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

构巢曲霉中缺乏分隔起始网络的分隔形成

• 批准号: 0615892
• 负责人: Bo Liu
• 金额: --
• 依托单位: University of California-Davis
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0615892&HistoricalAwards=false
• 关键词: Septum; Formation; Absence; Septation; Initiation

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.

在自然界中，许多真菌以丝状形式存在。 他们的菌丝体的营养身体包含多核细胞。 因此，与酵母和大多数其他真核生物等未实核细胞的生物不同，细胞质或细胞因子的分裂并不总是与这些真菌中的有丝分裂相结合。 该项目的长期目标旨在理解调节细胞因子的分子机制，被称为真菌中的细胞因子，使用曲霉尼古拉（Aspergillus nidulans）作为模型生物体。 在包括A. nidulans在内的几种真菌物种中，据了解，被称为分离启动网络（SIN）的信号级联反应会触发分隔期间称为中隔的十字壁的形成。 在A. nidulans中，SIDB基因编码一种激酶，该酶的功能依赖于由MOBA基因编码的新型蛋白质，该蛋白都是罪的必不可少的成分。 Liu博士的早期研究的结果表明，在A. nidulans中，犯罪是分离和分生的，而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生存。 Liu博士利用了这一特征，并隔离了SMO（MOBA的抑制剂）突变，这些突变在固定途径被灭活时恢复了分隔和分生。 这些SMO突变位于基因组的五个基因座，称为SMOA-E。 结果表明，由SMOA-E基因编码的蛋白质拮抗罪调节分隔。 SMOA基因已被克隆，它编码了一种新型的核蛋白，该核蛋白只有在丝状真菌中发现的同源物。 基于这些发现，刘博士提出了一个有效的假设，即SMOA和其他SMO蛋白会负调节隔膜形成所需的蛋白质的活性，以便在尼古拉菌菌丝体中形成多核细胞。 为了检验这一假设，计划在三个特定目标中进行实验。 首先，将表征SMOA的功能，以通过仅限于细胞质中的活性来了解SMOA核定位的重要性。 为了揭示SMOA与其他分离调节剂之间的潜在联系，通过表位tag缩，随后是亲和力色谱法，将与SMOA相互作用。 还将检查SMOA和LSKA之间的潜在相互作用，该核中的另一个分隔调节剂也将被检查。 第二个目标致力于识别和表征SMOB基因。 SMOB基因将通过DNA转化介导的互补来克隆。 一旦确定了SMOB，SMOA和SMOB蛋白是否直接或间接相互作用在体外和体内进行测试。 最终目标旨在将罪和SMO蛋白与分隔机械联系起来。 由于SIDB蛋白是一种激酶，并在分离位点起作用，因此它很可能磷酸化其隔膜组装所需的底物。 为了识别底物，将鉴定出功能丧失的SIDB突变的多拷贝抑制基因。 他们编码的蛋白质的功能以及它们与罪和SMO蛋白的关系将通过遗传和细胞生物学手段进行检查。 该项目的更广泛影响可以在两个方面预期。 首先，在nidulans的研究中获得的结果将使所有丝状真菌中分离的基本机制进行见解。 其次，除了在以发现为导向的研究中的作用外，A。Nidulans还成为本科教室中的宝贵教学材料。 虽然研究生和博士后研究员接受了真菌遗传学和细胞生物学的培训，但参与的高中生和本科生将具有“动手”研究经验。 他们还将能够从自己的实验中从视觉上理解基本的经典和分子遗传学。 目的是激发更多的年轻学生从事科学职业。