Zvonimir Dogic, Ph.D. - US grants

INTELLECTUAL MERIT: The proposal has four experimental aims organized around the common themes of entropy and chirality and how these affect interactions between, and self-assembly of, biopolymers. The first aim will focus on measuring the mesoscopic potential between a pair of actin filaments held together in part by entropic (depletion) forces. The goal is to determine the contribution of entropic fluctuations to the attraction between the filaments. Two alternative approaches will be used. In one, a laser tweezer is used to pull a pair of filaments apart in combination with a fluorescence microscope to image the system simultaneously. The attractive forces can be extracted directly from this experiment. An alternative approach analyzes images of isolated filaments which fold on themselves in hairpin fashion. The adhesion energy is determined by recognizing that it is balanced against the elastic energy stored in the folded filament to determine the size of the hairpin loop. The second aim is to study the self-assembly of monodisperse non-rodlike viruses into colloidal membranes, a phenomenon recently observed in the PI's laboratory. These virus particles do not exhibit the polarized head-tail amphilicity characteristic of most membrane formers, and this membrane assembly process is thus hypothesized to be driven by depletion forces. The third experiment examines the effects of chirality on the formation of colloidal membranes and twisted ribbons using both chiral and achiral filamentous viruses. This work explores the hypothesis that formation of ribbons is driven by the molecular chirality of the constituent rods. The final project examines extensions of the theories of the excluded-volume-driven transition of rodlike particles to a nematic phase at modest rod densities. Here it is planned to examine the role of chirality in the rods to explain the observed chiral nematic (cholesteric) phase often observed in these systems. In all of these studies the primary experimental tool is a multimode microscope that can acquire images in differential interference contrast and fluorescence modes. This is coupled with a laser tweezer to exert force on colloidal beads or other structures with high refractive index and determine the position of the beads with nanometer precision on a microsecond timescale.

BROADER IMPACTS: The proposal is highly interdisciplinary in nature and will provide new design principles for assembling complex biomaterials. The PI is developing a new advanced laboratory course in quantitative biology instrumentation, which will focus on the design and application of modern microscopy. This course is also the centerpiece of a larger effort at Brandeis, partially supported by the Howard Hughes Medical Institute and an NSF-IGERT grant, to establish an interdisciplinary graduate program in Quantitative Biology. The project will provide a platform for training of two graduate students. Funds are also included in the award to support two undergraduate students per year on the project.

DESCRIPTION (provided by applicant): The cytoskeleton is a highly dynamic structure that is capable of rapid assembly and disassembly. Actin, one of the primary building blocks of the cytoskeleton, can exist in either monomeric or polymeric form. Using a highly conserved set of approximately 20 to 30 proteins, cells attain remarkable spatial and temporal control of the nucleation of actin filaments, their elongation and their eventual disassembly into monomeric proteins. Formins have recently emerged as important nucleators of actin assembly. After inducing nucleation, formins remain attached to the growing end of a filament and thereafter guide insertion of new subunits. In budding yeast, formins are localized at the bud tip and bud neck and from there they direct the assembly of actin cables which are highly dynamic bundles of actin filaments. Actin cables play an important role in establishing cell polarity and cell morphogenesis. The goal of this mentored quantitative research development award is twofold. From a research perspective, it will provide new insight into mechanisms by which formins accelerate growth rates of actin filaments. The specific aims of the proposal are to (1) use single molecule fluorescence imaging to elucidate the mechanism by which the FH1 domain of formins enhances the polymerization rate of actin filaments and (2) to use yeast extracts to reconstitute the formation of dynamic actin cables and measure the dependence of actin turnover on various regulatory proteins using advanced optical microscopy. These two processes will be described with quantitative models which will be rigorously tested against experimental data. From a training perspective, the PI of this proposal will gain valuable hands on knowledge in techniques of molecular biology and biochemistry by working in the laboratory of his mentor (Bruce Goode). Additionally, the PI will also acquire formal knowledge of molecular biology and biochemistry by taking graduate level courses and through the attendance of seminars and journal clubs. In this proposal we will use budding yeast S. cerevisiae as a model organism. The majority of actin associated proteins that regulate the assembly of actin structures in yeast have direct counterparts in mammalian cells, with strikingly similar functions. Therefore, the knowledge gained from studies of yeast has direct relevance to our understanding of normal human cell biology and how mutations of various actin regulatory proteins give rise to diseases such as cancer.

0923057
Dogic
Brandeis U.

"This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)."

Technical Summary: This project aims to develop a multimode optical microscope which will simultaneously reveal the structure and dynamics of soft and biological materials from various perspectives and across multiple lengthscales. The proposed instrument will be centered around an inverted optical microscope and will have the following four mutually compatible modalities: (1) holographic optical tweezer based on an infrared laser, (2) epi-fluorescecence microscope capable of localizing single molecules with millisecond temporal resolution and nanometer spatial resolution, (3) quantitative polarization microscopy which will reveal either 2D birefringence maps at the highest spatial resolution attainable with visible light or 3D birefringence maps at a reduced spatial resolution and (4) dual view imaging. The new multimode microscope will be used to examine a broad range of soft and biological materials including self-assembled non-amphiphilic membranes whose formation is driven by chiral interactions, spontaneously oscillating eukaryotic flagella, dynamics and structure of semi-flexible polymers absorbed onto a hard wall and synthesis and expression of biopolymer collagen in vivo. The proposed instrument will be developed in a close collaboration between Brandeis University and Woods Hole Institute and the operation of the proposed instrument will be supported by the Brandeis MRSEC center. Special effort will be taken to develop a novel experimental course focused on optics and microscopy, which will enable undergraduate and graduate students to effectively use the multimode microscope.

Layman Summary: Optical microscopy uses visible light to directly image the structure and dynamics of various soft materials such as paints, foams, gels and liquid crystals. Over the past few decades various specialized modalities of optical microscopy have been developed, with each modality revealing certain aspects of the material under examination. For example, brightfield microscopy reveals the structure and dynamics of entire micron sized assemblages, fluorescence microscopy yields the dynamics of individual components within such an assemblage, polarization microscopy indicates the orientation of molecules within any given structure and laser tweezers enable studies of how these assemblages respond to external force. So far most soft materials have been examined with either single or dual mode microscopes. This project aims to develop a novel multimode microscope which will enable simultaneous examination of materials with all four different modalities: brightfield microscopy, fluorescence microscopy, quantitative polarization microscopy and holographic laser tweezers. Special care will be taken to ensure that the performance of each module is not compromised by the presence of other modalities. Development of the multimode microscope will enable experiments that will correlate behavior of materials across multiple length scales and will thus provide essential insight into how collective properties of a material such as its equilibrium shape and elasticity arises from interactions of individual components. The proposed instrument will be developed in a close collaboration between Brandeis University and Woods Hole Institute and the operation of the proposed instrument will be supported by the Brandeis MRSEC center. Special effort will be taken to develop a novel experimental course focused on optics and microscopy, which will enable undergraduate and graduate students to effectively use the multimode microscope.

ID: MPS/DMR/BMAT(7623) 0955776 PI: Dogic, Zvonimir ORG: Brandeis University

Title: CAREER: Hierarchial Self-Assembly of Biopolymers

INTELLECTUAL MERIT: Biopolymer-based materials are frequently organized into complex hierarchical structures that span multiple length scales ranging from nanometers to millimeters. The goal of this research is to elucidate pathways by which biopolymers self-assemble into macroscopic materials and relate the structure, mechanical and optical properties of these macroscopic materials to the underlying mesoscopic interactions and microscopic architecture of the constituent biopolymers. The proposal has two specific aims. The first aim is to determine how the continuum mesoscopic properties of individual biopolymers are affected by the mutations of the constituent proteins at the microscopic level. The second aim is focused on understanding the structure and interactions of macroscopic colloidal membranes. Here, attractive depletion interactions are used to assemble thousands of rods into an equilibrium membrane-like structure composed of a monolayer of aligned rods. These two experimental aims are related to each other in several important ways: They both require use of the same biopolymer model systems. They also involve similar experimental techniques and technologies. Finally, they explore the central theme of hierarchical assembly, with each aim focusing on a different level of hierarchy ranging from single filaments to assemblages containing thousands of filaments. Understanding biopolymer assemblages at any one level of hierarchy represents a significant advance in the field. However, only the combined understanding of structure and dynamics at all relevant length scales will result in general design principles required for a ?bottom up? engineering of novel nanostructured materials with predefined structural and mechanical properties. From a techniques perspective special emphasis is being placed on directly visualizing biopolymer assemblages at all levels of hierarchy. Using electron microscopy the PI will determine 3D conformations of biopolymers with nanometer resolution. With optical microscopy he will simultaneously visualize dynamics of individual filaments within an assemblages as well as the structure and dynamics of entire assemblages.

BROADER IMPACTS: The research and education plans are seamlessly joined together through their mutual emphasis on visualization techniques and interdisciplinary approach to science. The proposed educational plan contains four related aims. Specifically, (1) the PI and collaborators will continue development of an interdisciplinary laboratory course focused on optical microscopy. Since microscopy is used in numerous disciplines; they will ensure that the course is accessible to students from all scientific backgrounds. (2) They will integrate students at all levels of education, ranging from high school students to advanced graduate students, into the ongoing vigorous research program. (3) They will develop a virtual online laboratory focused on the quantitative analysis of microscopy images. Students anywhere will be able to remotely download actual experimental data and perform all the subsequent image analysis and data reduction steps using custom written software. (4) Finally, they will develop a permanent exhibit at The Discovery Museum in Acton, Massachusetts, which will use optical microscopy to provide a glimpse into a highly dynamic world at microscopic length scales. Successful implementation of these four aims will result in a new generation of students who are better trained in the fundamentals of microscopy. It will also motivate young minds to pursue science related careers and increases awareness of the importance of scientific research and optical microscopy amongst the general audience.

The objective of this award is to assemble self-limited structures and material that undergo large-scale structural changes in response to changing environmental conditions such as temperature, solution properties, or imposed force. As a basic building block the project will utilize bacterial flagella, a helical polymer which acts as a nanoscale spring and exhibits non-monotonic force-extension relationship that is not commonly found in synthetic materials. To achieve the goal the project will utilize a combination of experimental, computational and theoretical tools to elucidate the behavior of flagella based materials at three levels of hierarchy. First, the project will characterize and model the mechanics of individual flagella, as well as chimeric flagella engineered to achieve new polymorphic switching responses. Second, will be to generate, characterize, and control attractive interactions between a pair of flagella. Finally, these same attractive interactions will be used to assemble flagellar bundles or ropes consisting of thousands of flagella.

This study holds the promise to generate nano-structured, responsive fibrous materials with well-controlled mechanical and structural properties, in which conformational changes of individual monomeric units at Angstrom scales cooperatively cascade across multiple lengthscales to produce macroscopic mechanical changes in millimeter-sized materials. The educational and research component of this project will be integrated by expanding an REU program to hold an annual "Theory Meets Experiment" symposium for undergraduates participating in summer research at Brandeis and University of Massachusetts, to expose novice researchers to the distinct and complementary methods of theoretical and experimental research in soft materials. In addition, a partnership will be explored with Jennifer Sabin, architect, designer, and lecturer at the University of Pennsylvania School of Design in order to explore the relationship between complex biopolymer assembly motifs and a state-of-the-art textile design and fabrication process.

Molecular motors are nanoscale machines that convert chemical energy from ATP hydrolysis into mechanical movement along a filamentous track. Over the past two decades experimental advances have yielded remarkable images of single molecular motors taking nanometer-sized steps. Furthermore, state-of-the-art technology also makes it possible to pull on such nanomachines with optical traps thus determining the maximal force they can exert. Taken together these efforts have provided essential insight into biochemical mechanisms driving the motility of isolated molecular motors. However in many instances, ranging from contraction of a skeletal muscle to spontaneous beating of a biological cilium, motors do not act in isolation. Instead, thousands of motors coordinate their activity to produce large scale molecular motion. Despite its importance, little is known about the emergent collective properties of molecular motor ensembles. The first goal of this project is to develop an experimental assay that will quantify contractile sliding forces between a pair of aligned microtubules, exerted by molecular motor clusters that simultaneously bind and move along both filaments. Such structural motifs drive numerous complex processes in a biological cell. Using this information, the project will build microtubule bundles that are clamped at their base and driven by molecular motors. Theoretical models predict that such structures will spontaneous beat, thus mimicking the dynamical behavior of biological cilia. Experimental efforts will directly verify this prediction and provide insight into the mechanisms that control beating patterns of biological cilia. In parallel with experimental efforts, this project will also pursue development of computer simulation models for collective behavior of molecular motors; these will bridge the gap between existing theoretical models and experimental data.

Broader Impacts: The research and outreach efforts will be seamlessly integrated through a mutual emphasis on visualization and microscopy. Movies of dynamical biological structures obtained via microscopy capture the imagination and interest of scientists and non-scientists alike. The PIs will organize outreach activities at The Discovery Museum in Acton, MA, in which optical microscopes and specimens will be available at the museum, allowing visitors to peer into the microscopic world and directly visualize biological motion. The PIs will also disseminate practical knowledge of optical microscopy by teaching an intensive hands-on one-week summer course. Investigator involvement with the highly successful science Posse program will further enhance undergraduate and graduate student involvement from historically underrepresented groups.

Non-technical: Amphiphilic molecules consist of water loving and oil loving components that are permanently linked to each other, thus frustrating bulk separation. When added to water these molecules self-assemble into membranes, thin flexible sheets with remarkable properties that play an essential role in biology, physics, and material science. It is commonly believed that the antagonistic nature of the amphiphilic molecules is an essential requirement for membrane assembly. In this proposal PI will study a fundamentally different method for assembly of colloidal based membrane-like materials. Assembly of the novel colloidal membranes takes place in a simple mixture of chemically homogeneous filamentous viruses, which have the shape of billiard cues, and polymers, which resemble billiard balls. Besides providing fundamental insight into universal aspects of all membrane-like materials, results will also describe a new and easily scalable process for assembly of rod-like nanoparticles into novel nanostructures that could act as efficient photovoltaic devices. The PI's research and education plans are seamlessly joined together through their mutual emphasis on visualization techniques and an interdisciplinary approach to science. Specifically, the PI will continue to build upon existing successful collaborations with The Discovery Museum in Action, in order to organize biannual visits to the museum for hands-on demonstrations of various concepts in optical microscopy and materials science. The PI will also extend connections with the local elementary and middle schools by participating in Science Fairs, by organizing class visits, and hosting students in his laboratory. Finally, the PI will continue to provide opportunities to undergraduate students to pursue research projects and will continue to build upon existing local and national connections in order to recruit students form groups that are underrepresented in STEM fields.

Technical: Colloidal membranes are comprised from a one-rod-length thick liquid-like monolayer of aligned rods that is held together by the osmotic pressure of the enveloping polymer solution. The goal of this proposal is to elucidate the fundamental laws that govern the assembly processes of colloidal membranes and to use colloidal membranes as a basis for engineering of a new generation of spatially heterogeneous, shape-changing functional materials. The PI will first use colloidal membranes as a robust platform for assembly of well-defined mesoscopic clusters and macroscopic 2D materials with predetermined heterogeneities. In parallel, the PI will devise new methods to measure the mechanical properties of colloidal membranes, and use this knowledge to engineer shape-changing 3D materials. Colloidal membranes are interesting in their own right as they offer a unique opportunity to organize complex 2D materials on micron scales. Furthermore, although distinct on molecular scales, the continuum deformations of colloidal monolayers and lipid bilayers are described by the same free energy expressions. Thus they provide a unique opportunity to gain insight into universal membrane processes that are mainly determined by the fundamental symmetries of the system.

Non-Technical Abstract

This award supports the 2017 Gordon Research Conference and Gordon Research Seminar on "Soft Condensed Matter Physics: In and out of equilibrium". The award is jointly funded by the CMP and CMMT programs in the Division of Materials Research. This Gordon Research Conference will bring together leading scientists from a variety of fields in a collegial atmosphere that supports open discussion to discuss issues at the cutting edge of soft matter physics. This meeting will focus on two topics at the forefront of Soft Condensed Matter Physics: the mechanisms lead to self organization of materials though both thermal and athermal processes. Many The conference will focus on recent advances in the design of colloidal and liquid crystalline materials. An equally important goal is to illustrate growing connections between soft matter physics and related interdisciplinary fields, including those of biology, mathematical topology, fluid dynamics, nanotechnology and material science. In addition, this conference will be preceded by a Gordon Research Seminar that provides a unique opportunity for young researchers to share in the GRC experience and build informal networks with their peers.

Non-technical abstract:

Elucidating the rules by which thin flat sheets fold into complex three dimensional structures is of fundamental interest to a broad range of fields ranging from physics, and materials science to biology. For example, the process by which a flat 2D bilayer with exposed edges folds into an edgeless 3D vesicle is essential for transport across cellular membranes and also for developing new methods of drug delivery. However, visualizing and quantifying such folding processes using nanometer-sized lipid bilayers is currently not feasible. This project will use an analogy between lipid bilayer and colloidal monolayer membranes to quantitatively test foundational models of vesicle formation and other folding processes. Colloidal membranes will allow one to measure all the parameters and directly visualize the folding process with optical microscopy, to a degree that is not possible with conventional materials. Besides visualizing closed vesicle formation the team will also study processes by which flat sheets fold into topologically more complex mathematical structures, such as catenoids and Gyroid-like structures. In parallel, the team will pursue outreach activities focused on (1) providing rigorous training in interdisciplinary sciences to graduate and undergraduate students, (2) encouraging underrepresented groups to pursue work in STEM related fields, (3) and raising general awareness of the importance of scientific research to broader communities. This project will actively support undergraduate research and will build on a strong record of recruiting students from diverse backgrounds. They will also continue their involvement with the Cal-Bridge program, a NSF-funded program whose mission is to increase the number of underrepresented minority and female students entering doctoral program in physics. Finally, each year they will also participate in about half a dozen outreach activities at local elementary and middle schools in order to promote science education.

Technical abstract:

Colloidal membranes, which are comprised of one-rod-length thick liquid-like monolayer of aligned monodisperse rods, offer a unique opportunity to explore fundamental aspect of thin sheets with vanishing in-plane shear modulus. So far, studies have mainly examined the behavior of colloidal membranes in the regime were they remain flat 2D structures. The goal of the current project is to elucidate the fundamental laws by which 2D colloidal membranes fold into diverse and topologically distinct 3D architectures. Three specific aims are proposed. In the first aim they will develop active and passive fluctuations-based techniques to measure the mean and Gaussian elastic moduli of colloidal membranes, the phenomenological constants that determine the energetic cost required to bend a membrane from its minimum energy state. In the second aim they will tune microscopic constituents of colloidal membranes to investigate regimes were flat colloidal membranes are inherently unstable and spontaneously fold into complex structures. In one direction they will reduce the bending rigidity of colloidal membranes to induce folding of flat 2D membranes into edgeless 3D vesicles and visualize and quantify the pathways of this transition. In a complementary direction they will explore the regime were doping colloidal membranes with miscible short rods induces formation of remarkably diverse and topological complex surfaces with negative Gaussian curvatures. Using state of the art imaging technique they will elucidate the molecular mechanisms that drive these instabilities. In the final aim they will develop robust experimental tools that will enable spatiotemporal external control of the membrane curvature, thus allowing one to actively intervene in the ongoing folding processes and guiding formation of topological assemblages of predetermined final shape.

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.

Nontechnical Abstract:
Biological systems exhibit remarkable behaviors, including the ability to heal, reproduce, and be self-motile. Conventional synthetic materials do not display such properties because they are composed of inanimate molecules. In contrast, biological systems consist of active, energy transducing molecules which continuously push and pull on each other, producing active stresses. These active stresses make biological systems examples of active matter, and are ultimately are responsible for many of their unusual behaviors. Creating artificial active matter opens up a new route towards constructing materials with lifelike functionalities. However, a significant obstacle to realizing this possibility is an absence of understanding of how active stresses are produced: given specific elemental building blocks, there are not currently tools to either measure or theoretically predict the magnitude, type, or even the sign of the active stresses. The project will elucidate the foundational principles of active stress generation using a synergistic combination of experiments, simulations, and theory. The work will result in the creation of new experimental tools for measuring active stresses, new synthesis procedures for creating active materials, and new multi-scale modeling techniques for studying active matter. An integrated outreach program will bring the excitement of active matter to K-12 education, undergraduates, and graduates by combining interactive demonstrations, advanced interdisciplinary training, and intensive summer courses.

Technical Abstract:
The research focuses on studying assemblages of microtubules and kinesin-14 molecular motors. The work addresses three fundamental challenges in understanding active stresses generated by such active materials. First is the development of tools to measure active stresses at different length scales using optical tweezers, fluid flow measurements, microfluidics, and 3D printing, in combination with theory and simulations. Second, a novel system will be created in which active stress generation will be modulated by directly affixing kinesin-14 to microtubules in predetermined patterns. Theory and simulations will be used to guide which patterns to create, and conversely, measuring the resulting active stress generation in these designed materials will provide a stringent test of theory and simulations. Third, a multi-scale modeling framework will be developed and tested. These will be subsequently related to each other through a combination of analytical calculations and simulations. The resulting models will be made with continual feedback from experiments. Taken together, this work will establish new paradigms to study, understand, and engineer active stresses in active materials.

This DMR grant supports research to understand the assemblages of microtubules and kinesin-14 molecular motors with funding from the Condensed Matter Physics (CMP) and Biomaterials (BMAT) Programs in the Division of Materials Research of the Mathematical and Physical Sciences Directorate.

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.

Nontechnical Description:
Optical microscopy provides an essential window into the micrometer scale structure and dynamics of diverse systems ranging from simple colloidal suspension and biological cells to tissues and entire developing organisms. Most optical microscopy studies are limited to two-dimensional regions of interest. A typical method of characterizing three-dimensional structure relies on confocal point scanning techniques, which are very slow. Due to lack of appropriate techniques, high-spatial resolution imaging of fast dynamics of macro-sized samples remains uncharted territory. This project paves the way for overcoming this barrier. The resulting novel imaging technique named light-field light-sheet microscopy is tested and optimized with diverse samples ranging from quantifying dynamics and topological structure of three-dimensional active liquid crystals to imaging the whole-brain of a fruit fly during food-search. The design principles of the microscopy and the associated software analysis code are shared with scientific community. The imaging technique is also popularized through its inclusion in the Annual Santa Barbara Summer School in Quantitative Biology, where the participants receive the training in using the instrument.

Technical Description:
By seamlessly merging the existing techniques of the multi-view selective plane illumination microscopy and the light-field microscopy, scientists are developing a new optical microscope capable of imaging millimeter-sized samples with isotropic point spread function. When compared to the fastest imaging times accessible to state-of-the-art selective plane illumination microscopes, the technique being developed increases the temporal resolution by almost two orders of magnitude. This opens up the possibility of three-dimensional, high-spatial-resolution imaging of macroscopic samples at frequencies as high as 5 Hz. The microscope is used to study a variety of samples that range from materials science, cell biology and neuroscience. In particular, scientist use the microscope to (1) elucidate the interplay between the topological structure and dynamics of motile disclination loops in three-dimensional active nematic liquid crystals, (2) quantify temporal propagation of strain deformations in active gels where stresses are generated by single molecular motors, (3) perform whole-brain imaging during food-search in D. melanogaster larvae, and (4) characterize a whole nervous system as it forms and activates in the embryogenesis of D. melanogaster.

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.

Liquid-liquid phase separation is a pervasive phenomenon that plays a central role throughout physics, material science, engineering, biology, and everyday life. Two liquid phases are separated by soft interfaces that are greatly affected by gravitational forces and are easily deformed either by thermal fluctuation or by external flows. Everyday experience demonstrates how gentle shaking induces waves of a soft interface, while more rigorous shaking reinitializes the phase separation dynamics. Active matter provides an alternative method of generating fluid flows through the collective motion of microscopic energy-consuming constituents. It remains an open question of how active matter-based internally-generated flows couple to the soft interfaces and the associated liquid-liquid phase separation. Ground-based research indicates intriguing behaviors, but gravitational forces suppress large-scale deformation and full exploration of the possible behaviors. This project is focused on developing a quantitative understanding of the active interfaces. From a broader perspective, this project will train undergraduate students in interdisciplinary research, and provide support for developing a curriculum-enriching Saturday course for high school students that is centered around the topic of soft materials, liquid-liquid phase separation, and microgravity research.<br/><br/>The research goals of this project are divided into three complementary aims, which will greatly increase understanding of active liquid-liquid phase separation and associated active interfaces. In the first aim, millimeter-sized soft interfaces will be created that separate a bulk active fluid from a passive polymer suspension. The research will determine how gravity influences active fluctuations and associated traveling waves, which are visible by the naked eye. The project will also study how a macroscopic interface disintegrates to generate an active emulsion, a transition that is suppressed by gravity but should be observable in a microgravity environment. In the second aim, the project will start with a uniform initial state and study the dynamics of coarsening active droplets. Microgravity will render our samples truly two-dimensional. Finally, in the third aim, gravity affects will be studied to determine how the active wetting transition that occurs when active fluid climbs along a boundary. The acquired data will rigorously test theoretical models of how active stresses couple to soft deformable surfaces. Such models are essential for building synthetic mechanical protocells and might have relevance to our understanding of biology.<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.