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

哺乳动物的发育和随后的器官维持依赖于在正确的时间将正确的细胞运送到正确的地点的能力。这一高度协调的过程依赖于对干细胞中细胞命运转变的适当调控。然而，目前人们对细胞命运转换是如何调控的知之甚少。如何调控过渡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，基因组的空间组织以及它在细胞中的变化方式，有助于控制基因何时以及如何被激活。核外发现的蛋白质，被称为核膜蛋白，将特定的基因拴在细胞核的外围，并在调节这些基因方面发挥重要作用。作为它们重要性的证据，核膜蛋白的突变通常会导致发育障碍。突变的核膜蛋白与一系列人类病理有关，例如过早衰老(例如Hutchinson-Gilford早衰症)和神经肌肉疾病(例如Emery-Dreifuss肌营养不良症)。因此，重要的是要了解这些蛋白质如何影响哺乳动物的发育，不仅要阐明这些疾病是如何出现的，而且要开发将干细胞分化为特定选择谱系的治疗潜力。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，研究中的核膜蛋白也被证明对力敏感，这种力在发育和器官维持过程中普遍存在。此外，干细胞对这些力量有很高的反应能力，改变基因的定位和表达。在这个提议中，我们假设作用于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，这种力介导的基因调控是命运转换的一个重要方面。为了检验我们的假设，我们将集中在两个问题上。首先，核膜蛋白如何调节干细胞中的基因表达？第二，在干细胞分化过程中，作用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，不仅是为了更好地理解干细胞中基因表达的空间调控，也是为了了解干细胞如何响应和利用环境中的力量。在这里，我们将利用下一代测序的最新进展以及能够跟踪活细胞中单个蛋白质和基因的最先进的显微镜来研究核膜蛋白如何影响早期哺乳动物的发育。我们将首先识别在干细胞分化过程中由核膜蛋白控制的基因(目标1)。然后，我们将使用高分辨率显微镜跟踪单个基因的“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它们沉默的核外围，还是通过向这些基因招募特定的蛋白质来影响它们的表达(目标2)？最后，为了了解哪些核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和目标2中描述的方法来确定基因响应这些外力所需的特定核膜蛋白(目标3)。这些结果将阐明核膜蛋白在人类发育和疾病中的作用，增强对力量在发育中的作用的理解，并改进将多能干细胞分化为特定再生医学谱系的策略。

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.