Investigating How Active Fluctuations Drive Immune Receptor Dynamics and Signaling

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Forces are important in driving many cellular functions from cell motion, cell division and cell growth to the proper functioning of the immune response. Cell generated forces have been implicated in playing a role both at the molecular scale for changing the conformation of key signaling molecules to initiate signal transduction pathways to the cellular and tissue scale for large scale morphological deformations and collective motion. In particular, it is becoming increasingly clear that forces in cells are not static, rather they show strong fluctuations driven by the collective dynamics of actin and microtubules cytoskeleton and molecular motors which are highly non-equilibrium in nature. Most experimental and theoretical work has focused on demonstrating the existence of active fluctuations in living systems and characterizing the physical implications. However, little is known about how active fluctuations regulate cell function in a physiological context. In this project the PI will determine the role of active cytoskeletal and force fluctuations in the context of the immune response for B cell receptor and T cell receptor mediated signaling. The PI's results will have broad implications in the field of force mediated molecular transduction processes in biology such as during cell adhesion, migration and development. The interdisciplinary nature of the proposed work will enhance our understanding of the fundamental physics underlying the function of living systems and have significant impact both in the fields of active matter and cell biology. The PI will work to integrate key aspects of the research with teaching and outreach activities. The PI will offer an undergraduate course on critical thinking and research practice using hands on research modules on relevant themes such as cellular force generation. The PI will introduce concepts from soft matter and cell biology into a course for education majors, to instill in them an appreciation for physics and its connection to biology, which they can take to elementary school classrooms: a potentially powerful way to inspire children to STEM careers. The PI will give talks focused on the physical aspects of cells and soft matter in local elementary schools as part of Science Day programs. The PI will actively recruit a diverse group of undergraduate students and participate in programs for attracting high school students to biological physics research as part of the established Summer Girls program and a newly proposed Biophysics Day on campus. The PI also proposes to enhance outreach by working on the intersection of Science and Art with an exhibition of science-inspired photographs by scientists and paintings by artists to disseminate these ideas to the general public.This project is a first step in elucidating the biological relevance of non-equilibrium fluctuations for functional aspects of living systems. In this project the PI will use state of the art imaging and computational analyses to explore the role of force fluctuations in the immune response using two examples. In Objective 1, the PI will examine receptor clustering in B cells, which is an essential first step in antigen gathering. A current model posits that the actin cytoskeleton acts as diffusive barriers to BCR movement. Antigen binding and signaling leads to dissolution of these barriers and diffusion-limited growth of the clusters. However, the PI's preliminary studies suggest that actin dynamics may have an active role in this process. Theoretical models of active membrane-actin interactions have been shown to be able to form non-equilibrium structures that can support the formation of macromolecular assemblies of receptors. The PI will use single molecule imaging, analysis and modeling to test whether BCR clustering requires active stirring by actin dynamics. In Objective 2, the PI will study how force fluctuations originating from cytoskeletal and motor dynamics inside cells are transmitted to the extracellular environment and drive signal transduction. Experimental studies and theoretical work have shown that the non-equilibrium cytoskeletal fluctuations are clearly athermal in character with a Lorentzian power spectrum. On the other hand, most studies of TCR signaling consider the dynamics of TCR-antigen bonds under static loads. The PI hypothesizes that force fluctuations arising from stochastic motor activity and actin dynamics facilitate antigen discrimination and amplify signaling and will use traction force microscopy, optogenetics and biophysical studies to map the spatial structure of force fluctuations and correlate it with the kinetics of signaling, enabling her to elucidate the role of these non- equilibrium fluctuations for immune cell activation. More generally the work will have implications in understanding the fundamental principles underlying how active living systems function.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.