Khuloud Jaqaman, PhD - US grants

Project Summary/Abstract The overall goal of this project is to understand the cell biophysical mechanisms regulating transmembrane signal transduction, i.e. signal transfer from ligand to receptor to downstream effector. There is mounting evidence that cell surface receptors exhibit a high degree of dynamic organization, yet little is known about the mechanisms underlying this organization and its consequences for receptor signaling. This project will focus on vascular endothelial growth factor receptor 2 (VEGFR2), a key receptor in endothelial cells promoting angiogenesis, the process of sprouting new blood vessels from the existing vasculature. Angiogenesis is critical for growth and development, and goes astray in many diseases, from cancer to ischemia. In addition to the pathophysiological importance of angiogenesis, there are many commonalities between VEGFR2 signaling and other signaling pathways. Thus the knowledge gained from the proposed studies is expected to be applicable beyond the specifics of angiogenesis. This project will focus on the following major questions: (1) What mechanisms regulate the cell surface spatiotemporal organization and signaling of VEGFR2? The goal here is to determine the membrane and cytosolic factors that regulate VEGFR2 spatiotemporal organization on the cell surface (i.e. its dynamics, oligomerization state and spatial distribution), and to test the hypothesis that these factors provide a mechanism for the cell to modulate VEGFR2?s response to VEGF. (2) How do inter-receptor interactions regulate VEGFR2 signaling in response to its ligand VEGF? The goal here is to quantitatively characterize VEGFR2 interactions with other receptors, starting with the antagonistic anti-angiogenic receptor CD36, and to test the hypothesis that these interactions contribute to the integration of pro- and anti-angiogenic signals. (3) What are the spatiotemporal characteristics of signal transfer from VEGFR2 to downstream effectors? The goal here is to determine the nanoscale spatial relationship and kinetics of signal transfer from VEGFR2 to its downstream effectors, starting with phosphoinositide-3-kinase (PI3K). This will allow us to quantitatively link the spatiotemporal organization of VEGFR2 to its functional consequences. These questions will be addressed by developing integrative approaches combining cellular light microscopy (single-molecule, super-resolution and activity biosensor imaging) with novel analytical tools (computational image analysis, statistical data analysis and mathematical modeling). These analytical tools are necessary to extract quantitative, complete information from each imaging modality and to rigorously multiplex the complementary information that the different modalities reveal. Together they will enable the monitoring of VEGFR2 spatiotemporal organization and interactions down to the single-molecule level, and quantitatively link it to VEGFR2 signal initiation in its native cellular context. These integrative approaches and tools will be also broadly applicable, contributing to the biomedical research community beyond this specific project.

The goal of this project is to deepen our understanding of how cells regulate the process of sensing and responding to their environment. This will be achieved by investigating the interplay between cell surface proteins, called receptors, which allow cells to sense and respond to their environment, and the actin cortex, a component of the cell skeleton that is subjacent to the cell surface and that is a major regulator of cell surface receptors. The research will take one particular receptor, called CD36, as a model system, as it is likely representative of many cell surface receptors that are influenced by the actin cortex without directly binding to it. Using novel integrative microscopy and computational analysis tools, the investigator’s research team will (i) quantitatively characterize the influence of the actin cortex on nearby cell surface receptor behavior, (ii) shed light on the molecular mechanisms underlying this influence, and (iii) investigate how this influence may render receptor function sensitive to the stiffness of the cell’s environment, because of the sensitivity of the actin cortex to environmental stiffness. Through participation in the described research, as well as through a companion entry-level summer course on computational image analysis for undergraduate students, this project will train graduate, undergraduate and high school students in cutting-edge quantitative microscopy, an increasingly critical technique for modern biological research.

CD36 is an integral membrane protein that is expressed on the surface of many cell types and that binds diverse ligands. As for many receptors, CD36 signaling requires clustering, for which the actin cytoskeleton plays an important role. However, there still is a lack of mechanistic and quantitative understanding of how the actin cytoskeleton, and in particular the actin cortex, regulates the organization and signaling of cell surface receptors. To fill this gap, this project will pursue three specific aims: (i) Determine the influence of the actin cortex on CD36 signaling through a quantitative understanding of its influence on cell surface CD36 organization. (ii) Test the hypothesis that integrins and tetraspanins form the molecular link between cell surface CD36 and the actin cortex. (iii) Test the hypothesis that the actin cortex mediates crosstalk between microenvironmental stiffness and CD36 signaling. For this, the investigator’s research team will employ a novel approach combining simultaneous high spatiotemporal resolution imaging of cell surface receptors and cortical actin in live cells with statistical analysis of the relationships between them, as well as mutagenesis of CD36 and exposure of cells to microenvironments of different stiffness. Altogether, these studies will help reveal general principles of the mechanisms and signaling consequences of actin-mediated cell surface receptor organization.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

My lab seeks to understand the cell biophysical mechanisms regulating transmembrane signal transduction, i.e. signal transfer from ligands to receptors to downstream effectors. There is mounting evidence that cell surface receptors and associated proteins exhibit a high degree of dynamic organization at the plasma membrane (PM), which is critical for their ligand binding and signaling. However, many fundamental questions remain unanswered. Toward filling this gap in our knowledge, my lab will pursue two research directions over the next five years. The first direction will investigate how focal adhesions, the actin cortex, and interactions with integrins regulate the spatiotemporal organization and signaling of the endothelial cell receptor VEGFR2. VEGFR2 is the main promoter of angiogenesis (the formation of new blood vessels from existing vessels) in both normal physiology and disease. Thus there is great interest and need to understand the mechanisms that regulate its signaling. These studies will reveal mechanisms that underlie the activation of multiple pathways downstream of VEFR2, and that underlie the integration of multiple external signals at the level of the PM. As VEGFR2 belongs to the large family of receptor tyrosine kinases, our studies are expected to reveal general principles of the regulation and signaling of this important family of receptors. The second direction will focus on the novel organizational principle of liquid-liquid phase separation (LLPS) for proteins at the PM, using the transmembrane protein LAT as a model system. LAT is critical for the activation and immune function of T cells upon encountering an antigen presenting cell. Recent in vitro reconstitution work suggests that signaling clusters composed of LAT and its downstream effectors are formed through LLPS. The cellular environment is however much more complex than an in vitro reconstituted system. Thus we will investigate to what extent LAT microclusters at the PM of T cells are formed through LLPS, and the mechanisms that regulate LAT cluster composition. These studies will shed light on the role of LLPS for protein organization within the cellular environment in general. Both research directions ? by their very nature ? require probing molecular activities with high specificity and resolution in their native cellular environment. To achieve this, we will develop integrative approaches based on live-cell single-molecule, super-resolution and activity biosensor imaging, combined with cutting-edge mathematical and statistical analysis tools to extract quantitative information from the experiments and to multiplex the complementary information that the different imaging modalities provide. These analytical tools will be critical for our studies because single-cell and single-molecule microscopy often reveal molecular and cellular heterogeneity that is difficult to digest without such tools. We expect our novel analytical tools to be broadly applicable to other systems studied via similar experimental approaches. The knowledge gained from our studies will provide the building blocks for deepening our understanding of cell signaling from the single-molecule level to the systems level.