Benjamin Machta, Ph.D - US grants

Theoretical physics is the search for concise mathematical models of Nature. It has had great success in dealing with the inanimate world: we can now predict in quantitative detail what the most sensitive experiments will observe inside the nucleus and in the cosmos at large. By contrast, even as our ability to observe and measure improves dramatically, the phenomena of life remain largely unpredictable, even in their most qualitative aspects. In this project, a group of theoretical physicists will engage with students and postdoctoral scholars in an effort to close this gap; in short, to construct a theoretical physics of biological systems. The proponents have explored phenomena that span the tree of life: from metabolism in bacteria, through the determination of cell fate in embryonic development, to coding and computation of sensory information in brain. They have identified broad theoretical problems which cut across the traditional biological divisions of organism and system: Do living organisms operate near the limits set by the laws of physics as they gather and process information? Can we learn the detailed microscopic "model" of an organism, its "wiring diagram", from the finite set of observations we can make on how it behaves? How do organisms set the parameters that govern their function (i.e. how do they learn from experience)? These questions can all be given a mathematical form which guides a search for answers in terms of general principles, in the tradition of physics that will apply across disparate biological domains. The time is right to bring the beautiful phenomena of life under the powerful predictive umbrella of theoretical physics. Just as cosmology has progressed, in roughly one generation, from wild speculation to a precise framework for analyzing a rapidly expanding set of observations, the proponents believe that the intimate interaction between theory and experiment can lead to a new and deeper physics of biological systems. It is the creation of this scientific culture, where theory and experiment are equal partners in the exploration of life that is the fundamental intellectual merit of the project. It is not just the boundaries of academic disciplines, but our view of ourselves, which is at stake. A very important aspect of this project will be the training of a new generation of physicists for whom the development of a theoretical understanding of biological systems is a central part of their discipline. The graduate students and postdoctoral scholars who pass through the group will learn by example how to pursue that goal in a way consistent with the intellectual rigor and traditions of physics. They will eventually move on to faculty positions of their own, where they will transmit this attitude to new generations of students. More broadly, all project personnel are deeply engaged with new educational initiatives, addressing levels from the first year of college to advanced PhD students, which provide a more complete guide to the evolving, multidisciplinary intellectual landscape.

The participants in the project will assemble into subgroups to attack instances of these problems. The individual projects will have unusual scope: as an example, the question whether we can capture the complex statistics of biological behavior in a learnable mathematical model can be asked in very similar terms both of spiking retinal neurons, and of the antibody sequence repertoire of individual zebrafish. If the answer is yes and the models have similar mathematical structure, one will have learned something novel and deep about what makes evolved, living, systems different from the inanimate world. Since these questions can only be answered in the light of accurate data, the work will involve a close partnership with many experimental groups in fields ranging from bacteriology to human perceptual psychology. The product of these interactions will be the design of novel experiments and the creation of novel data analysis methods in order to address clearly formulated mathematical questions of broad significance.

This project is being jointly supported by the Physics of Living Systems program in the Division of Physics, the Cellular Cluster and the Systems and Synthetic Biology in the Division of Molecular and Cellular Biosciences, and the Neural Systems Cluster in the Division of Integrative Organismal Systems.

Project Summary Information in the nervous system is relayed mostly at synapses, where neurotransmitter is released with great temporal precision from a presynaptic terminal on to a post-synaptic cell via the fusion of membrane bound synaptic vesicles (SVs) with the cell membrane, in a process called exocytosis. The components of these SVs are subsequently retrieved via endocytosis and recycled for reuse. This grant aims to understand the interplay between SV recycling and membrane tension gradients and associated membrane flows. In neurons and neuroendocrine cells, both exocytosis and endocytosis are influenced by osmotic swelling or shrinking, suggesting they are influenced by membrane tension, ?. Conversely, membrane addition to the presynaptic terminal via exocytosis is expected to lower ?, while endocytosis should restore it. In addition, membrane tension has been suggested to be one of the possible signals for coupling exocytosis to endocytosis. However, despite these key roles, there are no measurements of membrane tension in synaptic terminals and how tension changes are related to exo-endocytosis is not known, mainly due to technical difficulties. The best method to probe ? is to pull a thin membrane tether from the cell surface using optical tweezers, manipulating a 1-3 ?m diameter bead as a handle. The bead's displacement from the trap center provides the tether force, which reflects ?. However, most terminals are small and are tightly coupled to post-synaptic structures, making tether pulling impractical. We overcome this challenge using goldfish bipolar cells which possess giant terminals, in a setup that combines optical tweezers with electrophysiology (to control stimulation and/or measure capacitance changes) and with high-resolution fluorescence microscopy (to label and identify sub- cellular structures and calcium imaging). We aim 1) to characterize the tether force response to electrical and mechanical perturbations that occur at a presynaptic terminal during activity. After stimulation, membrane added at an exocytic site needs to flow (and the associated tension perturbation propagate) over the terminal surface, then through the tether to produce a change in the measured tether force. We will characterize membrane flows in double-tether experiments and calibrate the tether response to step- changes in tether length. We will confirm that ? changes we observed in preliminary experiments (a drop ~1 s after stimulation, followed by recovery in ~10 s) are due to exo-endocytosis, and characterize rapid voltage- induced tether force changes. These will enable a quantitative understanding of measured ? changes associated with stimulation. Next, we will 2) characterize how membrane tension is regulated at a presynaptic nerve terminal. Combining pharmacological interventions with live imaging and ? measurements, we will test the hypothesis that F-actin is a major regulator of ? at the nerve terminal. We will manipulate ? and calcium independently to dissect calcium and ? requirements for SV turnover. These measurements will help generate a model of feedback between membrane trafficking and ? at the nerve terminal.

Abstract The cellular environment is heterogeneous. Some intracellular domains are now thought to arise due to weak attractive interactions between lipids, proteins and nucleic acids, leading to emergent domains much larger than individual components. Such phase-like behavior has been implicated in a range of processes from synapse structure, to signal transduction and transcriptional regulation. While it is appealing to speculate that these domains play a role in integrating signals and other cellular information, we cannot presently formulate the precise contributions these domains make to function. My group seeks to understand the new physical and biological principles that determine function in the gap in scale between all-atom molecular dynamics and established coarse-grained systems biology approaches. We will seek to understand the thermodynamics and physical underpinnings of domains in the cellular environment. We will build minimal models for domain forming membranes interacting with cytoplasmic droplets, motivated by synapses, and the diverse signaling platforms which often involve partitioning and localization of both membrane bound and cytoplasmic factors in close proximity. To this end we will use and extend the established theory of wetting transitions, which has mostly been developed in the context of solid homogeneous surfaces, and we will connect our theoretical predictions to experiments being performed by collaborator Veatch?s laboratory, and to other results in the literature. We will also investigate the ramifications of the high dimensionality of the space of protein and lipid abundances for phase behavior in cells, seeking strategies cells could use to navigate this space, and making predictions that can be verified with lipidomics and proximity labeling techniques. We will also investigate how driving from active cellular components can alter domain forming behavior. We will also investigate how thermodynamically driven domain can shape biological function at the level of individual proteins and interaction networks, combining established thermodynamic simulation techniques with non-equilibrium kinetic networks. We will investigate how the presence of domains can alter the function of established interaction motifs as well as strategies that explicitly depend on the propensity of the cellular environment to phase separate. We will quantify how domains could aid in the amplification and distribution of small signals and investigate whether domain formation could act as an effective analogue to digital converter. We will apply these ideas to the regulation of ion channels by membrane domains, especially in synapses, comparing with experiments in progress by Veatch. These projects will be significant in that they will provide deep insight into the role that phase behavior plays in determining cellular heterogeneity and in shaping function. The proposed work will have broad impacts for many researchers seeking to navigate a rapidly expanding field.