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。 其菌丝体营养体含有多核细胞。 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。 本项目的长期目标是以构巢曲霉为模式生物，了解调节胞质分裂的分子机制，称为真菌中的分隔。 在包括A.在nidulans中，已经了解到称为分隔起始网络（SIN）的信号级联在分隔期间触发称为隔膜的横壁的形成。 以.在nidulans中，sidB基因编码激酶，其功能依赖于由mobA基因编码的新蛋白，这两者都是SIN的必需组分。 刘博士早期的研究结果表明，在A. SIN是nidulans孢子分离和分生孢子形成所必需的，但不是菌丝延伸和菌落形成所必需的。 因此，这种真菌没有分隔而存活。 刘博士利用了这一特点，分离出smo（mobA抑制基因）突变，当SIN途径失活时，这些突变恢复了分隔和分生孢子。 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基因编码的蛋白质拮抗SIN调节分隔。 已克隆了smoA基因，它编码一种新的核蛋白，其同源物仅在丝状真菌中发现。 基于这些发现，Liu博士提出了一个工作假设，即SMOA和其他SMO蛋白负调节隔膜形成所需蛋白质的活性，从而在A.巢菌菌丝体 为了验证这一假设，实验计划在三个具体目标。 首先，SMOA的功能将被表征，以通过仅在细胞质中限制其活性来了解SMOA的核定位的意义。 为了揭示SMOA和其他分隔调节因子之间的潜在联系，将通过表位标记然后亲和层析分离与SMOA相互作用的蛋白质。 SMOA和LSKA之间的潜在相互作用，另一个分隔调节细胞核，也将被检查。 第二个目标是致力于识别和表征的smoB基因。 将通过DNA转化介导的互补来克隆smoB基因。 一旦鉴定出smoB，将测试SMOA和SMOB蛋白在体外和体内是否直接或间接相互作用。 最后的目标是连接SIN和SMO蛋白与分隔机制。 由于SIDB蛋白是一种激酶，作用于分隔位点，因此它最有可能磷酸化隔膜组装所需的底物。 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和细胞生物学手段进行检查。 该项目的广泛影响可以从两个方面来预测。 首先，A. nidulans将使人们深入了解调节所有丝状真菌分隔的基本机制。 第二，除了在发现导向研究中的作用外，A。nidulans也成为本科课堂上的宝贵教材。 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，但参与的高中生和本科生将在研究方面拥有"动手"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.