Dissecting the role of SPIN90 in cellular morphogenesis

活细胞最显著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细胞膜下方的一层薄薄的生物聚合物(称为皮质)中发生的机械变化决定的。皮质内的聚合物是由称为核蛋白的特殊蛋白质产生的。其中两个存在于皮层中，形成不同的网络组织：Arp2/3复合体形成树状网络，而mDia1产生线状细丝阵列。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，这些变化是由聚合物长度或网络组织的变化引起的。虽然我们对肌球蛋白活动如何改变大脑皮层机制知道很多，但我们对结构变化的影响却知之甚少。控制大脑皮层结构的一个潜在机制涉及对核因子的调节。然而，人们对核子之间的分子协调机制知之甚少。一类被称为成核促进因子(NPFS)的蛋白质参与了对核因子的调节。我们确定了几种皮质NPF，它们可以与多个核仁相互作用，使它们成为调节串扰的主要候选者。其中一种是SPIN90，它似乎在分裂和发育中是必不可少的，它深刻地改变了F-肌动蛋白网络的组织，控制着细胞的机制，其mRNA普遍表达。因此，它可能作为细胞形状改变的效应因子发挥普遍的作用。然而，我们目前对SPIN90在细胞形状改变中的作用地点和时间，它的相互作用，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SPIN90的功能。我建议研究SPIN90在细胞形状变化中的作用，主要集中在以下几个目标：1)确定SPIN90的相互作用；2)研究SPIN90在细胞形状变化过程中的时空作用；3)研究SPIN90如何控制皮质网络结构和细胞机械。由于我们在细胞皮质发生、细胞力学和细胞骨架组织方面的专业知识，我的实验室处于独特的位置来研究这些问题。为此，我们将SPIN90与生物素连接酶TurboID融合。这种酶产生一团活化的生物素，与SPIN90附近的蛋白质发生反应。生物素化的蛋白质可以通过质谱学进行分离和鉴定。尽管我们之前的工作表明，去除SPIN90会导致细胞死亡，但它在细胞形状变化期间何时何地起作用仍不清楚。目标2将利用荧光成像技术研究SPIN90在细胞形状改变过程中的作用。重要的是，我们将确定SPIN90何时何地与每个核仁相互作用，方法是只有在SPIN90与其相互作用的一个相互作用时才会出现荧光。我们公布的数据表明，耗尽SPIN90会导致细胞分裂中皮质硬度的显著增加。这种僵硬可能是由于皮质组织的变化所致。目的3将从分子水平上确定SPIN90的激活如何引起皮层结构的改变。为此，它将使用原子力显微镜，这是一种能够以纳米分辨率成像皮质和表征细胞力学的技术。我们将使用数值模拟来确定皮质结构的变化如何与力学的变化相关联。综上所述，该项目将确定SPIN90如何协调核因子活动来控制细胞形态变化的细胞机制。此外，我们将确定SPIN90如何在细胞分裂和迁移过程中与其他细胞骨架重塑途径整合。

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.