lipid raft component; 4) Dynamin can influence actin assembly; and 5) DIP-1, like DYN-1, regulates the establishment of cleavage furrow formation as well as completion. Based on these observations, the experimental focus of this project is on the role of DIP-1 in establishing the cleavage furrow and regulating DYN-1 function during cytokinesis in animal cells, using the nematode, C. elegans, as a model. Preliminary studies in dip-1 (RNAi) embryos show that (i) the spindle midzone microtubules assemble properly, but buckle and disappear during late anaphase (60%); (ii) the formation of cytoplasmic, spherical tubulin::GFP aggregates are observed (40%); (iii) the actin cortex is disorganized and membrane ruffling during cytokinesis is almost absent; and (iv) DIP-1 contains a BRCT motif commonly used to mediate interactions with other BRCT motif-containing proteins, such as BRCA2 and ECT2, known cytokinesis proteins. The strategy of this study is to: 1) Determine the cellular function of DIP-1; 2) Characterize the membrane-cytoskeletal consequences of DIP-1 depletion during cytokinesis; 3) Determine if DIP-1 directly binds to DYN-1; and 3) Determine the cell cycle localization of DIP-1::GFP and identify protein motifs and additional factors that regulate DIP-1::GFP localization during mitosis. These studies should provide new insight into the regulation and function of the membrane-cytoskeletal events that occur during cytokinesis with an understanding of the specific role of DIP-1 in this process. Broader Impact: This CAREER project also involves a significant, integrated educational component, designed to benefit both the students involved directly with the work and for the public, especially for local Native American communities. The high school, undergraduate and graduate students involved in or introduced to this research will gain hands-on experience using in vivo microscopy techniques in conjunction with genetics, biochemistry and molecular biology. Since live movies of cell division in C. elegans embryos are quite exciting and easy to comprehend, Dr. Skop's work is especially welcoming to all educational backgrounds. Since "systems biology" will be taught and performed in the laboratory, the introduction to these techniques will be skills that are highly sought after in the fields of genomics, proteomics and biology. Information about Dr. Skop's work and her "systems biology" course (Genetics 875) will be disseminated to the academic and high school communities through online resources related to her laboratory and courses.