Joshua W. Shaevitz - US grants

In this CAREER proposal the PI proposes to study a fundamental question in biology: How do nanometer-scale proteins give rise to micron-scale forces at the cellular level? Specifically, the PI will determine the mechanisms of chromosome segregation and cell division in bacteria. The proposed experiments will investigate intracellular organization and force generation in bacteria through (i) imaging, (ii) mechanical perturbation and (iii) force measurement. Super-resolution, three-dimensional imaging will be used to quantify the geometric conformations of cytoskeletal proteins inside cells and how they respond to externally applied forces. The three-dimensional trajectories of actively segregated regions of DNA will be revealed using single particle tracking. In addition, the role of local cell curvature in the placement of the cell division plane and the ability of the contractile ring to generate mechanical force during cell division will be probed with atomic force microscopy. The proposed education plan provides learning opportunities for future biophysical scientists through combined (i) course development, (ii) hands-on observations and (iii) a multimedia cellular environment. The PI will develop and teach two new courses on Biological Physics that integrate research results in a broader biophysical context through his joint appointment in the Department of Physics and the Lewis-Sigler Institute for Integrative Genomics at Princeton University. To motivate the next generation of scientists, the PI will work with local middle school students as part of Princeton Universitys Science and Engineering Expo, introducing them firsthand to the physics of cellular locomotion through microscopy and modeling activities. An interactive software program, partly based on imaging data from this proposal, will be created that allows students to explore the crowded and fascinating three-dimensional world that exists inside cells.

The long term goal of the proposal is to develop novel methods for studying single molecule dynamics in living systems and to understand how different length and time scales are integrated for a living bacteria or cell to emerge. This project encompasses aspects of cell biology, molecular biophysics, physics, and computational biology. The students working on the project will be trained at the interface of several fields of science. The proposed research has all ingredients to be truly transformational. This project is jointly supported by the Physics of Living Systems Program Molecular in the Division of Physics at the Physical Sciences Directorate and by the the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.

This collaborative research project, consisting of four institutions (Rice, Yale, UIUC and Princeton) aims to continue the Physics of Living Systems Student Research Network (PoLS SRN). This network has been in existence for four years and has had a dramatic impact on many graduate students, both in the US and abroad, working on the application of physical science techniques to living systems. These students now can participate in a global community that can help deal with the many complex issues involved in conducting research in such a new and inherently multidisciplinary field. These issues range from proper training, to gaining a broad perspective, to accessing technical expertise that may not be available at their home institution. In addition to the obvious broader impacts related to training of a research workforce, there are other broad impacts of this plan. Via the interaction of one of the PoLS nodes (Rice) with the biomedical community in Houston, students and faculty will be exposed to possible avenues whereby physics can contribute to human health issues. Funds to attract students from under-represented groups to network meetings will be available through the new funds administered by the newly proposed network coordinator. Also deas vetted by the PoLS SRN will be adapted to create student networks in other areas of science and engineering.

There is by now little disagreement with the general notion that concepts and methods from physics have been a critical contributor to the increased understanding of the living world, and that its importance will be growing as the scientific world moves toward an ever more quantitative and predictive form of biology. Thus, the physics community clearly needs to train a new generation of scientists who can lead this effort, scientists who have the right mix of physics/mathematics rigor and broad knowledge of living systems from molecular scales on up. The PoLS SRN aims at creating a community of graduate students who can collectively help themselves and their mentors accelerate and enhance this training process. This is being done by a mix of in-person and virtual modes of communication, and this proposal is a plan to continue and expand these efforts; it will reach more students, improve the social networking portals, and make use of the complementary research agendas of the different network nodes to provide broad technical expertise. Doing all of this, will boost the intellectual level of the entire research field and convince the best students that the Physics of Living Systems is truly the most exciting research frontier in 21st century science.

This project is being jointly supported by the Physics of Living Systems program in the Division of Physics, the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences, the Chemistry of Life Processes program in the Division of Chemistry, and the Cellular Dynamics and Function Program in the Division of Integrative Organismal Systems.

DESCRIPTION (provided by applicant): Progress toward understanding and curing many human neurological diseases is hindered by a lack of methods for objective and quantitative assessment of the connections between genes and behavior. Traditional studies of animal behavior probe only a small subset of user-defined behaviors in low- dimensional data and are inappropriate for the discovery of subtle phenotypes or complex relationships between genes and behavior. To move beyond these simple representations of animal behavior, we propose to build a high-resolution, three-dimensional imaging system that can track single behaving animals. This system will quantitatively connect behavior to genetics using a data-driven method for the discovery of stereotyped motions. Our specific aims are to 1) develop a new standard for behavioral observation and automatic phenotyping to quantify the behavior of freely moving fruit flies, 2) demonstrate the sensitivity and accuracy of our new technology by studying Drosophila models of neurodegenerative diseases and the behavior of closely-related species of Drosophila fruit flies and 3) expand our system to enable simultaneous tracking and analysis of two independent flies in a single arena and test this system with assays of courtship behavior between male and female flies. The ability to measure subtle changes in behavior in a model genetic organism will bring us closer than we have ever been to understanding neurological diseases and behavioral disorders at the genetic and molecular level.

Animals, from insects to humans, are inherently social, and brains have evolved to be most sensitive to sensory cues that carry social information (for example, speech sounds or pheromones). Very little is known regarding how animal brains process information in the context of social interactions. This proposal seeks to address this complex issue by focusing on the relatively simple nervous system of the fruit fly Drosophila, and takes advantage of the wealth of tools available in this system to dissect the mechanisms underlying social behaviors. The three principal investigators (Murthy, Shaevitz, and Bialek) are experts with behavioral analysis, theory/modeling, and neural circuit analysis, and will use several new methods to study courtship, a robust social behavior that has been shown to involve a complex interaction of a male and a female. This work will not only uncover the mechanism by which sensory inputs and internal states interact to generate behavior, but also benefit studies of disorders (e.g., autism spectrum) that impact the social brain. The research project is complemented by outreach efforts targeted at educating undergraduates, and in particular young women, in modern methods in computational neuroscience.

Animals, from insects to humans, spend a majority of their time engaged in social behaviors, and brains have evolved to be most sensitive to these dynamics and timescales, as they are important for survival. Social interactions involve both sensory perception (detecting cues generated by another individual) and coordinating motor outputs (to generate social behaviors). Most studies examine sensory and motor pathways in an "open loop" framework; however, social interactions are inherently "closed loop", as data gathered through the senses of each individual is profoundly shaped by his/her own actions and those of the other individual. With new methods and new theoretical frameworks, this proposal aims to solve the closed loop aspect of sensory perception between animals using the fruit fly Drosophila melanogaster as a model system. The investigators have pioneered several new methods to facilitate these studies and are experts with behavioral analysis, theory/modeling, and neural circuit analysis. They will combine unbiased behavioral quantification, whole-brain imaging in behaving animals, controlled sensory stimuli, and theoretical modeling to uncover the neural circuit dynamics underlying social behaviors and decision-making. A detailed analysis of the simultaneous behaviors of two courting flies will lead to the first rigorous and quantitative analysis of the dynamic sensory cues and interactions between individuals that shape social behaviors. Theoretical work on these data will reveal the dynamic neural computations that must be active during courtship. Finally, neural circuit recordings in animals engaged in closed loop fictive social interactions will be used to link brain activity to specific courtship behaviors and decisions.

The ability of groups of individuals to form complex and dynamic spatial patterns is a key aspect of biological phenomena ranging from collective behavior to multi-cellularity to development. In a cellular context, this often involves complicated chemical signaling and chemotaxis strategies. However, the PI has recently discovered that some bacterial species have evolved to take advantage of an active-matter phase separation that generates patterns without the need for chemical signaling. This project strives to understand this process from a physicist's perspective, but is hindered by a lack of tools to physically probe the mechanical properties and interactions of groups of motile bacteria. The PI's work with the single-celled bacterium Myxococcus xanthus focuses on the molecular details of force generation and the interactions of cells at the start of collective starvation-induced fruiting body formation. In this project, the PI seeks to explain the process of fruiting body development as an active dewetting process, linking new theoretical models with cutting-edge experimental data. The PI's research goals are complemented by an outreach plan that aims to involve more undergraduate students in biological physics and to engage non-scientists through public lectures. The PI's goals over the next few years include (i) expanding the Integrated Science program for first year undergraduates, (ii) starting a summer school aimed at advanced undergraduates, and (iii) putting on a series of public lectures in New York City meant to convey the excitement and innovation of biophysics using examples relevant to everyday life.

The three aims below seek to determine the role of motility and adhesion in driving starvation-based dewetting. The PI's current models of Myxococcus xanthus aggregation rely on particle jamming in 2D. While these incredibly simple models capture some of the observed phenomena, the actual dynamics occur in 3D as the population dewets off the surface without jamming to form round droplets. This more complicated reality requires more sophisticated experimental data. The laboratory combines expertise in Myxococcus xanthus motility, cutting-edge imaging techniques, force microscopy, and computer vision analyses, making the group uniquely qualified to carry out the proposed research. Aim 1: To understand the forces that cells generate on each other and on a substrate, the PI will measure cell-cell and cell-substrate mechanical interactions using a custom-built optical trapping microscope and mutant strains that lack specific motor proteins and adhesion molecules. Aim 2: To probe the motility of cells inside a fruiting body, the group will track cells in 3D using confocal microscopy to (i) compare the motion over time and between different locations in the aggregate, and (ii) investigate the formation of layered structures and flows within the fruiting body. Aim 3: To probe the macroscopic mechanics involved in fruiting body formation, the group will (i) measure the development of droplet shape and rheology using confocal imaging and atomic force microscopy, and (ii) probe the forces generated on the substrate using traction force microscopy. At each step, data from the three aims will be used to test, and be tested by, the active-dewetting theories being developed by the group's collaborators. This project lies firmly within the goals of the Physics of Living Systems program at the NSF by using physical measurements and analyses to understand the dynamics of living cells across spatial scales.

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.

? DESCRIPTION (provided by applicant) Pseudomonas aeruginosa (PA) is a Gram-negative bacterial pathogen that infects a wide variety of host animals including humans. PA is a significant public health concern as a leading cause of hospital-acquired infections, burn-wound infections, and cystic fibrosis pathology. PA exhibits high tolerance for many commonly- used antibiotics, raising a clear need for a better understanding of PA virulence regulation. Historically, the field has focused on PA's response to chemical cues such as nutrients or signaling molecules. However, we have recently demonstrated that PA also regulates virulence in response to mechanical cues in its environment and that the ability to respond to mechanical forces may underlie PA's ability to target such a broad range of hosts. Here we propose to develop new technology to answer the key outstanding question: what is the physical mechanism by which PA senses mechanical force? We recently discovered that the retraction of type IV pili induce the expression of virulence factors through the Chp chemotaxis-like system. However, there is nothing known about how mechanical forces from retraction are transmitted to the Chp system. Therefore, the studies proposed here are intended to fill this void in our understanding of PA surface sensing. First, we will construct a combined optical-trapping, fluorescence microscope capable of applying controlled forces to single PA pili in live cells over long periods of time and monitoring the expression of key virulence factors. Second, we will use this system to define the effect of force on virulence factor expression in a variety of force ranges. Last, we will investigate whether motor driven retraction or tension within the pilus provides the signal that is read-out by the Chp system. The information gained by these studies will substantially further our understanding of PA as an opportunistic pathogen and how it senses solid surfaces as a way of identifying hosts. This knowledge will pioneer new interventions for treating this important pathogen.

Project  Summary    Flexible behavior is central to virtually all cognitive and social abilities. Recent technical advances have opened an  unprecedented opportunity to comprehensively dissect the neural circuit mechanisms of this ability across multiple  brain areas in freely behaving animals. This proposal focuses on the cerebellum, a structure that is a major site of  pathology in autism spectrum disorder. Damage to the cerebellum at birth leads to a 36­fold increase in the risk of  autism,  and  this  region  is  also  a  principal  site  for  co­expression  of  autism  risk  genes.  Thus  cerebellar  development  may  act  as  an  intermediate  mechanistic  step  in  transducing  inherited  autism  risk  into  neurodevelopmental  phenotypes.  In  this  project,  a  multidisciplinary  team  of  leading  experts  proposes  to  investigate  the  neural  basis  of  this  disorder  using  advanced  technologies,  including  unbiased  automatic  classification  of  behavior,  large­scale  cellular­resolution imaging in behaving rodents, mouse genetic models for  autism, and manipulation of neural activity in specific cerebellar areas and cell types. In genetic mouse models of  autism,  the  researchers  will  identify  modes  of  behavior  based  on  physical  poses,  and  relate  these  modes  to  classical  behavioral  tests,  such  as  eyeblink  conditioning,  and  to cerebellar circuit dysfunction. In adult wild­type  and autism model mice, the researchers will use optogenetic methods to perturb specific cerebellar lobules while  quantifying  the  effects  on  behavioral  dynamics  and  learning.  In  juvenile  model  mice,  the  researchers  will  use  chemogenetic methods to identify long­lasting patterns of behavioral disruption and relate these patterns across  behaviors to build a quantitative map of these perturbations. In addition, they will use in vivo dendritic imaging to  evaluate the influences of cerebellar perturbation on neocortical neuron structure. All of these results will inform  modeling  of  cerebellar­neocortical  interactions  to  better  understand how these differently wired regions interact  during learning and development. The long­term goal of this project is to arrive at a chain of explanation, centered  on  principles  of  convergent  neuroscience,  to  understand  causal mechanisms of neurodevelopmental disorders.  This  project  will  join  genetics  with  circuit  function,  local  cerebellar  anatomy  with  behavioral  outcomes,  and  classical  behavioral  tests  with  modern  unbiased  methods.  This project is expected to produce an accurate and  detailed  understanding  of  cerebellar  contributions  to  normal  and  aberrant  neurodevelopment.  In  addition,  the  proposed research will enable researchers to generate and test a variety of hypotheses about the neural basis of  flexible  behavior.  Taken  together,  these  achievements  will  represent  a  crucial  step  toward  a  mechanistic  understanding  of  how the brain develops its complex ability to respond flexibly to the environment, from birth to  adulthood.   

A physical and quantitative understanding of the processes of life and living systems must describe biological functions in different contexts and across a wide range of systems. Through this Physics Frontiers Centers award the Center for the Physics of Biological Function (CPBF) will probe the unifying physical principles that govern the emergence of essential biological functions. A joint effort of Princeton University and the Graduate Center of the City University of New York, the CPBF will bring together theorists and experimentalists to study biological functions in a quantitative and systematic manner across a range of diverse systems. From cells to organisms to groups of organisms the CPBF will explore functional behavior, the mechanics of biological networks, the impacts of limited physical information within systems, and how biological systems set efficient parameters. The CPBF will foster the training and development of a new and diverse generation of scientists at the interface of physics and biology. It will expand successful existing interdisciplinary programs for undergraduates and graduate courses, and develop a dedicated undergraduate summer school. In addition, the center will host seminars and symposia that will connect CPBF with the larger scientific community and will engage in public lectures in New York City.

The CPBF research activities are organized around four themes, each of which will involve close collaboration between theory and experiment. The first focuses on animal behavior from the development of organisms to locomotion. The second considers the emergence of collective phenomena in groups of molecules, genes, neurons, and organisms. The third theme studies the role of physical limits on information transfer and processing in the genetic code, neural circuits, cellular sensors, and genetic and biochemical networks. The fourth theme examines the mechanisms through which biological systems arrive at a particular operating point from protein number to adaptive immunity.

This Physics Frontiers Centers award is co-funded by the Division of Physics and the Division of Chemistry within the Directorate of Mathematical and Physical Sciences, and by the Division of Molecular and Cellular Biosciences and the Division of Emerging Frontiers within the Directorate for Biological Sciences as part of Understanding the Brain activities.

Project Summary/Abstract Social interactions across the animal kingdom, from courtship rituals and aggressive interactions to spoken conversation, are wondrously complex - they necessarily involve back- and-forth feedback between nervous systems transmitted through multiple sensory modalities and each animal's behavior. Typical experiments in this field observe only a tiny fraction of the activity in any neuronal circuit, and then only under a very limited range of behavioral conditions. To overcome this limitation, the proposed research leverages the compact nervous system of Drosophila melanogaster, combined with its wealth of genetic tools, to study the dynamic behavioral interactions and detailed neural mechanisms that underlie courtship between males and females. The project combines unbiased measurement of behavior, neural circuit manipulations, neural recordings in behaving animals, and sophisticated computational models. The specific aims include: i) elucidating the computations that the brain performs during courtship by mapping the sensorimotor transformations underlying male and female interactions over time via quantitative behavioral assays and the generation of predictive models; ii) combining models with neural perturbations to map the underlying circuits that govern the link between sensory inputs and behaviors; And, iii) testing the models of neural control during courtship by monitoring neural activity in behaving animals experiencing fictive courtship stimuli in a virtual-reality apparatus. This work will substantially advance our understanding of how two interacting brains process and transfer information, and will uncover general principles of neural circuit function that will inform studies of sensorimotor integration in more complex animals, such as rodents and humans. The project will also produce new experimental and theoretical tools for studying social behaviors. Finally, it will shed light on the mechanisms that go awry in several disorders, including Autism Spectrum Disorder (ASD), in which sensory perception becomes disentangled from motor outputs ? these disorders have profound effects on cognitive well-being and a major impact on public health.

The lungs are a key avenue of attack for the SARS-CoV-2 virus. Respiratory problems are primary symptoms of COVID-19, and early indication is that it does not behave like previous examples of Acute Respiratory Distress Syndrome (ARDS). A severe urgency exists to understand how to provide optimal care for patients requiring artificial ventilation, to minimize both mortality and adverse long-term effects on those patients who survive. The project will illuminate lung function under the stress of COVID-19 and provide open tools to engage the larger community to help understand this very urgent societal problem. The project output will include instrumentation advances, software and data, as well as models of lung function under the stress of COVID-19. The project will also inform the medical community as to how to treat COVID-19 patients, because COVID-19 differs notably from prior experience with ARDS.

Respiration and lung function is fundamentally a dynamical physical system amenable to traditional pressure/volume/flow relationships, with a quantity called "lung compliance." COVID-19 is unique, in that the underlying biology can lead to changes in the parameters of this dynamical system that are surprisingly fast, and different from previous ARDS cases, on the time scale of hours or days. Medical personnel need to navigate the evolving nature of the consequences of the viral infection as well as mechanical ventilation induced lung inflammation and potential injury, with outcomes ranging from recovery with varying impacts on post-illness lung function to death. This project consists of three related activities: (1) Instrumentation: Continued development of a low-cost, open-source ventilator monitor, including additional options for readily sourceable parts, and related documentation on calibrations. (2) Data: Development, with the broader community, of open datasets of breathing and ventilator data, including flow, pressure, O2 levels, and derived quantities of interest to enable innovation and machine learning in a space that otherwise lacks open data. (3) Models: Development of open simulations, visualizations, and models for mechanical ventilation and the breathing process, under stresses like COVID-19, that enable a physicist's understanding of the system, enable innovation, and can potentially aid the medical community.

This grant is being awarded using funds made available by the Coronavirus Aid, Relief, and Economic Security (CARES) Act supplement allocated to MPS.

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.

The ability of groups of individual organisms to form complex and dynamic spatial patterns is a key aspect of biological phenomena ranging from bird flocking and fish schooling to multicellularity and embryonic development. In bacterial populations, this often involves complicated signaling mechanisms between cells. However, some species have evolved to take advantage of a special kind of physics called active matter to perform specific biological functions without the need for chemical signaling between cells. In the social bacterium Myxococcus xanthus, the Shaevitz group have shown that cells can form multicellular groups that resemble bird flocks, ant trails, and liquid droplets. These cells can change the way they move in response to environmental cues to change the behavior of the population. This project lays out a series of experiments and theoretical analyses to understand these processes from a physicist’s perspective, using tools to measure the spatial orientations, flows, and forces in these populations. Results from this work will answer fundamental questions about the mechanism and function of M. xanthus collective behavior and inform our understanding of how groups of organisms act collectively. In the future, this may lead to new methods for developing micro- and nanotechnologies that self-assemble using the principles of active physics.<br/><br/>This project focuses on three questions: how three-dimensional droplets form during starvation, how a surface-wave instability aids predatory behavior, and how these behaviors manifest in naturalistic soil-like environments. To understand how 3D fruiting bodies emerge from 2D wetted layers, the investigators will track nematic order, cell motion, and mechanical stress throughout the population during development. To understand the instability that governs rippling behavior during predation, the investigators will measure the height of the population and track cell motion while perturbing motility, interactions between cells, and the environment. To probe M. xanthus phase transitions on more realistic substrates, the investigators will measure motility, fruiting body formation, and rippling on curved substrates and artificial colloidal models of hydrated soils.<br/><br/>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.