The growth of the germline ring canals during Drosophila melanogaster oogenesis - Project Summary To better diagnose and treat infertility, which affects millions each year, it is essential to understand the molecular pathways and structures required for normal gamete formation. Intercellular bridges (IBs) are found in developing sperm and eggs throughout the animal kingdom, including in humans; they provide an essential cytoplasmic connection between developing gametes or to supporting nurse cells. The IBs, or ring canals (RCs), that connect germline cells within the developing fruit fly egg chamber have emerged as the premier model system to study IB structure and stability. RCs are formed following remodeling of the midbody at the end of germline cell divisions; their formation and subsequent growth are essential for fertility. Many structural and regulatory RC proteins have been identified, but an integrated model has not been developed. The long-term goal of this work is to determine the molecular mechanisms that regulate RC formation, stability, and growth during oogenesis. The PI’s lab has demonstrated that the Misshapen (Msn) kinase localizes to the germline RCs and regulates their size and stability, but upstream activators and downstream effectors in the germline have not been identified. Msn can be directly phosphorylated and activated by the Tao kinase in other cell types, but it is not known whether this occurs in the germline. Altering Msn levels led to changes in the actin cytoskeleton and the adherens junction (AJ) protein, E-cadherin, but downstream targets of Msn in the germline have not been identified. Msn contains a citron homology domain, which can bind small GTPases; these molecular switches can coordinate actin remodeling and AJ turnover. The objective of this work is to test the hypothesis that Msn is activated by the Tao kinase to coordinate AJ turnover and actin remodeling via activation of small GTPases, ultimately promoting regulated RC growth. Aim 1 will determine whether Msn contributes to the spatial and developmental regulation of E-cadherin in the germline. Aim 2 will determine whether one or more small GTPases (Rho or Rap family) regulate RC size or stability, downstream of Msn. Aim 3 will test the hypothesis that in the germline, Tao phosphorylates and activates Msn to regulate RC size and stability. Because ring canal stability and regulated growth require adherens junctions and remodeling of the actin cytoskeleton, the study of Msn can provide significant insight into these essential germline structures. The proposed aims utilize undergraduate-appropriate techniques that are established within the PI’s lab. The PI’s strong track record of mentoring 42 undergraduate student researchers, her expertise in cell and developmental biology, and the power and accessibility of the fruit fly model system make her well-positioned to complete the proposed aims. Students will gain valuable training and experience that will increase their interest in STEM fields. The proteins, structures, and processes being studied are also utilized during normal morphogenesis and can be mis-regulated in disease; therefore, insight from this work will improve our understanding of normal development and disease in humans.
