How the ESCRT-III-like protein Vipp1 assembles polymeric super-structures to mitigate membrane stress

在所有的生命系统中，膜被用来将细胞内部与外部环境分开。膜也被用来在内部塑造细胞，以便不同的区域可以形成具有不同角色的专科隔室。在细胞中，膜是动态的，需要对许多过程进行持续的重塑，包括为生长而进行的细胞分裂或为运输货物而进行的膜运输。为了重塑细胞膜，细胞进化了专门的蛋白质家族来承担这一物理工作。最重要的膜重塑家族之一是ESCRT-III蛋白。它们在真核生物(像我们自己的细胞)中是普遍存在的。ESCRT-III蛋白是如此古老，以至于它们在一些古生物中有祖先，真核生物后来就是从这些古生物进化而来的。最近，在一个令人兴奋的发现中，我们发现ESCRT-III蛋白也存在于细菌(PSPA)和蓝藻(Vipp1)中。这一点很重要，因为它表明在所有细胞的最后一个通用共同祖先(LUCA)中存在ESCRT-III样蛋白，并且包括细菌、古生物和真核生物在内的所有进化领域自最早的生命尝试以来都依赖ESCRT-III样蛋白来形成膜。ESCRT-III样蛋白承担许多基本功能。在人类中，它们对于分裂细胞的最终分离和膜修复是必不可少的。它们还与许多疾病有关，包括病毒入侵、细菌感染、癌症和神经退化，如痴呆症和亨廷顿病。由于PSPA在膜保护中的作用，它是抗菌素耐药(AMR)和细菌致病的驱动力。在这项建议中，我们研究Vipp1，它存在于所有蓝藻、藻类和植物中。我们知道Vipp1很重要，因为基因敲除通常是致命的。这是由于进行光合作用的类囊体膜的异常形成。我们仍然不知道的是，Vipp1在细胞中起什么作用，它的膜重塑功能是什么。目前，我们认为Vipp1蛋白组装在一起构建超结构，包括环、螺旋细丝和扁平支架，这些支架以某种方式塑造和支撑膜，可能在高应力区域，膜的完整性受到物理或化学威胁。这项提案的总体目标是了解Vipp1如何建造这些上层结构并使用它们在膜上进行机械工作的机制。Vipp1也代表了一个易于处理的系统，它可以向我们展示PSPA和更复杂的ESCRT-III系统如何工作并导致疾病的普遍机制原理。最后，基因工程蓝藻中的Vipp1修饰促进了营养和抗炎用途的脂肪酸的高产量。在未来的生物技术应用中，类似的Vipp1修饰可能有助于在蓝藻中生产其他有用的分子，如生物燃料。目的：1)了解Vipp1如何在不同的超结构之间建立和切换，从而形成、稳定和修复膜。具体地说，一种强大的电子显微镜形式将使我们能够可视化Vipp1原子在螺旋细丝中的准确位置，这样我们就可以了解它们的3D结构和化学。当二维平面细丝附着在薄膜上时，将在较低的分辨率下推导出该结构。了解Vipp1如何构建不同的结构形式是其膜重塑能力的核心2)探索Vipp1超结构如何在简化的“体外”环境中塑造膜的能力。通过将Vipp1与膜和Vipp1结合蛋白(VBP)混合，我们的目标是重建任何膜切割、连接或稳定事件，这些事件可能代表Vipp1在细胞中的功能。3)在细胞中寻找与Vipp1结合并改变其功能的其他蛋白质。这种VBP可能会改变Vipp1建造或拆卸上层建筑的方式，以及它重塑膜的方式。

In all living systems, membranes are used to separate the inside of the cell from the outside environment. Membranes are also used to shape cells internally so that different areas can form specialist compartments with distinct roles. In cells, membranes are dynamic requiring continual remodelling for many processes including cell division for growth or membrane trafficking for the movement of cargo. In order to remodel the membrane, cells have evolved specialist protein families to undertake this physical work.One of the most important membrane remodelling families are ESCRT-III proteins. They are universal in eukaryotes (cells like our own). ESCRT-III proteins are so ancient that they have ancestors in some archaea from which eukaryotes later evolved. Recently, in an exciting discovery, we showed that ESCRT-III proteins also exist in bacteria (PspA) and in cyanobacteria (Vipp1). This is important as it showed that an ESCRT-III-like protein was present in the last universal common ancestor of all cells (LUCA) and that all evolutionary domains including bacteria, archaea and eukaryotes have depended on ESCRT-III-like proteins to shape membrane since the earliest attempts at life.ESCRT-III-like proteins undertake many essential functions. In humans, they are essential for the final separation of dividing cells and membrane repair. They are also implicated in many diseases including viral invasion, bacterial infection, cancer and neurodegeneration such as dementia and Huntington's disease. Due to its role in membrane protection, PspA is a driver of anti-microbial resistance (AMR) and bacterial pathogenesis.In this proposal we study Vipp1, which is found in all cyanobacteria, algae and plants. We know that Vipp1 is important as gene knockout is usually lethal. This is due to abnormal formation of the thylakoid membranes where photosynthesis is undertaken. What we still do not know is what Vipp1 does in the cell and what its membrane remodelling duties are. Currently, we think that Vipp1 proteins assemble together to build superstructures that include rings, helical filaments and flat scaffolds that somehow shape and support membrane possibly in regions of high stress where the integrity of the membrane is physically or chemically threatened. The overall goal of this proposal is to understand the mechanism for how Vipp1 builds these superstructures and uses them to do mechanical work on the membrane. Vipp1 also represents a tractable system which can show us the universal mechanistic principles underlying how PspA and more complicated ESCRT-III systems work and cause disease. Finally, Vipp1 modification in engineered cyanobacteria facilitates high yields of fatty acids for both nutritional and anti-inflammatory use. In future biotechnological application, similar Vipp1 modification may facilitate the production of other useful molecules such as biofuels in cyanobacteria.Aims:1) to understand how Vipp1 builds and switches between different superstructures so as to shape, stabilise and repair membrane. Specifically, a powerful form of electron microscopy will allow us to visualize the precise position of the Vipp1 atoms within helical filaments so we can learn about their 3D structure and chemistry. 2D planar filament architecture when attached to membrane will be deduced at lower resolution. Understanding how Vipp1 builds different structural forms lies at the heart of its membrane remodelling capabilities.2) to explore how Vipp1 superstructures have the ability to sculpt membrane in a simplified 'in vitro' environment. By mixing Vipp1 with both membrane and Vipp1 binding proteins (VBPs), we aim to reconstitute any membrane cutting, joining or stabilising events that may represent what Vipp1 does in the cell.3) to find other proteins in the cell that attach to Vipp1 and changes how it functions. Such VBPs may shift the way Vipp1 builds or disassembles superstructures and how it remodels membrane.