Microfluidic setup for quantification of multivalent interactions and massive parallelization of single-molecule force spectroscopy: Unraveling virus-receptor interactions

在许多生物过程中观察到多价相互作用，如病原体(如病毒)与细胞的附着。对这种过程的定量理解需要在单一相互作用层面上量化潜在的多价相互作用，这是一个尚未解决的问题。为了应对这一挑战，我们在上一个资助期建立了基于微流控和全内反射荧光(TIRF)显微镜的两种分析方法，使我们能够以单分子分辨率和高数据吞吐量探测微弱和多价(病毒-膜)相互作用。我们证明，膜结合病毒的迁移率提供了病毒价态(即在多价病毒-膜相互作用中参与的单一相互作用的数量)的度量，从而能够确定关键的结合属性，如病毒附着率、病毒价态分布的定性度量以及病毒从膜中释放的价态依赖的速率。尽管这些研究对病毒-膜相互作用的性质产生了新的基本见解，但对病毒价态的准确量化仍然是不可能的。这一限制是由于缺乏关于价值对连接到支撑的脂质双层(SLB)的颗粒的流动性的影响的实验数据。膜流体动力学的这一方面在理论上已经得到了很好的证实，但到目前为止还没有得到实验证实。在这个项目的最后阶段，我们的目标是使用最近建立的方法和系统来进行专门的迁移率研究，这将能够定量地确定SLB结合的纳米颗粒(蛋白质和病毒)的价态-迁移率关系。为此，我们将结合微流体、基于TIRF的单分子定位显微镜和单粒子跟踪(SPT)来探测霍乱毒素B亚单位(CTxB)、多瘤病毒SV40的病毒样颗粒以及甲型流感病毒在没有和存在剪切力的情况下的运动。一种双重标记方法，其中受体和纳米颗粒被荧光标记，将能够同时确定单个SLB结合纳米颗粒的价态(通过逐步漂白)和迁移率(通过SPT)。施加定义明确的流体动力剪切力将能够诱导单个受体的释放(参与多价相互作用)，从而根据价态来量化受体的失效率。这两种方法结合在一起，将提供关于支撑膜的流体动力学的新信息，并允许定量确定多价相互作用的价态。虽然这里主要应用于病毒，但本方法原则上可以扩展到在界面上形成的任何类型的多价相互作用(即一般的感染性病原体)。

Multivalent interactions are observed in a multitude of biological processes like the attachment of pathogens (e.g., viruses) to cells. A quantitative understanding of such processes requires the quantification of the underlying multivalent interaction on the single-interaction level, which is a yet unresolved issue. To address this challenge, we established in the previous funding period two assays based on microfluidics and total internal reflection fluorescence (TIRF) microscopy, which enabled us to probe weak and multivalent (virus-membrane) interactions with single-molecule resolution and high data throughput. We demonstrated that the mobility of membrane-bound viruses provides a measure for the virus valency (i.e., the number of single interactions engaged within a multivalent virus-membrane interaction), which enabled to determine key binding properties, such as the rate of virus attachment, a qualitative measure for the distribution of the virus valency as well as the valency-dependent rate of virus release from the membrane. Although these investigations yielded new fundamental insights on the properties of virus-membrane interactions, an accurate quantification of virus valency was not yet possible. This limitation is caused by a lack of experimental data on the impact of the valency value on the mobility of particles linked to a supported lipid bilayer (SLB). This aspect of membrane hydrodynamics is theoretically very well established, but was so far not experimentally confirmed. In the final period of this project, we aim to use recently established methods and systems to perform dedicated mobility studies, which will enable to quantitatively determine the valency-mobility relationship of SLB-bound nanoparticles (proteins and viruses). To this end, we will combine microfluidics, TIRF-based single-molecule localization microscopy, and single particle tracking (SPT) to probe the motion of the subunit B of cholera toxin (CTxB), of virus-like particles of the polyomavirus SV40 and of influenza A viruses in absence and presence of an applied shear force. A dual labeling approach, in which receptors as well as nanoparticles are fluorescently labeled, will enable to simultaneously determine the valency (by stepwise bleaching) and mobility (by SPT) of individual SLB-bound nanoparticles. Application of a well-defined hydrodynamic shear force will enable to induce release of individual receptors (engaged in a multivalent interaction) and hence to quantify the off-rate of receptors in dependence of the valency.Taken together, both approaches will provide novel information about hydrodynamics of supported membranes and allow for a quantitative determination of the valency of multivalent interactions. Although applied here mainly to viruses, the presented methodology can, in principle, be extended to any type of multivalent interaction that is formed at interfaces (i.e., to infectious agents in general).