Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Mol Biol Cell. 2012 Feb;23(3):423-32. doi: 10.1091/mbc.E11-10-0850. Epub 2011 Dec 7.
Septins are conserved GTP-binding proteins involved in membrane compartmentalization and remodeling. In budding yeast, five mitotic septins localize at the bud neck, where the plasma membrane is enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2)). We previously established the subunit organization within purified yeast septin complexes and how these hetero-octamers polymerize into filaments in solution and on PtdIns4,5P(2)-containing lipid monolayers. How septin ultrastructure in vitro relates to the septin-containing filaments observed at the neck in fixed cells by thin-section electron microscopy was unclear. A morphological description of these filaments in the crowded space of the cell is challenging, given their small cross section. To examine septin organization in situ, sections of dividing yeast cells were analyzed by electron tomography of freeze-substituted cells, as well as by cryo-electron tomography. We found networks of filaments both perpendicular and parallel to the mother-bud axis that resemble septin arrays on lipid monolayers, displaying a repeat pattern that mirrors the molecular dimensions of the corresponding septin preparations in vitro. Thus these in situ structures most likely represent septin filaments. In viable mutants lacking a single septin, in situ filaments are still present, although more disordered, consistent with other evidence that the in vivo function of septins requires filament formation.
septins 是保守的 GTP 结合蛋白，参与膜区室化和重塑。在芽殖酵母中，五个有丝分裂 septins 定位于芽颈处，那里的质膜富含磷脂酰肌醇-4,5-二磷酸 (PtdIns4,5P(2))。我们之前建立了纯化酵母 septin 复合物的亚基组织，以及这些异八聚体如何在溶液中和含有 PtdIns4,5P(2)的脂质单层上聚合成长丝。体外 septin 超微结构与固定细胞中通过薄切片电子显微镜在颈部观察到的含 septin 的纤维之间的关系尚不清楚。由于这些纤维的横截面较小，因此在拥挤的细胞空间中对这些纤维进行形态描述具有挑战性。为了研究原位 septin 组织，通过冷冻取代细胞的电子断层扫描以及 cryo-electron tomography 分析了正在分裂的酵母细胞的切片。我们发现与母芽轴垂直和平行的纤维网络，类似于脂质单层上的 septin 阵列，显示出与体外相应 septin 制剂的分子尺寸相匹配的重复模式。因此，这些原位结构很可能代表 septin 纤维。在缺乏单个 septin 的活突变体中，原位纤维仍然存在，尽管更加无序，这与其他证据一致，即 septin 的体内功能需要纤维形成。
