Asymmetric division as a morphological checkpoint for B. subtilis sporulation

描述（由申请人提供）：我的长期研究目标是发现不对称细胞分裂与细胞分化的耦合机制。不对称分裂是许多发育过程的关键步骤，包括枯草芽孢杆菌的孢子形成。活孢子的产生需要细胞特异性转录因子（CTF和CTE）的分裂依赖性激活。在这项研究中，我将评估不对称分裂在CTF和aE活性的时空调节中的作用。aF激活蛋白SpollE在分裂过程中定位于隔膜，因此是aF激活前感知分隔完成的理想候选者。此外，CTE的非活性形式pro-oE和将pro-aE加工成活性aE的spolga可能定位于隔膜，也受分裂的调节。细胞特异性定位提供了一种机制，通过这种机制，分离可以确保前孢子中CTF和母细胞中aE的适当时间和空间激活。我将开发一种新的方法来检测SpollE， spolga和pro-aE是位于间隔的前孢子侧还是母细胞侧。通过使用分裂GFP技术，我将能够可视化这些蛋白质是否仅存在于前孢子间隔，仅存在于母细胞间隔或间隔两侧。间隔处的蛋白-蛋白相互作用也可能调节心房纤颤和心房纤颤活性。使用光漂白后荧光恢复（FRAP）将识别蛋白质迁移率的变化，这是由于蛋白质相互作用的变化而变化的。我将在间隔的不同阶段、间隔的相对两侧以及与膜的其他区域相比，对SpollE、spolga和pro-aE的GFP融合进行FRAP。根据这些结果，我将建立SpollE， spolga和pro-aE如何响应分隔的机制模型。分离可以作为CTE激活的直接形态学检查点。我会进行基因调查来验证这种可能性。此外，我将在大肠杆菌中表达aE前及其已知的调节蛋白，以测定aE激活所需的成分。这项研究将揭示发育过程中的重要机制，并可能揭示发育过程中基因表达异常导致的疾病的基础。此外，本研究将进一步加深我们对常见土壤细菌枯草芽孢杆菌和其他内孢子形成细菌（如炭疽芽孢杆菌）产孢的认识。

DESCRIPTION (provided by applicant): The long-term goals of my research are to discover the mechanisms that couple asymmetric cell division to cell differentiation. Asymmetric division is a key step in many developmental processes, including sporulation of the bacterium Bacillus subtilis. The production of viable spores requires the division-dependent activation of the cell-specific transcription factors, sigma F (CTF) and sigma E (CTE). In this study, I will assess the role of asymmetric division in the temporal and spatial regulation of CTF and aE activity. The aF activating protein SpollE localizes to the septum during division and therefore represents an ideal candidate for sensing completion of septation, which is required before activation of aF. In addition, pro-oE, the inactive form of CTE, and SpollGA, which processes pro-aE into active aE, may localize to the septum and also be regulated by division. Cell-specific localization offers one mechanism by which septation may ensure proper temporal and spatial activation of CTF in the forespore and aE in the mother cell. I will develop a new method to test if SpollE, SpollGA and pro-aE localize to the forespore side or mother cell side of the septum. By utilizing split GFP techniques I will be able to visualize if these proteins are present only at the forespore septum, only at the mother cell septum or on both sides of the septum. Protein-protein interactions at the septum may also regulate aF and aE activity. The use of Fluorescence Recovery After Photobleaching (FRAP) will identify changes in protein mobility, which varies as a result of changes in protein interactions. I will perform FRAP on GFP fusions to SpollE, SpollGA and pro-aE at different stages of septation, on opposing sides of the septum and at the septum in comparison to other regions of the membrane. From these results I will develop mechanistic models for how SpollE, SpollGA and pro-aE function in response to septation. Septation may function as a direct morphological checkpoint for activation of CTE. I will perform genetic investigations to test that possibility. In addition, I will express pro-aE and its known regulatory proteins in E coli to assay the necessary components for aE activation. This research will reveal important mechanisms in development and possibly uncover the basis of diseases that result from aberrant gene expression during development. In addition, this study will further our understanding of sporulation by the common soil bacterium Bacillus subtilis and other endospore forming bacteria, such as B. anthracis.