Higher-ordered assembly of septins in a cell-based system.

间隔蛋白形成高度保守的细胞骨架蛋白家族，其作用于膜和基于肌动蛋白和微管的细胞骨架的界面。已知异源和同源寡聚化成细丝和更高级的结构，如成束的细丝和环，隔蛋白支架其他蛋白质，并可以在不同的细胞结构中作为膜扩散屏障，如初级纤毛的基部，精子环和细胞分裂期间的分裂装置。人类基因组编码14个septin，而果蝇只有5个septin基因：sep 1，sep 2，sep 4，sep 5和peanut。我们已经发现果蝇Schneider的S2细胞形成细胞质，高度有序的管状结构，长度可达几微米，标记为Peanut和Sep 2，这是迄今为止测试的唯一septins。这些胞质管似乎不受细胞周期调节，也不与F-肌动蛋白或微管共定位。然而，当F-肌动蛋白聚合被抑制时，它们分解成环，表明与肌动蛋白细胞骨架的间接但功能性的相互作用。我们的长期目标是确定septins如何影响细胞结构。我们假设，更高层次的细胞质septin组件提供了一个独特的机会，了解septin的组织和功能的细胞。因此，我们的短期目标是开发和利用这种新的基于细胞的检测方法。我们将使用果蝇S2细胞模型，我们有丰富的经验，并结合遗传（RNAi，荧光蛋白的表达）和药理学工具，再加上活细胞荧光显微镜，定量图像分析和电子显微镜。我们将追求以下具体目标：1）定义Septin管和环的组成，装配原理和动态特性。2)测试肌动蛋白和隔蛋白细胞骨架之间相互作用的潜在机制。3)识别隔膜管组装/拆卸的其他调节器。这些研究将阐明septins在活细胞中组装成更高级结构的分子机制，并将阐明septins和肌动蛋白在间期细胞中无疑复杂的相互作用。这将进一步加深我们对后生动物细胞结构的基本理解。

Septins form a family of highly conserved, cytoskeletal proteins that act at the interface of membranes and the actin- and microtubule-based cytoskeletons. Known to hetero and homo oligomerize into filaments and higher-ordered structures such as bundled filaments and rings, septins scaffold other proteins and can act as membrane diffusion barriers in diverse cellular structures such as the base of the primary cilium, the sperm annulus, and the cleavage apparatus during cell division. The human genome encodes fourteen septins, while Drosophila has just five septin genes: sep1, sep2, sep4, sep5 and peanut. We have discovered that Drosophila Schneider's S2 cells form cytoplasmic, higher-ordered tubular structures up to several microns in length that label for Peanut and Sep2, the only septins thus far tested. These cytoplasmic tubes do not appear to be cell-cycle regulated and do not co-localize with F-actin or microtubules. However they disassemble into rings when F-actin polymerization is inhibited, indicating indirect, but functional interactions with the actin cytoskeleton. Hypothesis, Aims and Approach Our long-term aim is to determine how septins influence cellular architecture. We hypothesize that higher-ordered cytoplasmic septin assemblies provide a unique opportunity for understanding septin organization and function in cells. Our short-term aim is thus to develop and exploit this novel cell-based assay. We will use a Drosophila S2 cell model, with which we have extensive experience, and that combines genetic (RNAi, expression of fluorescent proteins) and pharmacological tools coupled with live-cell fluorescence microscopy, quantitative image analysis and electron microscopy. We will pursue the following specific aims: 1) Define the composition, principles of assembly and dynamic properties of septin tubes and rings. 2) Test potential mechanisms of interaction between the actin and septin cytoskeletons. 3) Identify other regulators of septin tube assembly/disassembly. These studies will elucidate the molecular mechanisms by which septins can assemble into higher-ordered structures in living cells, and will shed light on the undoubtedly complex interplay between septins and actin in interphasic cells. This will further our basic understanding of the architecture of the metazoan cell.