Vrabioiu Alina M, Mitchison Timothy J
Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
J Mol Biol. 2007 Sep 7;372(1):37-49. doi: 10.1016/j.jmb.2007.05.100. Epub 2007 Jun 9.
Septin filaments form ordered hourglass and ring-shaped structures in close apposition to the yeast bud-neck membrane. The septin hourglass scaffolds the asymmetric localization of many essential cell division proteins. However, it is unknown whether the septin structures have an overall polarity along the mother-daughter axis that determines the asymmetric protein localization. Here we engineered rigid septin- green fluorescent protein (GFP) fusions with various fluorescence dipole directions by changing the position of the GFP beta-barrel relative to the septin filament axis. We then used polarized fluorescence microscopy to detect potential asymmetries in the filament organization. We found that both the hourglass and ring filament assemblies have sub-resolution C(2) symmetry and lack net polarity along the mother-daughter axis. The hourglass filaments have an additional degree of symmetry relative to the ring filaments, most likely due to a twist in their higher-order structure. We previously reported that during the hourglass to rings transition septin filaments change their direction. Here we show that the filaments also undergo a change in their lateral organization, consistent with filament untwisting. The lack of net septin polarity along the mother-daughter axis suggests that there are no septin-based structural reasons for the observed asymmetry of other proteins. We discuss possible anisotropic processes that could break the septin symmetry and establish the essential bud-neck asymmetry.
Septin丝在紧邻酵母芽颈膜的位置形成有序的沙漏状和环状结构。Septin沙漏结构支撑着许多重要细胞分裂蛋白的不对称定位。然而,尚不清楚Septin结构在母-子轴上是否具有整体极性,该极性决定了蛋白的不对称定位。在此,我们通过改变绿色荧光蛋白(GFP)β桶相对于Septin丝轴的位置,构建了具有不同荧光偶极方向的刚性Septin-绿色荧光蛋白融合体。然后,我们使用偏振荧光显微镜检测丝组织中的潜在不对称性。我们发现,沙漏状和环状丝组装体都具有亚分辨率的C(2)对称性,并且在母-子轴上缺乏净极性。相对于环状丝,沙漏状丝具有额外的对称程度,这很可能是由于其高阶结构中的扭曲。我们之前报道过,在从沙漏状向环状转变的过程中,Septin丝会改变其方向。在此我们表明,这些丝在横向组织上也会发生变化,这与丝的解捻一致。沿母-子轴缺乏净Septin极性表明,其他蛋白所观察到的不对称性不存在基于Septin的结构原因。我们讨论了可能打破Septin对称性并建立重要芽颈不对称性的各向异性过程。