RNA localisation in endothelial cells: elucidating spatial regulation of gene expression to understand blood vessel growth and homeostasis

信使(M)RNA分子是基因和蛋白质之间的中间人。它们从DNA转录而来，然后用作翻译模板来合成功能蛋白质。大多数mRNAs并不均匀地分布在整个细胞中。根据不同的调节因素，这些分子倾向于在细胞的特定区域内积累，产生亚细胞分布模式。这种现象是称为RNA定位的一般机制的一部分，已经在几种细胞类型中进行了描述。利用关键的生物学模型，研究人员发现，mRNAs的定位对特定的细胞功能至关重要，如果没有它，细胞可能无法对刺激做出正确的反应，无法获得某些命运，甚至无法正确塑造。因此，人们认为RNA定位在动物发育中具有重要意义，它可能是一些人类疾病的基础。我们的实验室发现，RNA定位发生在内皮细胞中，内皮细胞是连接所有血管的构件。在这种情况下，将mRNAs分配到特定的亚细胞室，并在那里进行翻译，调节血管的生长。这项工作提供了新的证据，表明mRNAs的区隔对组织形成至关重要。然而，许多问题仍然没有得到回答。在内皮细胞中靶向mRNAs的确切分子机制是什么？定位机制是否也与血管功能的其他方面有关，如调节内皮细胞在完全形成的血管中形成的屏障？在这份提案中，我们将使用不同的模型和方法来回答这些问题。我们最新的发现导致了这一提议，表明mRNAs可以被重塑细胞骨架的蛋白质结合，细胞骨架是一种确保细胞结构和行为的结构。特别是，其中一些细胞位于细胞附着在底物或彼此之间的位置。这表明这些因素可能具有RNA定位作用，但尚不清楚。此外，考虑到它们在细胞运动和细胞间接触中的相关性，我们假设细胞骨架重塑蛋白在血管形成和内皮屏障功能过程中分布mRNAs。为了探索这些想法，我们将首先确定与细胞骨架重塑蛋白结合的确切mRNAs，然后描述它们的空间共同分布。我们将在运动的内皮细胞形成新血管时耗尽感兴趣的蛋白质，并使用最先进的显微镜分析信使核糖核酸定位模式的变化。我们还将调查这些蛋白质的丢失是否会改变局部靶基因的翻译。接下来，我们计划将重点放在血管内的内皮层。为此，我们将使用细胞外因子来破坏内皮层形成的屏障，并研究这些因素是否会改变mRNAs的定位。此外，我们将阻止细胞骨架重塑蛋白与其信使核糖核酸靶标的结合，以证明这一过程与内皮屏障生物学有关。鉴于到目前为止所描述的工作利用了在培养皿中模拟血管生物学的体外工具，在项目的最后阶段，我们将把我们的测试转移到小鼠视网膜上，这是研究体内血管形成和维护的优秀工具。这种方法对于理解RNA定位在复杂组织中的重要性至关重要。我们希望这个框架将帮助我们充分了解亚细胞mRNA的分布如何决定细胞行为和组织生物学。这将为精确医学开辟道路，不仅可以治疗血管相关的病理，还可以针对其他疾病中的细胞迁移和组织维护。

Messenger (m)RNA molecules are the middlemen between genes and proteins. They are transcribed from DNA and then used as translation templates to synthetise functional proteins. Most mRNAs are not equally distributed throughout the cell. Depending on distinct regulatory factors, these molecules tend to accumulate within particular regions of the cell, generating subcellular patters of distribution. This phenomenon is part of a general mechanism called RNA localisation, which has been described in several cell types. Using key biological models, researchers have found that the localisation of mRNAs is fundamentally important for particular cellular functions and without it, cells may fail to respond correctly to stimuli, to acquire certain fates or even to be properly shaped. Hence, it is thought that RNA localisation has major implications in animal development and it can be the basis of some human disorders.Our lab found that RNA localisation takes place in endothelial cells, the building blocks that line all blood vessels. In this context, distribution of mRNAs to particular subcellular compartments, where they are translated, regulates how vessels grow. This work provided new evidence that the compartmentalisation of mRNAs is crucial for tissue formation. However, many questions remain unanswered. What are the exact molecular mechanisms responsible for the targeting of mRNAs in endothelial cells? Are mechanisms of localisation also implicated in other aspects of vascular function, such as the regulation of the barrier formed by the endothelial cells in fully formed vessels? In this proposal, we will use distinct models and methodologies to answer these questions. Our most recent findings leading to this proposal indicate that mRNAs can be bound by proteins that remodel the cytoskeleton, a structure that ensures cell architecture and behaviour. In particular, some of these are located to sites where cells adhere to the substrate or to each other. This suggests that such factors could have RNA localisation roles yet to be described. Furthermore, given their relevance in cell movement and cell-cell contacts, we hypothesise that cytoskeletal remodelling proteins distribute mRNAs during blood vessel formation and endothelial barrier function.To explore these ideas, we will first identify the exact mRNAs bound by the cytoskeletal remodelling proteins and then characterise their spatial co-distribution. We will deplete the proteins of interest from motile endothelial cells as they form new vessels and use state-of-the-art microscopy to analyse shifts in mRNA localisation patterns. We will also investigate if the loss of these proteins alters local target translation. Next, we plan to focus on the endothelial layer within blood vessels. For this, we will use extracellular factors that disrupt the barrier formed by endothelial layers and investigate if these alter the localisation of mRNAs. In addition, we will block the binding of the cytoskeletal remodelling proteins to their mRNA targets to prove that this process is implicated in endothelial barrier biology. Given that the work described so far takes advantage of in vitro tools mimicking vascular biology in a petri dish, in the final stages of the project we will transpose our tests to the mouse retina, an excellent tool to study blood vessel formation and maintenance in vivo. Such approach will be crucial to understand the importance of RNA localisation in the context of a complex tissue. We hope that this framework will help us to fully grasp how subcellular mRNA distribution determines cell behaviour and tissue biology. This will open avenues in precision medicine not only to treat vascular related pathologies, but also to target cell migration and tissue maintenance in other disorders.