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.
