Asymmetric division as a morphological checkpoint for B. subtilis sporulation

描述(申请人提供)：我研究的长期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中的关键步骤，包括枯草芽孢杆菌的芽胞形成。活孢子的产生需要依赖于分裂的细胞特异性转录因子sigma F(CTF)和sigma E(CTE)的激活。在这项研究中，我将评估不对称分裂在CTF和AE活动的时间和空间调节中的作用。房颤激活蛋白SpollE在分裂过程中定位于隔膜，因此是检测房颤激活前所需的隔膜完成的理想候选者。此外，CTE的非活性形式PRO-OE和将PRO-AE加工成活动的AE的SpollGA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，分隔可以确保在前孔中CTF和在母细胞中AE的适当的时间和空间激活。我将开发一种新的方法来测试SpollE、SpollGA和Pro-AE是否定位于隔膜的前孔一侧或母细胞一侧。通过使用分裂的GFP技术，我将能够可视化这些蛋白质是否只存在于前孔隔膜、仅存在于母细胞隔膜或存在于隔膜的两侧。隔区的蛋白质-蛋白质相互作用也可能调节房颤和耳声发射的活动。使用荧光漂白后恢复(FRAP)将识别蛋白质迁移率的变化，这是蛋白质相互作用变化的结果。我将在隔膜的不同阶段、隔膜的对侧和隔膜的其他区域对GFP与SpollE、SpollGA和Pro-AE的融合进行FRAP。根据这些结果，我将开发SpollE、SpollGA和PRO-AE如何对间隔做出反应的机制模型。间隔可作为激活CTE的直接形态检查点。我会进行基因研究来测试这种可能性。此外，我还将在大肠杆菌中表达Pro-AE及其已知的调节蛋白，以检测激活AE所必需的成分。这项研究将揭示发育中的重要机制，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，本研究还将加深我们对土壤常见细菌枯草芽孢杆菌和其他内孢子形成细菌如炭疽杆菌产孢量的了解。

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.