Polarized growth and the role of apical sterol-rich domains in Aspergillus nidulans

丝状真菌的极化生长依赖于微管(MT)和肌动蛋白细胞骨架及其相关的运动蛋白。与TIP膜相关的标志性蛋白，即所谓的“细胞末端标记”，连接着两个细胞骨架。最近的研究结果表明，顶端富含甾醇的膜域(SRD)在细胞的极化生长和末端标记的定位中起着重要的作用。SRD是真菌特有的，被认为是由脂筏聚集形成的。在我们的初步研究中，我们揭示了FloA，它作为膜上形成筏子的支架，参与了SRD的形成。通过所提出的研究，我们的目标是以FloA为RAFT标记，利用超分辨显微镜详细分析SRDS(脂筏簇的分布)的定位。为了研究SRDS的形成机制，对一些候选基因(肌动蛋白-脂结合蛋白)进行了基因缺失和荧光蛋白标记的分析。为了确定与SRDS形成有关的新基因，将对鞘磷脂合成突变体的多拷贝抑制因子进行遗传筛选。为了分析SRD在细胞骨架完整性、细胞末端标记和生长机制中的作用，我们将分析它们在反向遗传和抑制分析中确定的基因的相应突变中的定位。

Polarized growth of filamentous fungi depends on the microtubule (MT) and the actin cytoskeleton along with their associated motor proteins. Tip membrane-associated landmark proteins, so-called “cell end markers” link the two cytoskeletons. Recent results indicate that apical sterol-rich membrane domains (SRDs) play important roles in polarized growth and localization of cell end markers. SRDs are specific to fungi and thought to be formed by lipid raft clustering. In our preliminary study, we revealed that a flotillin orthologue, FloA, which serves as scaffold for raft formation at the membrane, is involved in the formation of SRDs. With the proposed research we are aiming to analyze the localization of SRDs (distribution of lipid raft clusters) in detail by super-resolution microscopy using FloA as a raft marker. To investigate the formation mechanism of SRDs, some candidate genes (actin-lipid binding proteins) are going to be analyzed by gene deletion and fluorescent protein tagging. To identify new genes involved in SRDs formation, genetic screens for multi-copy suppressors of sphingolipid synthesis mutants will be performed. To analyze the roles of SRDs on the integrity of the cytoskeletons, cell end markers, and growth machinery, we will analyze their localization in corresponding mutants of the genes identified in the reverse-genetic and the suppressor analysis.