Analysis of the protein composition of the spindle pole body during sporulation in Ashbya gossypii

The spores of fungi come in a wide variety of forms and sizes, highly adapted to the route of dispersal and to survival under specific environmental conditions. The ascomycete Ashbya gossypii produces needle shaped spores with a length of 30 μm and a diameter of 1 μm. Formation of these spores relies on actin and actin regulatory proteins and is, therefore, distinct from the minor role that actin plays for spore formation in Saccharomyces cerevisiae. Using in vivo FRET-measurements of proteins labeled with fluorescent proteins, we investigate how the formin AgBnr2, a protein that promotes actin polymerization, integrates into the structure of the spindle pole body during sporulation. We also investigate the role of the A. gossypii homologs to the S. cerevisiae meiotic outer plaque proteins Spo74, Mpc54 and Ady4 for sporulation in A. gossypii. We found highest FRET of AgBnr2 with AgSpo74. Further experiments indicated that AgSpo74 is a main factor for targeting AgBnr2 to the spindle pole body. In agreement with these results, the Agspo74 deletion mutant produces no detectable spores, whereas deletion of Agmpc54 only has an effect on spore length and deletion of Agady4 has no detectable sporulation phenotype. Based on this study and in relation to previous results we suggest a model where AgBnr2 resides within an analogous structure to the meiotic outer plaque of S. cerevisiae. There it promotes formation of actin cables important for shaping the needle shaped spore structure.真菌孢子的形态与尺寸丰富多样，高度适配其传播路径与特定环境下的存活需求。子囊菌棉阿舒囊霉（Ashbya gossypii）可产生长度为30 μm、直径为1 μm的针状孢子。该类孢子的形成依赖于肌动蛋白（actin）与肌动蛋白调控蛋白，其机制与肌动蛋白在酿酒酵母（Saccharomyces cerevisiae）孢子形成中所发挥的次要作用截然不同。本研究借助对荧光蛋白标记蛋白的活体荧光共振能量转移（FRET）测量，探究了促进肌动蛋白聚合的成蛋白（formin）AgBnr2在孢子形成过程中如何整合进入纺锤体极体（spindle pole body）的结构。同时，我们还研究了棉阿舒囊霉中与酿酒酵母减数分裂外层斑块蛋白Spo74、Mpc54及Ady4同源的蛋白在该菌孢子形成过程中的功能。研究发现，AgBnr2与AgSpo74的FRET信号最强。进一步实验表明，AgSpo74是将AgBnr2靶向至纺锤体极体的核心因子。与上述结果相符的是，Agspo74缺失突变体无法产生可检测到的孢子；而Agmpc54的缺失仅会影响孢子长度，Agady4的缺失则未表现出可检测到的孢子形成表型。基于本研究结果并结合既往研究成果，我们提出了一个模型：AgBnr2定位于与酿酒酵母减数分裂外层斑块结构类似的区域，在此处促进形成对针状孢子结构塑形至关重要的肌动蛋白束。