Topological Assembly of Signalling Proteins in T cells

背景T细胞是人类免疫所必需的适应性免疫细胞，在病原体消除和肿瘤监视中发挥核心作用。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呈递细胞（APC）的表面。在抗原识别后，T细胞和APC之间的接触界面（称为免疫突触）发生巨大变化，触发一系列信号传导事件，最终导致多种T细胞应答，包括增殖、分化或细胞死亡。似乎对信号转导至关重要的一种机制是TCR和其他信号蛋白的空间组织（即，细胞表面受体、活化激酶和衔接蛋白）。荧光显微镜技术对于观察这种机制至关重要[1]。在活化的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），这是早期信号传导的重要位点。然后这些微簇聚结形成中央超分子活化簇，其被粘附蛋白和致密的皮质肌动蛋白网包围，以在T细胞和APC之间建立紧密接触[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在很大程度上是已知的，但介导蛋白质运输以在成熟突触中建立微簇形成的机制尚未完全理解。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通过在突触外周形成致密的网络起关键作用[3，4]。然而，目前对膜蛋白和簇集调控的确切机制还缺乏了解。瞄准我们的主要假设是，在较大的激活集群的微簇的合并可以解释与积极的拓扑缺陷的膜上的产生由于动态重组的肌动蛋白在抗原呈递细胞。肌动蛋白网络显示了向内生长的运输网络的所有特征，并且我们相信这种伪2D架构的几何限制可以影响质膜拓扑结构，这反过来又增加了蛋白质在局部环境中的停留时间，从而有助于微簇的形成。这个博士项目的总体目标是使用主动向列的数学形式（由自驱动的细长单元制成的活性系统，例如肌动蛋白丝[5]），以揭示T细胞免疫突触内控制信号蛋白组织的机制。事实上，在活性线虫中，拓扑缺陷自发出现并导致复杂的集体自组织行为[5]。实验将在Simoncelli的实验室中使用永生T细胞系Jurkat进行，其中开发了当前的T细胞信号传导模式。将使用LCN中可用的细胞培养设施培养Jurkat T细胞系（已可用），并在需要时（根据标准方案）用荧光表达载体瞬时转染。为了模拟T细胞-APC界面，将T细胞沉积在用能够活化T细胞的合适蛋白质和配体修饰的平面支撑的脂质双层上。将使用定制的TIRF显微镜（已经可用）记录不同T细胞蛋白（包括TCR、CD 45和LFA-1（在静息和活化条件下））的蛋白质扩散。目前，该设备配备了两条激光线（647 nm和488 nm），一个科学互补金属氧化物半导体（sCMOS）相机，以及适用于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.