Regulation of plant cell size coupled to DNA content

从细菌到人类，细胞大小对许多过程都很重要，包括营养物质和信号的交换、细胞分裂、细胞寿命和特化细胞的功能。尽管它很重要，但细胞如何实现并保持其基因控制的大小一直是一个长期存在的谜。在这一领域的一项突破中，我们最近发现植物细胞利用它们的DNA含量作为一种内部的、与大小无关的标准来调节自己的大小。该机制依赖于一种在视网膜母细胞瘤（RB）途径中起作用的蛋白质（KRP4），该途径控制从植物到人类等生物的细胞增殖。我们发现KRP4在细胞分裂前与染色体结合，而未结合的KRP4则被破坏。通过这种方式，无论新生细胞出生时大小如何，都会从染色体中释放一定量的KRP4。因此，这些细胞生长到相同的目标大小，KRP4被充分稀释以允许进行下一次细胞分裂。这一发现提出了进一步的重要问题。首先，这种机制依赖于KRP4在细胞分裂的不同阶段与染色体相互作用方式的变化，但这些变化发生的方式和原因的细节尚不清楚。揭示这些细节将有助于揭示不同细胞类型和不同生物体中细胞大小控制的共同和不同特征。为了实现这一目标，我们将鉴定在细胞分裂的不同阶段与KRP4相互作用的其他蛋白质，确定KRP4积累的基因组位点，并使用遗传方法来观察破坏这些相互作用的后果。其次，在发育过程中如何调整机制以产生不同大小的特化细胞？对于这个问题，我们将重点关注气孔（称为气孔）的形成，气孔调节叶片与大气之间的水和二氧化碳交换。我们关注气孔有几个原因：气孔细胞的大小非常均匀，对光合效率和水分利用非常重要；气孔的发育很容易进行实时成像，并且具有很好的遗传学特征。为了揭示气孔细胞大小是如何调节的，我们将使用定量实时成像来分析改变候选调节基因的后果，包括KRP4/RB通路中的基因。第三，一个多世纪以来，人们一直观察到，在许多生物体中，细胞的大小反映了它们基因组的大小，但其原因尚不清楚。使用DNA含量作为细胞大小控制的标准是否解释了为什么基因组较大的生物体往往具有较大的细胞？相反，在进化过程中，细胞大小的选择压力是否限制了基因组的大小？这些问题在植物中尤为重要，基因组复制在植物的进化和驯化中起着重要作用。为了解决这些问题，我们将测试KRP4/RB遗传途径的突变是否会使细胞大小与基因组大小脱钩。我们还将与瑞士的另一个研究小组（Kerstin Bomblies， ETH）合作，他们研究基因组复制在植物进化中的作用。在这次合作中，我们将测试基因组复制后重新调整气孔细胞大小的选择压力是否导致了KRP/ rb介导的细胞大小控制的重新校准。该项目的这一部分将使用群体基因组学方法和遗传分析结合实时成像来测试选择压力下的基因是否与细胞大小控制有关。

From bacteria to humans, cell size is important for many processes, including exchange of nutrients and signals, cell division, cell longevity and the function of specialised cells. Despite its importance, how cells achieve and maintain their genetically-controlled size has been a long-standing mystery. In a breakthrough in this field, we have recently found that plant cells adjust their own size using their DNA contents as an internal, size-independent standard. The mechanism relies on a protein (KRP4) that functions in the Retinoblastoma (RB), pathway, which controls cell proliferation in organisms ranging from plants to humans. We showed that KRP4 binds to chromosomes before cell division, whilst unbound KRP4 is destroyed. In this way, a fixed amount of KRP4 is released from the chromosomes of newly born cells, regardless of their size at birth. Consequently, these cells grow to the same target size, at which KRP4 is sufficiently diluted to allow progression to the next cell division. This discovery raises important further questions. First, the mechanism relies on changes in the way KRP4 interacts with chromosomes during different stages of cell division, but the details of how and why these changes happen are unknown. Revealing these details will help to uncover shared and divergent features of cell size control in different cell types and across different organisms. To achieve this aim, we will identify other proteins that interact with KRP4 at different stages of cell division, define the genomic sites where KRP4 accumulates, and use genetic methods to see the consequences of disrupting these interactions. Second, how is the mechanism adjusted during development to produce specialised cells with different sizes? For this question, we will focus on the formation of pores (called stomata) that regulate the exchange of water and carbon dioxide between the leaf and the atmosphere. We have several reasons to focus on stomata: the size of stomatal cells is remarkably uniform and important for photosynthetic efficiency and water use; the development of stomata is readily accessible for live imaging and is genetically very well characterised. To reveal how stomatal cell size is regulated, we will use quantitative live imaging to analyse the consequences of altering candidate regulatory genes, including those in the KRP4/RB pathway. Third, it has been observed for over a century that the size of cells reflects the size of their genome in a wide range of organisms, but the reason for this remains unclear. Does the use of DNA contents as a standard for cell size control explain why organisms with larger genomes tend to have larger cells? Conversely, do selective pressures on cell size constrain genome size during evolution? These questions are particularly important in plants, in which genome duplications have played a major role in evolution and domestication. To address them, we will test whether mutations in the KRP4/RB genetic pathway de-couple cell size from genome size. We will also collaborate with another group in Switzerland (Kerstin Bomblies, ETH), who study the role of genome duplication in plant evolution. In this collaboration, we will test whether selective pressure to re-adjust stomatal cell size after genome duplication has led to a re-calibration of KRP/RB-mediated cell size control. This part of the project will use population genomics methods and genetic analysis combined with live imaging to test whether genes under selective pressure are implicated in cell size control.