Role of mechanical signalling at the nuclear envelope in pluripotent stem cell differentiation

核膜机械信号在多能干细胞分化中的作用

• 批准号: BB/W000423/1
• 负责人: Richard Tyser
• 金额: $ 82.24万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW000423%2F1
• 关键词: Role; mechanical; signalling; nuclear; envelope

Mammalian development and subsequent organ maintenance relies on the ability to deliver the right cells to the right place at the right time. This highly coordinated process relies on the proper regulation of cell fate transitions in stem cells. However, at this time, how cell fate transitions are regulated is poorly understood. One aspect to how transitions are regulated is how the instructive genes are positioned and expressed in the nucleus. Indeed, the spatial organisation of the genome, and how it changes in a cell, helps to control when and how genes are activated. Proteins found at the outside of the nucleus, referred to as nuclear envelope proteins, tether specific genes to the periphery of the nucleus and play an important role in regulating those genes. As evidence of their importance, mutations in nuclear envelope proteins often result in developmental disorders. Mutant nuclear envelope proteins have been implicated in a range of human pathologies such as premature ageing (e.g. Hutchinson-Gilford progeria syndrome) and neuromuscular diseases (e.g. Emery-Dreifuss muscular dystrophy). It is therefore important to understand how these proteins influence mammalian development to shed light not only on how these diseases emerge, but also to exploit the therapeutic potential of differentiating stem cells towards specific lineages of choice. Nevertheless, how nuclear envelope proteins influence gene expression in stem cells during development remains unclear. At the same time, the nuclear envelope proteins in question have also been shown to be sensitive to forces, which are ubiquitous during development and organ maintenance. Furthermore, stem cells are highly responsive to these forces, altering gene positioning and expression in response to them. In this proposal, we hypothesize that forces on the nucleus alter the function of nuclear envelope proteins and thus gene positioning and expression. We further hypothesize that this force-mediated genetic regulation is an important aspect of fate transitions. To test our hypotheses, we will focus on two questions. First, how do nuclear envelope proteins regulate gene expression in stem cells? Second, how do forces affect nuclear envelope proteins and subsequent gene expression during stem cell differentiation? In the proposed research, we will investigate these questions to bring greater understanding not only to spatial regulation of gene expression in stem cells, but also to how stem cells respond to, and exploit, forces in their environment. Here, we address how nuclear envelope proteins influence early mammalian development by taking advantage of recent advances in next-generation sequencing that allow us to study how the genome folds at the level of a single cell and 'state-of-the art' microscopes capable of tracking individual proteins and genes in living cells.We will first identify genes that are controlled by nuclear envelope proteins during stem cell differentiation (Aim 1). We will then use high-resolution microscopes to track individual genes 'live', allowing us to determine how nuclear envelope proteins control expression of these genes: is it by positioning or tethering them at the nuclear periphery where they are silenced, or by recruiting specific proteins to these genes that influence their expression (Aim 2)? Finally, to understand which nuclear envelope proteins influence the ability of a cell to sense forces in the cell, we will use a bespoke cell stretcher to apply mechanical stress to cells and use approaches described in Aim 1 and 2 to determine which specific nuclear envelope proteins are required for genes to respond to these external forces (Aim 3).These results will shed light on the role of nuclear envelope proteins in human development and disease, enhance understanding of the role of forces in development, and also improve strategies for differentiating pluripotent stem cells towards specific lineages for regenerative medicine.

哺乳动物的发育和随后的器官维护依赖于在正确的时间将正确的细胞运送到正确的地方的能力。这种高度协调的过程依赖于干细胞中细胞命运转变的适当调节。然而，在这个时候，细胞命运转变是如何被调控的，人们知之甚少。过渡如何被调节的一个方面是指导基因如何在细胞核中定位和表达。事实上，基因组的空间组织及其在细胞中的变化，有助于控制基因何时以及如何被激活。在细胞核外发现的蛋白质，被称为核包膜蛋白，将特定的基因连接到细胞核的外围，并在调节这些基因方面发挥重要作用。作为其重要性的证据，核膜蛋白的突变经常导致发育障碍。突变的核包膜蛋白与一系列人类病理有关，如早衰（如Hutchinson-Gilford早衰综合征）和神经肌肉疾病（如Emery-Dreifuss肌营养不良）。因此，了解这些蛋白质如何影响哺乳动物的发育是很重要的，这不仅有助于揭示这些疾病是如何出现的，而且还有助于开发干细胞分化的治疗潜力，使其朝着特定的谱系发展。然而，核膜蛋白在干细胞发育过程中如何影响基因表达仍不清楚。与此同时，所讨论的核膜蛋白也被证明对力很敏感，而力在发育和器官维持过程中无处不在。此外，干细胞对这些力量有高度的反应，改变基因的定位和表达。在这个提议中，我们假设对细胞核的力改变了核膜蛋白的功能，从而改变了基因的定位和表达。我们进一步假设这种力介导的遗传调控是命运转变的一个重要方面。为了验证我们的假设，我们将关注两个问题。首先，核膜蛋白如何调节干细胞中的基因表达？其次，在干细胞分化过程中，力如何影响核膜蛋白和随后的基因表达？在拟议的研究中，我们将研究这些问题，不仅对干细胞中基因表达的空间调控有更深入的了解，而且对干细胞如何响应和利用其环境中的力量也有更深入的了解。在这里，我们通过利用下一代测序的最新进展来解决核膜蛋白如何影响早期哺乳动物的发育，下一代测序使我们能够研究基因组如何在单细胞水平上折叠，以及能够跟踪活细胞中单个蛋白质和基因的“最先进”显微镜。我们将首先确定在干细胞分化过程中由核膜蛋白控制的基因（目的1）。然后，我们将使用高分辨率显微镜来跟踪单个基因的“活”，使我们能够确定核膜蛋白如何控制这些基因的表达：是通过将它们定位或拴在沉默的核外围，还是通过向这些影响其表达的基因募集特定蛋白质（目标2）？最后，为了了解哪些核包膜蛋白影响细胞感知细胞内力的能力，我们将使用定制的细胞拉伸器对细胞施加机械应力，并使用目标1和2中描述的方法来确定基因需要哪些特定的核包膜蛋白来响应这些外力（目标3）。这些结果将揭示核膜蛋白在人类发育和疾病中的作用，增强对发育中力量作用的理解，并改进多能干细胞向再生医学特定谱系分化的策略。