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。它们的菌丝体营养体内含有多核细胞。因此，与酵母和大多数其他真核生物等具有单核细胞的生物不同，在这些真菌中，细胞质的分裂或胞质分裂并不总是与有丝分裂相结合。该项目的长期目标是以尼杜拉曲霉为模式生物，了解调节胞质分裂的分子机制，这种机制在真菌中被称为隔膜。在包括假单胞菌在内的几个真菌物种中，人们已经了解到，一个被称为隔膜起始网络(SIN)的信号级联在隔膜形成过程中触发了称为隔膜的横壁的形成。在弧菌中，sidB基因编码一种激酶酶，其功能依赖于由MOBA基因编码的一种新的蛋白质，这两种蛋白质都是SIN的重要组成部分。刘博士早期的研究结果表明，在结球拟青霉中，SIN是分离和分生孢子所必需的，但不是菌丝伸展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下来。刘博士利用了这一特性，并分离出了当SIN途径失活时恢复隔膜和分生孢子形成的SMO(MOBA抑制因子)突变。这些smo突变位于基因组中的五个基因座，称为SmoA-E。结果表明，由SmoA-E基因编码的蛋白质与SIN拮抗，从而调节隔膜。SmoA基因已经被克隆，它编码一种新的核蛋白，其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蛋白对隔膜形成所需蛋白质的活性进行负面调节，从而在多核拟青霉菌丝体中形成多核细胞。为了验证这一假设，实验计划在三个特定目标内进行。首先，将对SMOA的功能进行表征，以了解通过限制其仅在细胞质中的活性来定位SMOA的核的意义。为了揭示SMOA与其他隔膜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(S)。SMOA和LSKA之间的潜在相互作用也将被研究，LSKA是细胞核中的另一种隔膜调节因子。第二个目标是致力于鉴定和表征SmoB基因。将通过DNA转化介导的互补作用克隆SmoB基因。一旦SmoB被鉴定出来，SMOA和SMOB蛋白在体外和体内是否直接或间接相互作用将受到测试。最终目标是将SIN和SMO蛋白与分离机制联系起来。因为SIDB蛋白是一种激酶，作用于隔膜部位，它很可能使其底物(S)磷酸化，这是组装隔膜所需的。为了确定底物(S)，将确定功能丧失的SIDB突变的多拷贝抑制基因(S)。它们的编码蛋白(S)的功能以及它们与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。该项目的更广泛影响可以从两个方面预见。首先，从根结线虫的研究中获得的结果将使人们对所有丝状真菌中调节隔膜的基本机制有更深入的了解。其次，除了在以发现为导向的研究中发挥的作用外，尼杜兰还成为本科生课堂上无价的教材。虽然研究生和博士后研究员接受真菌遗传学和细胞生物学方面的培训，但参与培训的高中生和本科生将拥有研究方面的实践经验。他们还将能够从自己的实验中直观地了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追求科学事业。

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.