Learning principles from the pyrenoid, a phase-separated organelle

项目摘要 相分离是一种新兴的细胞内生物学组织原则。现在正在进行的流程 据了解，利用相分离包括遗传物质、基因表达、核糖体的存储 合成、信号、应激反应和新陈代谢。虽然每个相分离系统都有独特的 除了特点外，还有与所有这类制度有关的普遍主题，包括对阶段的管制 边界、凝析油内部和凝析油之间的混合动力学以及凝析油之间的相互作用 和他们周围的环境。为了揭示关于这些共同主题的一般原则，我们将重点放在 非常适合的模式生物和系统：遗传易驯化的藻类莱茵衣藻和 它的类蛋白核是一种无膜结合、相分离的细胞器，负责有效地固定碳。 类蛋白核具有许多实用的优点：1.它的相分离是由两个势垒驱动的。 鉴定的组分，刚性酶复合体Rubisco和柔性连接蛋白EPYC1，VIA 已知的特定结合界面；2.类蛋白核在体外的流动性是在体外复制的，没有能量 来源；3.体内组装/拆解可由外部线索控制；以及4.蛋白核是单一的， 足够大，足够稳定，可以系统地研究它与其他细胞的功能相互作用 组件。基于这些优势，类蛋白核已被证明是许多 发现包括一种灵活的多价联接子缩合刚性成分的能力，遗传 通过分裂获得非膜结合细胞器，通过保守的结合基序进行特异性募集，以及 魔数效应。我们将用这个系统解决的关键普遍问题是：角色是什么 决定凝析油性质的相互作用基元的价态、强度和间距？多么 小分子齐聚物的稳定性是否控制相界？是什么使凝析油保持在液体中？ 州政府？电池如何控制凝析油的数量、大小和位置，包括它们与其他 细胞结构？我们的方法将把理论和实验紧密结合起来，作为提供基础 这些问题的答案需要多学科的方法，将具体数据放在广泛的 理论框架。我们预计，我们对潜在生物物理机制的关注将有助于 我们的结果可推广到广泛的相分离的细胞内系统。

PROJECT ABSTRACT Phase separation is an emerging organizing principle for intracellular biology. Processes that are now understood to exploit phase separation include storage of genetic material, gene expression, ribosome synthesis, signaling, stress response, and metabolism. While each phase-separating system has unique features, there are universal themes relevant to all such systems, including regulation of the phase boundary, the dynamics of mixing within and between condensates, and the interactions of condensates with their surroundings. To uncover general principles regarding these common themes, we focus on a well-suited model organism and system: the genetically tractable alga Chlamydomonas reinhardtii and its pyrenoid, a non-membrane bound, phase-separated organelle responsible for efficient carbon fixation. The pyrenoid offers many practical advantages: 1. its phase separation is driven by two well- characterized components, the rigid enzyme complex Rubisco and the flexible linker protein EPYC1, via a known specific binding interface; 2. the pyrenoid’s in vivo liquidity is reproduced in vitro with no energy source; 3. in vivo assembly/disassembly is controllable by external cues; and 4. the pyrenoid is singular, large, and stable enough to systematically investigate its functional interactions with other cellular components. Based on these advantages, the pyrenoid has already proven to be a source of many discoveries including the ability of a flexible multivalent linker to condense a rigid component, inheritance of non-membrane bound organelles by fission, specific recruitment via a conserved binding motif, and a magic-number effect. The key universal questions we will address with this system are: What is the role of the valence, strength, and spacing of the interacting motifs in determining condensate properties? How does the stability of small oligomers control phase boundaries? What keeps condensates in a liquid state? How do cells control the number, size, and location of condensates, including their relation to other cellular structures? Our approach will closely integrate theory and experiment, as providing fundamental answers to these questions requires a multidisciplinary approach that places specific data within a broad theoretical framework. We anticipate that our focus on underlying biophysical mechanisms will facilitate generalizability of our results to a wide range of phase-separated intracellular systems.