Ned S. Wingreen - US grants

DESCRIPTION (provided by applicant): The long-term aim of this research is to explore the role played by Min-protein oscillations in bacterial cell division. The specific goal of this proposal is to develop and test a quantitative model for oscillations of the Min proteins in both rod-shaped cells such as Escherichia coli and round cells (cocci) such as Neisseria gonorrhoeae. The coupling of modeling and experiment for the Min proteins will help answer 2 fundamental questions: (1) How do Min-protein oscillations contribute to the reliability and extreme accuracy of cell division in E. coli and related bacteria? (2) Are Min-protein oscillations able to select-the longest axis of the cell in nearly round cocci in order to define the division plane? To address the first question theoretically, we will develop a particle-level simulation of Min-protein oscillations in E. coli. The simulation will follow the diffusion and interactions of thousands of individual MinD and MinE proteins (the particles) in a three-dimensional (3D) cell geometry. The simulation will build on existing models that consider protein densities rather than individual protein molecules. The particle-level simulation will capture several experimentally observed features of the Min system for the first time, including the helical assemblage of membrane-associated MinD polymers and the significant stochastic fluctuations of the oscillation pattern. To complement and extend our modeling results, we will collaborate to experimentally define the role of the Min system is cell-division accuracy by correlating oscillation period, the accuracy of medial divisions, and the onset of polar divisions (mini celling). To address the second question, we will extend the Min-protein simulations to cocci. To determine whether Min-protein oscillations are able to select the longest axis of nearly round cells, particle-level simulation will be applied to cells with a range of sizes and shapes. The results of these simulations, together with imaging experiments of Min oscillations in N. gonorrhoeae, will define a possible role for the Min system in determining the division plane in cocci. Answers to the above questions will contribute to an understanding of how cells recognize their own shape - an issue of importance for cell division, cell motility, and the creation of multicellular structures in both prokaryotes and eukaryotes.

DESCRIPTION (provided by applicant): In the bacterial environment, temperature variation and temperature gradients are as ubiquitous as chemical variation and gradients. For bacteria, locating the optimal combination of temperature and nutrients is crucial for maximizing growth. Indeed, bacteria perform chemotaxis over a large range of temperatures and perform directed motion in temperature gradients, i.e. perform thermotaxis. In Escherichia coli, chemotaxis and thermotaxis both exploit the same well-characterized signaling network. The specific aim of this research is to develop a predictive, quantitative understanding of the thermal properties of this network. Combining experiment with modeling will help answer several fundamental questions: What network features allow robust chemotaxis over a wide range of temperatures? How does the signaling network allow both chemotaxis and thermotaxis? How do multiple sensory stimuli such as attractants/repellants and temperature changes interact? Our preliminary results indicate that the individual steps of the chemotaxis pathway are temperature dependent, but that at the systems level, these dependencies compensate for one another. To quantify these observations, we will obtain temperature-dependent data on the individual steps of the E. coli chemotactic signaling pathway using single-tethered-cell measurements and multi-cell FRET studies. The existing theory for chemotaxis that we have helped develop will guide efficient data acquisition, and the same theory will be used as the basis for modeling. To determine the molecular mechanism(s) underlying E. coli thermotaxis, and to extend a preliminary thermotaxis model we have developed, the thermotactic response of E. coli will be systematically measured via tethered-cell and FRET studies. All experimental studies will exploit our large pre-existing collection of engineered mutant strains of E. coli. Our results are likely to have significance for many cellular signaling networks. Because temperature potentially affects all components of all signaling pathways, our results may reveal universal mechanisms used by cells to ensure faithful signaling over a range of temperatures. Also, since the chemotaxis network of bacteria has been implicated in infectivity, and is widespread and well conserved among bacteria but is not shared by humans, the pathway presents a potential target for the development of future antibiotics. PUBLIC HEALTH RELEVANCE: The aim of this research is to develop a deeper understanding of the network of proteins that allows bacteria to perform chemotaxis and thermotaxis, that is to sense and swim towards food or a preferred temperature, respectively. We propose to develop such an understanding through closely coupled experimental approaches and computational modeling. From a human health perspective, chemotaxis by bacterial pathogens has been implicated in infectivity and maintenance of disease, and thermotaxis may be implicated as well. Since the chemotaxis/thermotaxis network is widespread and well conserved among bacteria, but is not shared by humans, the pathway presents a potential target for the development of future antibiotics. In addition, as all signaling pathways within living cells must function in the context of fluctuating thermal environments, our efforts may reveal universal mechanisms by which cells ensure faithful signaling.

In this project the PIs will explore 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 the 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 the detailed microscopic model of an organism, its wiring diagram be understood from the finite set of observations that can be made on how it behaves? How do organisms set the parameters that govern their function (i.e. how do they learn from experience)? These questions will be given a mathematical form, which will guide a search for answers in terms of general principles, in the tradition of physics, that will apply across disparate biological domains. 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 the complex statistics of biological behavior can be captured in a learnable mathematical model will be asked in very similar terms both of spiking retinal neurons, and of the antibody sequence repertoire of individual zebrafish. These questions will be answered in the light of accurate data and 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. An important component of this project is 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.

Biofilms are complex living systems that are widespread in nature and technology. For example, biofilms play critical roles in water purification in streams and lakes, environmental quality during mineral processing (e.g. mine drainage), quality control of manufacturing processes, etc. Thus, understanding the structural and developmental features of biofilms has broad implications. In the simplest characterization, biofilms are aggregates of bacteria and polymer (the extracellular matrix), and they are commonly found directly attached to surfaces. Hence, biofilms are generally regarded as flat mats of cells. Nevertheless, filamentous biofilms, or bacterial streamers, that are attached to surfaces but with thread-like structures extending into a flow, have been observed in some systems. Little is known about the detailed structure of these bacterial streamers, or how they form and mature. In most bacterial species studied, proper biofilm formation relies on quorum sensing, a process of bacterial cell-cell communication that depends on the production, release, and population-wide detection of signal molecules called autoinducers. Since quorum sensing involves diffusible molecules, a flow should significantly impact various biochemical and biophysical processes through its impact on diffusion and convection processes. Moreover, one of the important questions relevant to quorum sensing and evolutionary stability is how quorum sensing and extracellular polymer production survive the emergence of "cheater" cells that sense but do not produce a signal. Therefore, this project will address the biophysical, mechanical and physicochemical features of biofilm streamers, the way these characteristics interact with a flow, and how quorum sensing, which is critical to bacterial behavior and development, interacts with the flow to form and organize the streamers. The project team combines expertise in engineering, molecular biology, and physics and the research will inform both the physics and microbiology of biofilm streamers, and the coupling of the two subjects.

Broader impacts:, In addition to publishing their findings in journals that span biology, engineering, and physics, the project team will continue their multi-faceted outreach activities, including hosting visitors from different disciplines and educational institutions, engaging undergraduate students in research and giving talks at conferences, leading professional development activities for undergraduate, graduate, and postdoctoral colleagues, and participating in mentoring programs for young researchers from under-represented groups. Also, members of the team have successfully incorporated research themes into "holiday" lectures for children and parents that they have given over the past 9 years and which they will jointly continue to develop and present. This research will have a direct impact on our understanding of bacterial communities in flow, which is of widespread interest in food processing, water treatment, agricultural management, and other settings where biomass is present in flowing systems.

DESCRIPTION (provided by applicant): To survive, organisms must continually adapt to changing conditions. While some of these responses represent relatively simple mechanisms of homeostasis, others are more complex, reflecting associative learning and even predictive ability. Such sophisticated responses are not solely the domain of multicellular organisms - bacteria have also evolved refined strategies to deal with their complex, changing environments, e.g. circadian rhythms and temperature/oxygen association. While such examples primarily reflect metabolic adaptation, there is recent evidence that bacterial sensory systems are also reshaped by the cell's growth environment. In particular, the chemotaxis network of Escherichia coli undergoes ~10-fold changes in protein levels and ratios in response to nutrient abundance, temperature, and cell density. The large number of assays available for this system and the existence of well-tested quantitative models of its operation make it an ideal target to explore the principles of history-dependent sensing. To carry out this exploration, we will grow E. coli cells under a wide range of physiologically relevant conditions, including nutrient type and abundance, temperature, pH, O2 levels, osmolarity, cell density, and the presence of multiple chemical signals. We will then characterize the chemotactic network at three levels: protein abundances, signaling response to stimulation (via fluorescence resonance energy transfer), and chemotactic behavior (via tracking of single cells swimming in microfluidic gradients). We will exploit the well-established model for chemotactic signaling to interpret our experimental results, and to develop a working model for how growth conditions reshape the chemosensory apparatus. The molecular mechanisms underlying history-dependent regulation, both known and newly discovered, will be characterized by assaying mRNA and protein levels/stability and by exploiting a variety of fluorescent reporters. Finally, we will extend the existing model for chemotactic signaling to determine how chemotactic performance depends on network composition, predict optimal scaling relations between protein levels and receptor cooperativity, and test these predictions with microevolution experiments. PUBLIC HEALTH RELEVANCE: We will investigate how cells of the model bacterium Escherichia coli remodel their sensory apparatus in response to a broad range of external conditions, including nutrients and temperature. It is advantageous for our purposes that the chemosensory system of Escherichia coli is the best-studied and most tractable sensory system of any living organism - it is therefore a natural place to look for general insights into how a cel's history shapes its strategies for survival in a complex and changing environment. We expect the results of our study to apply to a wide range of bacterial species - including major human pathogens - and also to help us understand the sensory strategies employed by eukaryotic cells such as our own.

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.

This INSPIRE award is partially funded by the Systems and Synthetic Biology program in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences, the Physics of Living Systems in the Division of Physics in the Directorate for Mathematical & Physical Sciences, and the Biotechnology, Biochemical, and Biomass Engineering program in the Division of Chemical, Bioengineering, Environmental, and Transport Systems, in the Directorate for Engineering. INTELLECTUAL MERIT: In their natural environments, bacteria primarily exist in multicellular, surface-bound communities called biofilms. While biofilms are desirable in the context of wastewater treatment, biofilms are notorious for causing undesirable problems such as chronic, medical device-associated, and hospital-associated infections, and persistent damage to surfaces of nearly all materials. Cells in biofilms display striking differences from cells that are free living, such as production of adhesive polymers, and a 1,000-fold increase in tolerance to antibiotics. Studies to date have been limited to investigations of biofilm formation, when only a few cells are present, or to overall characterization of the entire structure. The PIs made a recent breakthrough in microscopy: they resolved the individual cells in biofilms. This is the first time anyone has peered "into" a biofilm, to watch it develop, cell by cell, under conditions that model environmental, medical, and industrial systems. The PIs have also developed procedures to perturb the biofilm using genetic, mechanical, chemical and optical means. The PIs propose to characterize biofilms from the gene to the genome and from the cell to the collective. The project is based on a radically new approach that is essential to gain the understanding necessary to solve a fundamental problem with broad implications for science, engineering, and society. BROADER IMPACTS: This is a project at the interface of Physics, Biology and Engineering, and as such, offers to the students and postdoctoral researchers involved exceptional research educational opportunities. Moreover, each investigator has remarkable track records in teaching, outreach, and service.

PROJECT ABSTRACT In nature, bacteria primarily live in communities, specifically biofilms, which are surface-attached communities of cells embedded in an extracellular matrix. There are many advantages for the bacteria that adopt this communal lifestyle including adhesion to surfaces, resistance to antibiotics and predation, and collective processing of nutrient sources. From a human perspective, biofilms can be beneficial, e.g. in the context of waste-water processing and bioremediation. However, biofilms can also be problematic, e.g. biofilms cause major problems in medicine as they lead to chronic infections, and in industry biofilms foul surfaces and clog filtration devices. Because biofilms are three dimensional, heterogeneous, and rearrange over time, to date investigations have been limited to optical studies of biofilm formation when only a few cells are present or to gross characterization of the entire structure. We recently made a breakthrough, resolving individual cells in living, growing biofilms up to a depth of 30 microns, using customized spinning-disk confocal microscopy, fluorescent reporters, and automated cell-segmentation software. Biofilms can form clonally from a founder cell or by aggregation of many independent cells. In the first case, our analysis of a mature biofilm, grown from a single founder cell of the model pathogen Vibrio cholerae, revealed a striking transition during biofilm development from disordered cells to an orientationally ordered nematic state. In the second case, we found that autoaggregation of Escherichia coli relies on chemotaxis to a quorum-sensing signal produced, detected, and consumed by the cells themselves. Understanding these contrasting developmental processes and their ramifications for health and industry requires deeper mechanistic understanding. To this end, we will combine biophysical modeling with experiments to explore the role of cell-cell interactions, both physical and chemical, in the development of microbial communities. Experimentally, we will extend our studies of both V. cholerae and E. coli to include engineered signal and matrix-production mutants, and we will explore cellular heterogeneity within colonies using antibody labeling and fluorescent-reporter strains. On the theoretical side, our approach will combine agent- based and continuum models. Agent-based modeling will focus on single-cell behavior during ordering and aggregation processes. Continuum modeling, including a substantial extension of nematodynamics theory to describe 3D biofilm growth, will capture behavior over long distances and times. We expect the insights gained from this study and the modeling tools we develop to be applicable to bacterial community development over a wide range of organisms and conditions.

PROJECT ABSTRACT It is now understood that in their natural environments, bacteria primarily exist in multicellular, surface-bound communities called biofilms. Biofilms cause major problems in medicine as they are inherently resistant to antibiotics and cause chronic infections; in industry, biofilms foul surfaces and clog filtration devices. Cells in biofilms display striking differences from planktonic cells, such as extracellular matrix production, a 1,000-fold increase in tolerance to antibiotics, and unique gene expression patterns that are specific to particular locations within the biofilm. Because biofilms are three dimensional, heterogeneous, and rearrange over time, investigations have been limited to optical studies of biofilm formation when only few cells are present or to gross characterization of the entire structure. We recently made a research breakthrough: We resolved the individual cells in living, growing biofilms up to a depth of 30 microns, using customized spinning-disk confocal microscopy, fluorescent reporters, and automated cell-segmentation software. This is the first time anyone has peered into a biofilm, to watch it develop, cell by cell, in the presence of flow, under conditions that model environmental, medical, and industrial systems. Thus, we are in a position to use three-dimensional imaging, combined with key technological advancements they propose to make in photo-activation and optogenetics, to characterize biofilms from the gene to the genome and from the cell to the collective. Central questions to be addressed for the first time include how do quorum sensing and genome-wide expression profiles vary in space and time within growing biofilms? Experimental design and interpretation of measurements will be guided by biophysical modeling. We will launch the studies with the human pathogen Vibrio cholerae, known for rapid but transient biofilm formation. Specifically, we will pioneer a comprehensive examination of biofilm formation, development, and signal transduction from the single-cell to multi-cell levels and in realistic environments that mimic the spatial, temporal, and physical constraints found in nature. The interdisciplinary work will lead to understanding of gene regulation, cell-to-cell communication, and the spatial and temporal organization of biofilms, which in turn, dictate the large- scale features and ecological fitness of these multicellular systems. The proposal is unusually interdisciplinary: it teams Bassler, a microbiologist who is a leader in quorum sensing and biofilms, with Stone, an engineer whose focus is imaging, fluid dynamics, and the modeling of transport processes, and Wingreen, a theoretical biophysicist who models bacterial signaling circuits and biofilm development. The approach of direct imaging, beyond connecting genetics to biophysics, promises new insights relevant to understanding and manipulating biofilms with the goal of improving human health.

PROJECT ABSTRACT Phase separation is an emerging organizing principle for intracellular biology. Processes that are now understood to exploit phase separation include storage of genetic material, gene expression, ribosome synthesis, signaling, stress response, and metabolism. While each phase-separating system has unique features, there are universal themes relevant to all such systems, including regulation of the phase boundary, the dynamics of mixing within and between condensates, and the interactions of condensates with their surroundings. To uncover general principles regarding these common themes, we focus on a well-suited model organism and system: the genetically tractable alga Chlamydomonas reinhardtii and its pyrenoid, a non-membrane bound, phase-separated organelle responsible for efficient carbon fixation. The pyrenoid offers many practical advantages: 1. its phase separation is driven by two well- characterized components, the rigid enzyme complex Rubisco and the flexible linker protein EPYC1, via a known specific binding interface; 2. the pyrenoid?s in vivo liquidity is reproduced in vitro with no energy source; 3. in vivo assembly/disassembly is controllable by external cues; and 4. the pyrenoid is singular, large, and stable enough to systematically investigate its functional interactions with other cellular components. Based on these advantages, the pyrenoid has already proven to be a source of many discoveries including the ability of a flexible multivalent linker to condense a rigid component, inheritance of non-membrane bound organelles by fission, specific recruitment via a conserved binding motif, and a magic-number effect. The key universal questions we will address with this system are: What is the role of the valence, strength, and spacing of the interacting motifs in determining condensate properties? How does the stability of small oligomers control phase boundaries? What keeps condensates in a liquid state? How do cells control the number, size, and location of condensates, including their relation to other cellular structures? Our approach will closely integrate theory and experiment, as providing fundamental answers to these questions requires a multidisciplinary approach that places specific data within a broad theoretical framework. We anticipate that our focus on underlying biophysical mechanisms will facilitate generalizability of our results to a wide range of phase-separated intracellular systems.