Abstract: How the endocytic network mediates specificity of cell signaling Receptor crosstalk – the cooperation between two or more receptors to modulate cell responses – is a key signaling mechanism. It enables cells to generate a large combinatorial repertoire of specific signaling with a limited variety of receptors. Receptor crosstalk plays an essential role in cell physiology. Consequently, dysfunctions in receptor crosstalk are associated with many human diseases, such as infectious diseases (including COVID-19), cancer, and cardiovascular diseases. To understand the physical mechanisms by which signals from different receptors are integrated in crosstalk, studies have exclusively focused on receptor interactions at the plasma membrane. In contrast, how the crosstalk signals are transduced with high fidelity from the plasma membrane to the nucleus is poorly understood and scarcely explored. The overall goal of this research is to establish the functional role of the endocytic network in transducing and regulating receptor crosstalk. Under the prior ESI-R35 support, our group has made pioneering discoveries in support of the central hypothesis that the endocytic network is where extracellular chemical and physical stimuli intertwine to regulate receptor crosstalk. Specifically, we reported a new model in which receptors can crosstalk by forming overlapping interfaces between discrete signaling clusters, challenging the prevailing view that receptors oligomerize to crosstalk. Importantly, such spatially organized interaction between receptors at endosomes and plasma membranes is modulated by extracellular physical cues, and directly regulates cell inflammatory responses. These prior discoveries and the plethora of new approaches we developed for studying endosome functions laid a critical and unique foundation for us to address the knowledge gap: how does the endocytic network mediate receptor crosstalk? We will address how the endocytic network orchestrates chemical cues from receptor crosstalk (Direction 1), and transduces extracellular physical cues to refine the specificity of crosstalk signaling (Direction 2). To address the first direction, we will define the physical mechanisms by which endocytic sorting, collective endosome-organelle interactions, and endosome-specific activation modulate crosstalk signaling originated from the plasma membrane. To address the second direction, we will integrate experiments with computational modeling to determine the feedback loop between the endocytic network and cell-matrix interactions that regulate receptor crosstalk. This project will establish a mechanistic and predictive understanding of how the endocytic network mediates the spatiotemporal specificity of receptor crosstalk and cell signaling in general; a topic that is poorly understood. It will also lower the technical barrier that has impeded research on this topic, by establishing novel quantitative toolsets for dissecting the dynamics and ...