Dissecting the role of SPIN90 in cellular morphogenesis

剖析 SPIN90 在细胞形态发生中的作用

• 批准号: BB/V007483/1
• 负责人: Guillaume Charras
• 金额: $ 60.36万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV007483%2F1
• 关键词: Dissecting; role; SPIN90; cellular; morphogenesis

One of the most striking properties of living cells is their ability to change shape to fulfil their function, such as when they divide, migrate, and differentiate. Cell shape changes are governed by mechanical changes that occur in a thin layer of biopolymer situated below the membrane, known as the cortex. Polymers within the cortex are produced by specialized proteins, known as nucleators. Two of these are present in the cortex forming distinct network organisations: the Arp2/3 complex makes arborescent networks while mDia1 generates linear arrays of filaments. Changes in cortical mechanics can originate from changes in motor protein activity or cortex architecture, which arise from changes in polymer length or network organization. While we know a lot about how myosin activity alters cortex mechanics, we know much less about how changes in architecture do. One potential mechanism to control cortex architecture involves regulation of nucleators. Yet, little is known about the molecular mechanisms of coordination between nucleators. One class of proteins called Nucleation Promoting Factors (NPFs) is involved in regulating nucleators. We identified several cortical NPFs that can interact with multiple nucleators, making them prime candidates to mediate crosstalk. One of these, SPIN90, appears essential in division and development, profoundly alters the organisation of F-actin networks, controls the mechanics of cells and its mRNA is ubiquitously expressed. Thus, it may play a general role as an effector of cell shape change. However, we currently know very little about where and when SPIN90 acts in cell shape change, its interactors, and how it induces cortical reorganization. Indeed, to date, only ~30 papers have examined its function.I propose to investigate SPIN90 function during cell shape change focusing on the following aims:1) Determine the interactors of SPIN90 2) Investigate the spatiotemporal role of SPIN90 in processes involving cell shape change3) Examine how SPIN90 controls cortical network architecture and cell mechanicsMy lab is uniquely placed to investigate these questions because of our expertise on the genesis of the cell cortex, cell mechanics, and cytoskeletal organisation.Objective 1 will identify interactors of SPIN90. For this, we will fuse SPIN90 to a biotin ligase, TurboID. This enzyme generates a cloud of activated biotin that reacts with proteins in close proximity to SPIN90. Biotinylated proteins can be isolated and identified by mass spectrometry. Although our previous work showed that removal of SPIN90 led to cell death, where and when it acts during cell shape changes remains unclear. Aim 2 will examine a role for SPIN90 in processes of cell shape change using fluorescence imaging. Importantly, we will identify when and where SPIN90 interacts with each nucleator using approaches in which fluorescence only occurs upon interaction between SPIN90 and one of its interactors.Our published data demonstrates that depleting SPIN90 leads to a significant increase in cortical stiffness in dividing cells. This stiffening may result from changes in cortex organisation. Aim 3 will establish how SPIN90 activation induces changes in cortex architecture at the molecular scale. For that, it will use atomic force microscopy, a technique which enables imaging of the cortex with nm-resolution and characterization of cell mechanics. We will use numerical simulations to determine how changes in cortex architecture correlate with changes in mechanics. In summary, this project will determine how SPIN90 coordinates nucleator activity to control cell mechanics for cell shape change. In addition, we will identify how SPIN90 integrates with other cytoskeleton remodelling pathways during cell division and migration.

活细胞最引人注目的特性之一是它们能够改变形状以实现其功能，例如当它们分裂，迁移和分化时。细胞形状的变化是由位于膜下的生物聚合物薄层（称为皮层）中发生的机械变化控制的。皮质内的聚合物是由称为成核剂的特殊蛋白质产生的。其中两种存在于皮层中，形成不同的网络组织：Arp 2/3复合物形成树状网络，而mDia 1产生丝状物的线性阵列。皮质力学的变化可能源于运动蛋白活性或皮质结构的变化，这些变化源于聚合物长度或网络组织的变化。虽然我们知道很多关于肌球蛋白活动如何改变皮质力学，我们知道的很少关于结构的变化。控制皮层结构的一个潜在机制涉及核因子的调节。然而，很少有人知道的成核剂之间的协调的分子机制。一类称为成核促进因子（NPFs）的蛋白质参与调节成核因子。我们确定了几个皮质NPF，可以与多个成核剂相互作用，使其成为介导串扰的主要候选人。其中之一，SPIN 90，似乎在分裂和发育中必不可少，深刻地改变了F-肌动蛋白网络的组织，控制细胞的力学，其mRNA普遍表达。因此，它可能作为细胞形状改变的效应器发挥一般作用。然而，我们目前对SPIN 90在细胞形状变化中的作用位置和时间、它的相互作用物以及它如何诱导皮质重组知之甚少。事实上，到目前为止，只有大约30篇论文研究了它的功能。我建议研究SPIN 90在细胞形状变化过程中的功能，重点是以下目标：1）确定SPIN 90的相互作用2）研究SPIN 90在细胞形状变化过程中的时空作用3）研究SPIN 90如何控制皮层网络结构和细胞力学我的实验室是唯一的位置来研究这些问题，因为我们的专业知识在细胞皮层的起源，细胞力学和细胞骨架组织。目标1将确定SPIN 90的相互作用。为此，我们将SPIN 90与生物素连接酶TurboID融合。这种酶产生一团活化的生物素，与SPIN 90附近的蛋白质反应。生物素化的蛋白质可以通过质谱法分离和鉴定。虽然我们以前的工作表明，去除SPIN 90会导致细胞死亡，但在细胞形状变化期间，它在何处以及何时起作用仍不清楚。目的2将使用荧光成像检查SPIN 90在细胞形状变化过程中的作用。重要的是，我们将确定何时何地SPIN 90与每个成核剂相互作用的方法，其中荧光只发生在SPIN 90和它的interactors之一之间的相互作用。我们发表的数据表明，耗尽SPIN 90导致分裂细胞皮质硬度显着增加。这种硬化可能是由于皮层组织的变化。目的3将确定SPIN 90激活如何在分子水平上诱导皮质结构的变化。为此，它将使用原子力显微镜，这是一种能够以nm分辨率对皮质进行成像并表征细胞力学的技术。我们将使用数值模拟来确定皮层结构的变化如何与力学变化相关联。 总之，本项目将确定SPIN 90如何协调成核剂活性以控制细胞形状变化的细胞力学。此外，我们将确定如何SPIN 90整合与其他细胞骨架重塑途径在细胞分裂和迁移。