The regulation and function of cytoplasmic foci in quiescent cells

描述(申请人提供)：我们对真核细胞在不利于继续生长的条件下进入的类G0休眠状态的生物学感兴趣。我们的目标是定义在这些静止期诱导的过程，并确定它们如何共同促进细胞生存。这项建议扩展了这一分析，并研究了在静息细胞的细胞质中形成的一种特殊的核糖核蛋白(RNP)复合体，称为加工小体或P小体。P-小体是相对较大的聚集结构，包含非翻译的mRNAs和一组不同的蛋白质成分，包括参与这些转录本加工的一些酶。有趣的是，P小体只是G0细胞中形成的大量类似细胞质结构中的一个。这种流行表明，这些复合体对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潜在原因知之甚少。这些细胞质结构似乎对人类健康也很重要，因为与之相关的RNP复合体，应激颗粒，参与了神经疾病的病理，如肌萎缩侧索硬化症(ALS)和脊髓小脑性共济失调2型。总之，这些数据表明，这些细胞质病灶在真核生物学中发挥着重要的和不同的作用。因此，至关重要的是，我们必须更好地了解这些结构的功能及其组装受到管制的方式。这项建议通过研究一个模型RNP复合体，酵母的P-体，酿酒酵母来解决这些更广泛的问题。P-小体从酵母到人类都是保守的，我们对这些RNP结构的大部分了解都来自于对这种萌芽酵母的研究。然而，尽管进行了广泛的努力，人们对P小体组装的调节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。我们最近的研究为这两个问题提供了有趣的答案。特别是，我们已经确定cAMP依赖的蛋白激酶(PKA)是P小体组装的关键调节因子，并发现更大的P小体焦点似乎是静止细胞长期生存所必需的。我们的数据表明，PKA直接磷酸化P体的保守核心成分Pat1，从而破坏更大聚集体结构的形成。这里提出的实验旨在确定PKA控制的机制细节，并阐明较大的病灶如何促进细胞存活。最后，我们将进一步探索有趣的观察结果，即信号分子，如PKA本身，也存在于P小体和相关的复合体中。有待检验的基本假设是，随后恢复生长所需的特定蛋白质和mRNAs被储存在静止细胞中的P体灶内并受到保护。该方案的具体目的是：(1)确定PKA信号在P体灶形成中的调节作用；(2)确定P体灶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.