Topological Assembly of Signalling Proteins in T cells

背景资料。T细胞是人类免疫必不可少的获得性免疫细胞，在清除病原体和肿瘤监测中发挥着核心作用。为了识别有害抗原，T细胞依靠其膜抗原受体(TCR)快速有效地扫描抗原提呈细胞(APC)的表面。在抗原识别后，T细胞和APC之间的接触界面发生了巨大的变化，称为免疫突触，触发了一系列信号事件，最终导致多种T细胞反应，包括增殖、分化或细胞死亡。一种似乎对信号转导至关重要的机制是在T细胞激活过程中TCR和其他信号蛋白(即细胞表面受体、激活激酶和适配器蛋白)的空间组织。荧光显微镜技术是观察这一机制的关键[1]。在激活的T细胞中，信号蛋白在磷酸化的TCR周围形成微簇(多分子聚集体)，这是早期信号传递的重要位置。然后，这些微簇结合在一起形成一个中心超分子激活簇，该簇被黏附蛋白和致密的皮质肌动蛋白网所包围，从而在T细胞和APC之间建立了密切的联系[2]。虽然免疫突触内的主要信号蛋白、它们的相互作用和空间组织已知很多，但在成熟突触中介导蛋白质运输以建立微簇形成的机制尚不完全清楚。肌动蛋白细胞骨架重塑被广泛认为在免疫突触启动过程中起着关键作用，它在突触外围形成致密的网络[3，4]。然而，对调控膜蛋白和聚集性的确切机制缺乏了解。瞄准。我们的关键假设是，在较大的激活簇中，微簇的结合可以用由于肌动蛋白对抗原提呈细胞的动态重组而在膜上产生正的拓扑缺陷来解释。肌动蛋白网络显示出向内生长的运输网络的所有特征，我们认为这种伪2D结构的几何限制可以影响质膜拓扑，从而增加蛋白质在有助于微簇形成的局部环境中的停留时间。这个博士项目的总体目标是使用主动向列学(由自动驱动的细长单元组成的主动系统，如肌动蛋白细丝[5])的数学形式来阐明控制T细胞免疫突触内信号蛋白组织的机制。事实上，在主动向列学中，拓扑缺陷自发地出现并导致复杂的集体自组织行为[5]。实验将在西蒙塞利的实验室里进行，实验对象是不朽的T细胞系Jurkat，目前的T细胞信号传递模式就是在Jurkat中开发出来的。Jurkat T细胞系(已有)将被培养，并在需要时(根据标准方案)使用LCN提供的细胞培养设施瞬时转染荧光表达载体。为了模拟T细胞-APC界面，T细胞将沉积在平面支撑的脂质双层上，上面装饰着能够激活T细胞的合适的蛋白质和配体。不同T细胞蛋白的蛋白质扩散，包括TCR、CD45和LFA-1(在静止和激活条件下)将使用定制的TIRF显微镜(已有)进行记录。该装置目前包括两条激光线路(647 nm和488 nm)，一个科学互补的金属氧化物半导体(SCMOS)相机，以及适合超分辨率成像的适当光学元件。

Background. T cells are adaptive immune cells essential for human immunity, playing a central role in pathogen elimination and tumour surveillance. To recognise harmful antigens, T cells rely on their membrane antigen receptors (TCRs) to scan the surface of antigen-presenting cells (APCs) rapidly and efficiently. After antigen recognition, large changes occur in the contact interface between T cells and APCs, known as the immunological synapse, triggering a cascade of signalling events that ultimately lead to multiple T cell responses, including proliferation, differentiation, or cell death. One mechanism that seems essential for signal transduction is the spatial organisation of TCRs and other signalling proteins (i.e., cell surface receptors, activating kinases and adaptor proteins) during T cell activation. Fluorescence microscopy techniques have been paramount to observe this mechanism [1]. In activated T cells, signalling proteins form microclusters (multi-molecular aggregates) around phosphorylated TCRs, which are important sites for early signalling. These microclusters then coalesce to form a central supramolecular activation cluster which is surrounded by adhesion proteins and a dense cortical actin mesh to establish an intimate contact between the T cell and the APC [2]. Whilst the main signalling proteins, their interactions and spatial organisation within the immunological synapse are largely known, the mechanisms that mediate protein trafficking to establish microcluster formation in the mature synapse are not completely understood. Actin cytoskeletal remodelling is widely thought to play a key role during the immunological synapse initiation by forming a dense meshwork at the synapse periphery [3,4]. However, there is a lack of understanding of the exact mechanism responsible for modulating membrane protein and clustering. Aim. Our key hypothesis is that the coalescence of microclusters in larger activation clusters can be explained with the generation of positive topological defects on the membrane due to the dynamic reorganisation of actin in response to antigen-presenting cells. The actin network shows all the characteristic of an inward-growing transportation network, and we believe that the geometrical confinement of this pseudo-2D architecture can influence the plasma membrane topology, which in turns increases dwell time of proteins in local environments aiding microcluster formation. The overall goal of this PhD project is then to use the mathematical formalism of active nematics (active systems made of self-driving elongated units, such as actin filaments [5]) to shed light into the mechanisms that govern the organisation of signalling proteins within the T cell immunological synapse.In fact, in active nematics, topological defects spontaneously emerge and lead to complex collective self organisation behaviours [5].Methodology. Experiments will be performed in Simoncelli's lab with the immortal T cell line Jurkat, in which the current T cell signalling paradigm was developed. The Jurkat T cell line (already available) will be cultured and transiently transfected with fluorescently expressing vectors when required (according to standard protocols), using the cell culture facility available in the LCN. To mimic the T cell-APC interface, T cells will be deposited on planar supported lipid bilayers decorated with suitable proteins and ligands capable of T cell activation. Protein diffusion of different T cell proteins, including the TCR, CD45 and LFA-1 (under resting and activating conditions) will be recorded using a custom-built TIRF microscope (already available). The set up currently counts with two laser lines (647 nm and 488 nm), a scientific complementary metal-oxide-semiconductor (sCMOS) camera, and appropriate optical elements suitable for super-resolution imaging.