Regulation of Cytokinesis by Microtubules in Aspergillus Nidulans

一个细胞分裂成两个细胞是生命的关键功能之一。为了确保亚细胞成分（如细胞器和染色体）在子细胞中的适当分布，在空间和时间上精确调节细胞分裂是至关重要的。细胞分裂并不一定总是对称的；在真菌、植物和动物中可以发现许多不对称分裂的例子，这些通常是分化和有机体形态发生所必需的。从各种酵母和丝状真菌的研究中，人们对真菌细胞分裂（也称为细胞质分裂或分裂）有了大量的了解。在这些真菌中，分裂是由分裂酵母中的分裂起始网络（SIN）等信号转导途径调节的。SIN包括一个MAP-激酶样的激酶级联，这个级联的激活会引发分裂。SIN网络激活的结果是激活了收缩内侧环上尚未识别的成分，主要由肌动蛋白微丝和肌球蛋白以及包括肌动蛋白结合蛋白在内的各种其他蛋白质组成。这个环的最终功能是在适当的位置启动由长链多糖（β -葡聚糖和几丁质）组成的隔膜的组装。虽然所有的SIN分子都定位于酵母的纺锤极体，但末端的Sid2p激酶及其相关的Mob1p部分转移到分裂位点以激活分裂。该项目解决了这些SIN分子（Sid2p和Mob1p）如何从纺锤极体移动到分裂位点的问题。微管在SIN分子的定位中起着至关重要的作用。这一假设将在丝状真菌——空心曲霉身上得到验证。对于这些研究来说，拟南芥是一个特别好的模式生物，因为有大量的实验工具，其基因组的完整序列预计很快就会完成。在A. nidulans中，AnMOB1蛋白不仅定位于纺锤体极体和分裂位点，还定位于中央纺锤体。定位依赖于微管。细胞质动力蛋白是一种在细胞分裂过程中与纺锤体相关的微管马达，已被证明在隔膜定位中发挥作用。动力蛋白突变体将用于检测AnMOB1的定位是否依赖于细胞质动力蛋白。实验还将测试AnMOB1的主轴极体定位是否依赖于KLPA中的负端定向运动。我们将探讨BIMC中正端定向运动蛋白在AnMOB1定位到中心纺锤体中的作用。此外，将采用药理学和遗传学方法来测试微管动力学是否在AnMOB1定位中起作用。这些研究将深入了解微管动力学、微管马达和微管组织中心在SIN分子胞内运动中所起的作用，并最终在细胞分裂中发挥作用。更广泛的影响：细粒曲霉不仅是一种有价值的研究模型，而且作为本科和高中学生的教学材料也很有用。刘博士用活的曲霉向参观他实验室的当地初中学生和在家学习的学生演示细胞生物学现象（细胞分裂、细胞器运动和微管动力学）。他还在本科生物学入门课程和研究生课程《植物细胞和分子生物学的常用方法》中使用曲霉作为教学模型。刘博士还指导了几名在他的实验室进行研究的本科生；这些学生已经进入研究生院或从事生物技术研究工作。

Division of one cell into two is one of the key critical functions of life. It is essential that cell division be precisely regulated, both spatially and temporally, in order to ensure appropriate distribution of subcellular components such as organelles and chromosomes to the daughter cells. Cell division is not always necessarily symmetrical; numerous examples of assymetrical division can be found among fungi, plants and animals, and these are often essential for differentiation and organismal morphogenesis. A great deal has been learned about fungal cell division (also termed cytokinesis or septation) from studies of various yeasts and filamentous fungi. In such fungi, septation is regulated by signal transduction pathways such as the septation initiation network (SIN) in fission yeast. SIN includes a MAP- kinase-like kinase cascade, and activation of this cascade initiates septation. The consequence of activation of the SIN network is to activate as-yet-unidentified components at the contractile medial ring, composed mainly of actin microfilaments and myosin plus various other proteins including actin-binding proteins. The ultimate function of this ring is to initiate the assembly of the septum, composed of long chain polysaccharides (beta-glucan and chitin), at the right place. While all SIN molecules localize to the spindle pole body in yeast, the terminal Sid2p kinase and its associated Mob1p partially translocate to the septation site to activate septation. This project addresses the question of how these SIN molecules (Sid2p and Mob1p) travel from the spindle pole body to the septation site. It is proposed that microtubules play essential roles in the localization of SIN molecules. This hypothesis will be tested in the filamentous fungus, Aspergillus nidulans. A. nidulans is a particularly good model organism for these studies because there exists a substantial armamentarium of experimental tools, and completion of the full sequence of its genome is anticipated shortly. In A. nidulans, the AnMOB1 protein localizes not only to the spindle pole body and the septation site, but also to the central spindle. The localization is dependent on microtubules. Cytoplamic dynein, a microtubule motor associated with the spindle during cell division, has been demonstrated to play a role in septum positioning. Dynein mutants will be used to test whether AnMOB1 localization is dependent on cytoplasmic dynein. Experiments will also be performed to test whether spindle pole body localization of AnMOB1 is dependent on minus end-directed kinesin KLPA. The roles of the plus end-directed kinesin BIMC in AnMOB1 localization to the central spindle will be explored. In addition, pharmacological and genetic approaches will be used to test whether microtubule dynamics plays a role in AnMOB1 localization. These studies will provide insights into the roles played by microtubule dynamics, microtubule motors, and the microtubule-organizing center in the intracellular movement of SIN molecules, and ultimately in cytokinesis.Broader Impacts: Aspergillus nidulans is not only a valuable research model, but it is also useful as teaching material for undergraduate and high school students. Dr. Liu uses living Aspergillus to demonstrate cell biological phenomena (cell division, organelle movement, and microtubule dynamics) to local Junior High School students and home-schooled students who visit his laboratory. He also uses Aspergillus as a teaching model in an undergraduate Introductory Biology course and in a graduate level course, Common Approaches in Plant Cell and Molecular Biology. Dr. Liu has also mentored several undergraduates who have performed research in his laboratory; these students have gone on to graduate school or to careers in biotechnology research.