Septum Formation in the Absence of the Septation Initiation Network in Aspergillus Nidulans

在自然界中，许多真菌以丝状形式存在。它们的菌丝营养体含有多核细胞。因此，与酵母菌和大多数其他真核生物等具有单核细胞的生物不同，这些真菌的细胞质分裂或细胞质分裂并不总是伴随着有丝分裂。本项目的长期目标是了解调节细胞质分裂的分子机制，即真菌的分裂，以细粒曲霉为模式生物。在一些真菌物种中，包括a . nidulans，已经了解到一个被称为分隔起始网络（SIN）的信号级联触发分隔过程中称为隔膜的交叉壁的形成。在a . nidulans中，sidB基因编码一种激酶酶，其功能依赖于mobA基因编码的一种新蛋白，这两种蛋白都是SIN的重要组成部分。刘博士早期的研究结果表明，在A. nidulans中，SIN是分离和分生所必需的，但不是菌丝延伸和菌落形成所必需的。因此，这种真菌在没有分离的情况下存活。刘博士利用了这一特点，分离出了在SIN通路失活时恢复分离和条件作用的smo （mobA抑制因子）突变。这些smo突变位于基因组中的五个位点，称为smoA-E。结果表明，smoA-E基因编码的蛋白可拮抗SIN调控分离。smoA基因已经被克隆，它编码一种新的核蛋白，这种蛋白的同源物只在丝状真菌中发现。基于这些发现，刘博士提出了一个可行的假设，即SMOA和其他SMO蛋白负调控隔膜形成所需蛋白质的活性，从而在空心芽孢杆菌菌丝体中形成多核细胞。为了验证这一假设，实验计划有三个具体目标。首先，我们将对SMOA的功能进行表征，了解其核定位的意义，将SMOA的活性限制在细胞质中。为了揭示SMOA与其他分离调节因子之间的潜在联系，将通过表位标记和亲和层析分离与SMOA相互作用的蛋白。SMOA和LSKA（细胞核中另一种分裂调节因子）之间的潜在相互作用也将被研究。第二个目标是致力于识别和表征烟雾基因。smoB基因将通过DNA转化介导的互补克隆。一旦确定了smoB，将测试SMOA和smoB蛋白是否在体外和体内直接或间接相互作用。最终目的是将SIN和SMO蛋白与分离机制连接起来。由于SIDB蛋白是一种激酶，作用于分离位点，因此它很可能使隔膜组装所需的底物磷酸化。为了鉴定底物，将鉴定功能丧失sidB突变的多拷贝抑制基因。其编码蛋白的功能及其与SIN和SMO蛋白的关系将通过遗传学和细胞生物学手段进行研究。这个项目的广泛影响可以从两个方面来预测。首先，从A. nidulans的研究中获得的结果将带来对所有丝状真菌中调节分离的基本机制的见解。其次，除了在发现导向的研究中发挥作用外，A. nidulans也成为本科生课堂上宝贵的教材。研究生和博士后接受真菌遗传学和细胞生物学方面的培训，而参与研究的高中生和本科生将有“实践”的研究经验。他们也将能够从自己的实验中直观地理解基本的经典和分子遗传学。其目标是激励更多的年轻学生从事科学事业。

In nature, many fungi exist in filamentous forms. Their vegetative body of the mycelium contains multinucleate cells. Thus, unlike organisms with uninucleate cells like yeasts and most other eukaryotes, the division of the cytoplasm, or cytokinesis, is not always coupled with mitosis in these fungi. The long-term goal of this project aims at understanding molecular mechanisms that regulate cytokinesis, termed as septation in fungi, using Aspergillus nidulans as a model organism. In several fungal species including A. nidulans, it has been learnt that a signaling cascade known as the septation initiation network (SIN) triggers the formation of the cross wall called the septum during septation. In A. nidulans, the sidB gene encodes a kinase enzyme whose function relies on a novel protein encoded by the mobA gene, which are both essential components of the SIN. Results from Dr. Liu's earlier studies indicate that in A. nidulans the SIN is required for septation and conidiation, but not for hyphal extension and colony formation. Thus, this fungus survives without septation. Dr. Liu has taken advantage of this feature, and isolated smo (suppressor of mobA) mutations that restored septation and conidiation when the SIN pathway was inactivated. These smo mutations are located at five loci in the genome, termed as smoA-E. The results suggest that proteins encoded by smoA-E genes antagonize against the SIN to regulate septation. The smoA gene has been cloned, and it encodes a novel nuclear protein with homologs found only among filamentous fungi. Based on these findings, Dr. Liu formulated a working hypothesis that SMOA and other SMO proteins negatively regulate activities of proteins required for septum formation so that multinucleate cells are formed in the A. nidulans mycelium. In order to test this hypothesis, experiments are planned within three specific objectives. First, the function of SMOA will be characterized, to learn the significance of the nuclear localization of SMOA by limiting its activity only in the cytoplasm. To reveal potential connection between SMOA and other septation regulators, protein(s) interacting with SMOA will be isolated by epitope-tagging followed by affinity chromatography. The potential interaction between SMOA and LSKA, another septation regulator in the nucleus, will also be examined. The second objective is devoted to identifying and characterizing the smoB gene. The smoB gene will be cloned by DNA transformation-mediated complementation. Once smoB is identified, whether SMOA and SMOB proteins interact directly or indirectly with each other in vitro and in vivo will be tested. The final objective aims at linking the SIN and SMO proteins with the septation machinery. Because the SIDB protein is a kinase and acts at the septation site, it most likely phosphorylates its substrate(s) required for the assembly of the septum. To identify the substrate(s), multi-copy suppressor gene(s) of a loss-of-function sidB mutation will be identified. The function of their encoded protein(s) and their relationship with the SIN and SMO proteins will be examined by genetic and cell biological means. The broader impacts of this project can be anticipated in two aspects. First, results garnered from the study in A. nidulans will bring insights into basic mechanisms that regulate septation in all filamentous fungi. Second, in addition to its role in the discovery-oriented research, A. nidulans also becomes an invaluable teaching material in undergraduate classrooms. While graduate students and postdoctoral fellows are trained in fungal genetics and cell biology, participating high school students and undergraduate students will have "hands-on" experience in research. They will also able to visually understand basic classical and molecular genetics from their own experiments. The goal is to inspire more young students to pursue a career in science.