The regulation and function of cytoplasmic foci in quiescent cells

描述（由申请人提供）：我们对真核细胞在条件不利于持续生长时进入的 G0 样静息状态的生物学感兴趣。我们的目标是定义在这些静止期间诱导的过程，并确定它们如何共同促进细胞存活。该提案扩展了这种分析，并检查了在静息细胞的细胞质中形成的特定核糖核蛋白（RNP）复合物，称为处理体或 P 体。 P-体是相对较大的聚集体结构，包含非翻译 mRNA 和一组独特的蛋白质成分，包括参与这些转录物加工的许多酶。有趣的是，P 体只是 G0 细胞中形成的大量相似细胞质结构之一。这种普遍性表明这些复合物对于静息细胞的生物学很重要，但目前对这种大规模大分子隔离的根本原因知之甚少。这些细胞质结构似乎对人类健康也很重要，因为相关的 RNP 复合物（应激颗粒）与肌萎缩侧索硬化症 (ALS) 和 2 型脊髓小脑共济失调等神经系统疾病的病理有关。总而言之，这些数据表明这些细胞质灶在真核生物学中发挥着重要且多样的作用。因此，我们必须更好地了解这些结构的功能及其组装的调节方式，这一点至关重要。 该提案通过检查模型 RNP 复合体（酿酒酵母的 P 体）来解决这些更广泛的问题。 P-体从酵母到人类都是保守的，我们对这些 RNP 结构的了解大部分来自对这种芽殖酵母的研究。然而，尽管付出了大量努力，但人们对调节 P-body 组装的机制以及在静止细胞中形成的较大病灶的生理作用知之甚少。我们最近的工作为这两个问题提供了有趣的答案。特别是，我们已经确定 cAMP 依赖性蛋白激酶 (PKA) 是 P 体组装的关键调节因子，并发现较大的 P 体焦点似乎是静止细胞长期存活所必需的。我们的数据表明，PKA 直接磷酸化 Pat1（P-体的保守核心成分），从而破坏更大聚集结构的形成。这里提出的实验旨在定义 PKA 控制的机制细节，并阐明较大的焦点如何促进细胞活力。最后，我们将进一步探索一个有趣的观察结果，即信号分子（如 PKA 本身）也存在于 P 体和相关复合物中。待测试的基本假设是，随后恢复生长所需的特定蛋白质和 mRNA 在静止细胞的 P 体病灶内储存和保护。该提案的具体目标是：（1）确定PKA信号在P体灶形成调节中的作用； (2) 确定 P 体焦点在静止细胞中的生理作用。

DESCRIPTION (provided by applicant): We are interested in the biology of the G0-like resting states that eukaryotic cells enter when conditions are not conducive to continued growth. Our goal is to define the processes that are induced during these periods of quiescence and to determine how they collectively contribute to cell survival. This proposal extends this analysis and examines a particular ribonucleoprotein (RNP) complex that forms in the cytoplasm of resting cells, known as a Processing-body, or P-body. P-bodies are relatively large aggregate structures that contain non-translating mRNAs and a distinct set of protein constituents, including a number of enzymes involved in the processing of these transcripts. Interestingly, the P-body is just one of a large number of similar cytoplasmic structures that form in the G0 cell. This prevalence suggests that these complexes are important for the biology of the resting cell, but little is presently known about the underlying reasons for this large-scale sequestration of macromolecules. These cytoplasmic structures also appear to be important for human health as a related RNP complex, the stress granule, has been implicated in the pathology of neurological disorders, like amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type 2. In all, thes data suggest that these cytoplasmic foci play important and diverse roles in eukaryotic biology. It is therefore critical that we develop a better understanding of the functions of these structure and the manner in which their assembly is regulated. This proposal addresses these broader issues by examining a model RNP complex, the P-body of the yeast, Saccharomyces cerevisiae. P-bodies have been conserved from yeast to humans, and much of what we know about these RNP structures has come from studies with this budding yeast. However, despite extensive effort, little was known about the mechanisms regulating P-body assembly and the physiological role of the larger foci that form in quiescent cells. Our recent work has suggested interesting answers to both of these questions. In particular, we have identified the cAMP-dependent protein kinase (PKA) as a key regulator of P- body assembly, and found that the larger P-body foci appear to be required for the long-term survival of quiescent cells. Our data indicate that PKA directly phosphorylates Pat1, a conserved core constituent of P- bodies, and thereby disrupts the formation of the larger aggregate structures. The experiments proposed here aim to define the mechanistic details of this control by PKA and to elucidate how the larger foci promote cell viability. Finally, we will explore further the intriguing observation that signaing molecules, like PKA itself, are also found in P-bodies and related complexes. The underlying hypothesis to be tested is that specific proteins and mRNAs required for the subsequent resumption of growth are stored and protected within P-body foci in quiescent cells. The specific aims of this proposal are: (1) to determine the role of PKA signaling in the regulation of P-body foci formation; and (2) to determine the physiological role of P-body foci in quiescent cells.