Revealing the nanoscale organization of B lymphocyte surface receptors and their lipid environment using super resolution imaging approaches

B细胞是适应性免疫系统的关键要素，因为它们具有识别特定病原体并产生保护性抗体的能力。外来分子（抗原）的检测是由B细胞抗原受体（BCRs）进行的，它嵌入细胞质膜内。BCR受辅受体的严格调控，辅受体可增强或降低其活性，从而促进或抑制抗体的产生。这些不同的细胞表面受体的相互作用是一个分子决策过程，进一步涉及周围的质膜脂。然而，所有这些成分的空间组织以及它们共同作用以允许适当的细胞反应的方式仍然不清楚。最近，我们开发了一种方法来确定细胞表面受体的组织具有高精度和精密度，依赖于高分辨率成像。该方法基于精确标记的高亲和小分子探针结合超分辨率显微镜对质膜定位分子的有效染色。该方法将用于破译不同BCR同型的拓扑结构，这些同型与调节性辅助受体CD19、CD22和CD45有关。成像方法将伴随着功能信号研究和突变分析，以获得最终控制抗体产生的不同细胞表面受体之间分子相互作用的详细图像。此外，我们将通过不同的超分辨率成像技术阐明信号活性质膜脂的分布和组织。为此，我们将采用同位素标记亲和探针和脂质代谢标记，利用纳米二次离子质谱（nanoSIMS）成像确定它们与B细胞表面受体的定位。我们的工作将大大扩展我们对免疫受体和质膜成分的相互作用和相互作用的认识，从而将推进我们对控制体液免疫和自身免疫的分子决策过程的理解。

B cells are key elements of the adaptive immune system as they have the capability to recognize specific pathogens and to produce protective antibodies. The detection of foreign molecules (antigens) is carried out by B cell antigen receptors (BCRs), which are embedded within the cellular plasma membrane. The BCR is critically regulated by coreceptors that either enhance or reduce its activity and thus can either promote or repress the production of antibodies. The interplay of these different cell surface receptors is a molecular decision-making process that furthermore involves circumambient plasma membrane lipids. However, the spatial organization of all of these components as well as the way by which they act together to allow for an appropriate cellular response remain unclear. Recently, we have developed a methodology to determine the organization of cell surface receptors with high accuracy and precision that relies on high-resolution imaging. The method is based on the efficient staining of plasma membrane-localized molecules with precisely labeled high-affinity small molecular probes in combination with super-resolution microscopy. This approach will be used to decipher the topology of different BCR isotypes in relation to the regulatory coreceptors CD19, CD22 and CD45. The imaging approaches will be accompanied by functional signaling studies and mutational analyses to obtain a detailed picture of the molecular interactions between the different cell surface receptors that ultimately control antibody production. Furthermore, we will elucidate the distribution and organization of signaling-active plasma membrane lipids by using different super-resolution imaging techniques. To this end, we will employ isotope-labeled affinity probes and the metabolic labeling of lipids to ascertain their localization in relation to B cell surface receptors using nanometer secondary ion mass spectrometry (nanoSIMS) imaging. Our work will substantially extend our knowledge about the interplay and interactions of immune receptors and plasma membrane constituents and thus will advance our understanding of the molecular decision-making processes that control humoral immunity and auto-immunity.