Amy S. Gladfelter - US grants

Dr. Gladfelter will investigate how cell division is regulated spatially by septins in cells with several nuclei. For these studies, a multinucleated, filamentous fungus called Ashbya gossypii in which nuclei divide asynchronously is used. Asynchronous nuclear division enables cells to restrict responses to external environmental signals in a spatial manner. Septins, which are conserved, filament-forming proteins that assemble into cortical rings, appear to function in A. gossypii nuclear division by establishing mitosis promoting zones in the cell. Preliminary data demonstrates that nuclei divide near septin rings in A. gossypii and that the spatial pattern of nuclear division is disturbed in cells lacking septins. Thus, the septins appear to give spatial directions to nuclei that determine the position of division. A. gossypii septins assemble into a variety of morphologically distinct organizations and it is unclear whether all septin structures in the cell can promote mitosis and in which phase of the nuclear cycle septins act. Dr. Gladfelter hypothesizes that septins accelerate progression through G2 of the division cycle and that only a subset of septin structures have the power to direct mitosis. To test these hypotheses, the dynamics of nuclear division and changes in septin organization will be observed simultaneously in living cells using time-lapse microscopy. Strains will be generated in which components of the septin ring, the spindle pole body (SPB, fungal centrosome) and the nucleus are labeled with different fluorescently tagged proteins to detect septins and nuclear division progression in individual cells. These experiments will define how morphologically distinct septin structures arise and mature and how the kinetics of nuclear progression depends upon the presence of different septin organizations. The results of these experiments will provide a foundation for studying both the mechanisms of septin ring assembly in multinucleated cells and the basis for how the septins communicate with the cell cycle machinery.
Broader Impacts:
This work will be performed with the assistance of undergraduate woman at Dartmouth College who participate in the Woman In Science Program (WISP).

Proteins at the cell cortex link the cell interior and exterior by transmitting and responding to a variety of signals. Thus, the cell cortex is an information processing center where cells can receive and react to diverse stimuli in their environment or within the cell. A conserved family of proteins called septins organizes and regulates structures and processes at the cortex of eukaryotic cells. In addition to performing essential functions specific to certain cell types, septins are also important for normal cell division; they can act as "molecular fences" in membranes to keep proteins in specific locations and they are thought to act as protein "scaffolds" to assemble specific groups of proteins at the cell cortex. In most cells, septin proteins self-assemble into filaments that further assemble into complex higher-order structures such as rings and fibers. Despite the widespread distribution and varied functions of septins, little is known about the molecular mechanisms that direct septin assembly into filaments and the higher order organization of these filaments. A major goal of this project is to determine how septins assemble into an array of morphologically different forms within a single cell. The PI and her colleagues will investigate how septin structures are assembled, reorganized and regulated in the multinucleated, filamentous fungus, Ashbya gossypii. In this filamentous fungus, septins assemble into many morphologically distinct and dynamic structures, making it an excellent model for understanding the molecular controls of complex septin organization. The simple lifestyle and small genome of the fungus facilitates generation and analysis of mutants in regulatory proteins. Additionally, septin proteins can be visualized in living cells by linking the septin proteins to fluorescent proteins that are visible under the microscope. The specific goals of this project are to: 1. Identify the composition and dynamics of different septin complexes that coexist in one A. gossypii cell. 2. Establish the regulatory pathways that control the assembly of different septin rings. 3. Determine if the nuclear division cycle directs changes in the septin cortex in a multinucleated cell. These goals will be achieved using a combination of time-lapse fluorescence microscopy, electron microscopy and biochemistry for mutant analysis. This work is significant because the septins are a nearly ubiquitous, highly conserved protein family, yet major gaps exist in our knowledge of how they assemble and function in complexes in living cells. Furthermore, little is known about how internal or external signals lead to the assembly of elaborate types of septin structures. Knowledge gained from this work will provide a mechanistic foundation for understanding septin organization in other eukaryotic cells. The project will have substantial broader impact on education and training. The PI will mentor graduate and undergraduate students from multiple programs in research projects; undergraduate students will perform experiments, attend weekly lab meetings and present their data at Dartmouth's undergraduate research symposia.

DESCRIPTION (provided by applicant): In organisms ranging from single-celled yeasts to mammals, genetically identical cells exhibit variable cell division cycle times even when growing in the same environment. The sources and benefits of variability in a process such as the cell cycle that is wired for accuracy are unknown. In particular, it is not understood whether cell cycle timing variability is purely stochastic or whether it may be regulated as part of the design of cell cycle circuits. Variability in other cell processes has been shown to be beneficial, so cell cycle variability may in fact be an adaptive trait. The multinucleate, filamentous fungus, Ashbya gossypii, is a unique model system to study cell cycle timing variability because nuclei divide asynchronously within a common cytoplasm. Such asynchrony in a syncytium requires variable timing and nuclear autonomy in cell cycle signaling. As all proteins are translated in a common cytoplasm, it is mysterious how multiple, out of sync, cell cycle oscillators can coexist. We are taking advantage of the asynchronous division cycle in this model system to discover whether variability is programmed into the cell cycle and to learn how nuclear autonomy can be established. Knowledge of the molecular basis for variability is necessary for a complete understanding of cell cycle control and the pathologies influenced by a misregulated cell cycle. Population level variability in cell cycle decisions can impact processes as diverse as fungal pathogenesis and tumor cell behavior, and may be a factor influencing the efficacy of pharmacological treatments. While some cell-to-cell variability can be attributed to molecular noise in transcription, it is certain that other, as yet unidentified, cellular reservoirs of non-genetic individuality exist. In this proposal, we combine live cell imaging with computational and molecular genetic approaches to identify sources of variability in the cell cycle and determine how nuclear autonomy is established. With this model fungal system, we are well positioned to identify conserved sources of cell cycle variability and learn how cell signaling processes can be insulated within a common cytoplasm. The specific aims of the project are: 1) To determine whether variability in G1 duration is stochastic or regulated. 2) To test the hypothesis that nuclear size controls cell cycle timing and variability. 3) To test the hypothesis that spatially restricted protein movement creates nuclear autonomy. Timing variability exists in nearly all cell division cycles and knowing the basis of heterogeneity is essential for a complete understanding of the cell cycle. In this project, we will determine if timing variability is programmed in the cell division cycle, learn how nuclear size controls timing and how the cytoplasm can be functionally compartmentalized to maintain asynchrony. PUBLIC HEALTH RELEVANCE: In organisms ranging from single-celled yeasts to mammals, genetically identical cells take different amounts of time to divide even when growing in the same environment. This cell-to-cell variability in division timing can impact processes as diverse as fungal pathogenesis and tumor cell growth, and may be a factor influencing the efficacy of pharmacological treatments. In this work we will identify molecular sources of timing variability that will have relevance to the diverse diseases influenced by a misregulated cell division cycle.

This Major Research Instrumentation award to Dartmouth College funds the acquisition of a confocal microscope with a hybrid scanner, spectral unmixing and components that allow for Fluorescence Lifetime Imaging (FLIM) and Fluorescence Correlation Spectroscopy (FCS) measurements. This system will significantly advance basic science research and teaching infrastructure at Dartmouth and throughout New Hampshire and Vermont. The research enabled by the new system addresses fundamental questions in cell biology and developmental biology, and utilizes a diverse array of model systems (bacteria, fungi, algae, plants, worms, flies and mice). In all cases, the new confocal system will substantially improve and expand the imaging capabilities and allow Dartmouth investigators to continue to conduct leading-edge research and to train the next generation of life scientists. Each of the faculty using the new microscope is committed to teaching science at all levels and to preparing successful future scientists and science teachers. The new system makes state-of-the-art imaging technology more accessible to undergraduate students, graduate students and post-doctoral scholars, as well as students and teachers in our local secondary schools. Furthermore, Dartmouth faculty members have an extensive history of including undergraduate interns in their research programs and incorporating the latest scientific approaches and data into their courses. The results of these research and teaching efforts will be broadly disseminated through abstracts and peer reviewed publications, as well as by active participation of students and faculty at professional meetings.

Intellectual merit
The cytoskeleton is made of protein-based polymers that are required for proper cell structure and function. These polymer networks serve as machines and tracks to segregate cell components, change cell shape, and act as protein and membrane scaffolds. The septins are a highly conserved component of the cytoskeleton important for processes such as cell division, membrane compartments, and secretion. The septin proteins are widely expressed from fungi to mammals. Septins come together in cells to build higher-order structures such as rings, bars, and fibers. We use a genetically tractable model filamentous fungus called Ashbya gossypii to study septins because its persistent, dynamic and large septin-based assemblies are especially amenable to microscopy and biochemical analysis. The objectives of this project address three key aspects of septin architecture that are presently not understood: 1. Determination of how septin filaments polymerize and link together in living cells; 2. Identification of what factors control where septins are assembled together in cells; and 3. Evaluation of the dynamics of septin filaments. The research employs a combination of novel single molecule and polarized fluorescence imaging, genetic mutant analysis and in vitro biochemical approaches. The experiments in this project tackle a general cellular engineering problem: how to erect persistent, yet dynamic, structures of a constant size at specific locations in the cell. This project will discover basic rules cells use to organize themselves in space to carry out functions of life such as growth and division.
Broader Impacts
The PI is committed to training undergraduates and graduate students and communicating science in the community. As the course director for undergraduate Cell Biology, the PI will expand on an inquiry-based lab unit developed by her and her research group using Ashbya as a model system. After writing and receiving a grant for "clickers" to use in Biology classes, she has developed a series of questions to change the lecture based cell biology class into an active learning space and will continue to expand and share this tool kit with other faculty in future offerings. The PI has trained seven undergraduates in the lab in the last five years, including two female math and computer science majors and one male computer science major. One undergraduate was awarded an REU in the summer of 2010 to work with the PI at the Marine Biological Lab in Woods Hole. Two Dartmouth undergraduates completed honors theses in the PI's lab this year and both now have had their work published as first authors. She has mentored two female PhD students, one female post-doc and one female technician and has had five female rotation students, two of whom are Latina. The PI speaks about issues facing women in science and balancing science and motherhood at many venues on campus including various sorority faculty dinners, at the Biology undergraduate society, and the undergraduate Women in Science Program. The PI was the keynote speaker at the American Society for Cell Biology Annual Meeting Special Session for undergraduate participants in 2010. The PI has mentored two teacher-scholar post-doctoral fellows to facilitate their development as teachers by co-teaching with them and providing weekly feedback on their teaching. The PI speaks at evening seminars in her community about the importance of basic science research and has spoken to middle school science classrooms about what it is like to be a scientist. Graduate students in the PI's lab regularly teach local high school students and teachers about the principles of light microscopy and the research in our lab. The PI will weave training, mentoring and local community involvement into her research program through the continuation of these described activities.

DESCRIPTION (provided by applicant): The goal of this proposal is to acquire a total internal reflection (TIRF) microscope capable of simultaneous two-color acquisition (green, red) and near-simultaneous acquisition of a third color (far-red). The microscope would also be equipped with photobleaching/photoconversion capability. This equipment would be used as a primary research tool for quantitative analysis of dynamic processes, both biochemically and in live cells. There are five major users in the Department of Biological Sciences (Dartmouth College) and Department of Biochemistry (Geisel School of Medicine at Dartmouth) whose research projects are aimed at understanding molecular mechanisms of cellular processes such as actin dynamics, septin dynamics, intraflagellar transport during cilliary function, COPII transport from endoplasmic reticulum exit sites, and the function of a novel cytoskeletal element (Pil1 filaments). In all cases, multiple cellular elements are undergoing dynamics at sub-second timescales, including cytoskeleton and cellular membranes. Attainment and expansion of the goals of the funded research by these investigators requires the ability to monitor the dynamics of multiple molecules at high spatial and time resolution, and at single-molecule sensitivity. By limiting the excitation depth, TIRF microscopy has become a widely used technique to attain high spatial resolution and increased detection sensitivity. The iXON cameras and associated equipment allows simultaneous capture of two fluorescent signals, a necessity for processes with sub-second dynamics. In addition, a third fluorescent signal can be detected within 0.2 seconds of the other two signals. The EM-CCD camera allows single molecule sensitivity at moderate laser powers, minimizing photobleaching. This instrument would be the only one of its kind at Dartmouth and would serve the entire Dartmouth life science community.

This proposal requests partial support to facilitate the attendance and participation of early career scientists to the Gordon Research Conference on Cellular and Molecular Fungal Biology to be held at the Holderness School, June 19-24, 2016. Fungi are major causes of morbidity especially among immunocompromised patient populations and yet the arsenal of treating fungal infections is limited. Insight into the mechanisms of fungal reproduction and interactions with hosts and other microbes is essential for the advancement of treatment against fungal infections. The broad and long-term goal of the conference is to disseminate information about fungal pathogenesis and biology among an interdisciplinary group of researchers, and to increase our collective understanding of basic fungal biology and its application to medically important problems. The specific aim of this meeting will be to convene 52 speakers who represent the leading edge of fungal research, including established leaders in the field and up-and-coming early career researchers. A total of ca. 130-140 participants, most of whom will present posters, will gather for a five-day conference in a setting with few distractions. The oral and poster sessions are designed to emphasize discussion and networking, and evaluations from past conferences demonstrate the effectiveness of this format. The 2016 meeting will combine the core topics that underpin this area of research with newly emerging research topics, such as sessions focusing on biophysics and mathematical modeling, pathogenesis, symbiosis, environmental stress responses and fungal community interactions. These topics are all integral to understanding fungal pathogenesis and to develop novel therapeutics. The significance of this application is the demonstrated effect of this conference in accelerating research in the national and international fungal biology community, particularly in the areas of fungal-host interactions, evolution, and microbial communities that directly impact on endeavors such as fungal disease treatment and the development of anti-fungal therapeutics.

? DESCRIPTION (provided by applicant): (PI Gladfelter, AS) Macromolecules can partition by liquid-liquid phase separation without membrane compartmentalization. We found this mechanism is critical in the large, multinucleate cells of the fungus Ashbya gossypii whose nuclei divide asynchronously in a common cytoplasm and many sites of polarity coexist. Both nuclear asynchrony and polarity rely on spatially organized cytosol and serve as powerful functional measures of regionalized cytoplasm. We discovered that specific mRNA-protein assemblies promote formation of distinct cytoplasmic compartments not delimited by membranes. These assemblies behave as phase-separated liquid droplets that control the localization of mRNAs encoding key proteins (cyclins and formins). One driving force for phase separation is the polyQ-containing and low complexity sequences (LCS) found in two mRNA-binding proteins, Whi3 and Puf2. Our work revealed novel physiological functions for polyQ tracts outside of pathological contexts in generating cellular phase separations. A key feature of our model system is the clear functional read-outs for disruption of cytoplasmic partitioning, enabling us to link biophysical changes in phase-separated compartments to cellular function. Our proposed work addresses how discrete physiological RNA- protein (RNP) droplets assemble and how their biophysical properties contribute spatial organization to cytosol. We combine quantitative, live cell imaging in cells with in vitro reconstitution and mathematical modeling. We exploit multiple functionally relevant readouts of RNP droplets including cell cycle regulation and polarity initiation. The work spans multiple size scales by addressing the molecular mechanism of droplet assembly from the level of single molecules up to functional roles in translation in whole cells. Our specific aims are as follows: Aim 1. Determine how mRNA controls RNP droplet assembly, properties and function; Aim 2. Determine how cytoplasmic signals lead to variable droplet assembly and function; Aim 3. Determine mechanisms by which RNP droplets spatially regulate translation. This fundamental work impacts diverse cell processes, as phase separation of macromolecules is a conserved mechanism of patterning cytosol in distinct cell types. It is hypothesized that many proteins that are linked to toxic amyloid or aggregated states exist in liquid or phase separated states for normal function, and that the liquid state is a step in the assembly path of mature amyloids. Thus, understanding mRNP droplet regulation is important for understanding how cells manage the balance point between physiological and pathological aggregates that are the hallmark of many neurodegenerative diseases. PI-Gladfelter AS

Cell shape is intimately tied to cell function. The skeleton of a cell, called the cytoskeleton, is made of a network of polymers, which drive shape changes. The dynamic assembly and rearrangement of the cytoskeleton allow cells to react in both time and space to specific signals. A fundamental challenge for a cell is that the molecules of the cytoskeleton are much smaller than the size of the whole cell. A goal of this project is to understand how information about scale is transmitted in a cell so that small building blocks (nanometer in size) can carry out shape changes that are orders of magnitude larger. This work will reveal the scaling blueprint for one part of the cytoskeleton. The experiments will determine how structures built from filaments coming together in different arrangements and patterns leads to differences in cell shape and function. This work is important because it will reveal basic principals of self-assembly in living systems. Understanding these principals is essential to design synthetic cells that will likely be used for solving a variety of current and future problems such as in computing, bioenergy or bioremediation.


The septin cytoskeleton is an especially powerful polymer network to analyze how cells scale and shape protein structures because septins assemble into diverse forms in cells. Septin filaments can be knit together into bundles, gauzes, rings and lattices depending on the cell context. These different forms can be hundreds of nanometers to micrometers in size. In many cases these assemblies form on membrane surfaces where they function as scaffolds and membrane barriers. It is not understood how assemblies of such varied geometry and size can be built out of the same fundamental building block of a septin filament. The objectives of this study address how cells regulate septins to build structures of versatile form and function. These objectives will be achieved through biophysical approaches, optogenetics and cell-free reconstitution. Cutting-edge, quantitative imaging will be employed including single-molecule and polarization fluorescence microscopy, high-speed atomic force microscopy together with cell free reconstitution and mathematical modeling. Septins are nearly ubiquitous in eukaryotes and yet there are major gaps in understanding how cells construct higher-order structures from septin filaments. The ultimate goal is to understand how variable function emerges from this variation in form. The experiments here address problems that are fundamental to how all cells are spatially organized as there are many examples of micrometer-scale assemblies that are built from nanometer-sized building blocks.

Cells containing multiple nuclei (syncytial cells) are common across the entire tree of life and are found in every biosphere from bone, muscle, placenta and embryos of animals to the complex networks formed by fungi, water- and slime-molds. Yet little is known about the advantages an organism receives from having multinucleated cells, or how multiple nuclei communicate and coordinate to control cell behavior. This project will use biological and mathematical tools to test whether and how nuclei within multinucleate cells of the filamentous fungus Neurospora crassa adopt different roles in response to changes in the fungus' environment - allowing for a flexible division of labor within the cell. In addition to revealing general rules for nuclear coordination, the project has potential to enhance fungal productivity for food production and biotechnology. The project will offer graduate and undergraduate students interdisciplinary training in mathematical modeling, cell biology, genetics and novel microscopic imaging technology. In addition, new K-12 outreach activities will be catalyzed, including training of teachers and creation of new lesson plans on real world applications of mathematics and the utility of quantitative analysis in biology. Finally, the syncytial cell research community will be stimulated by a workshop that brings together mathematicians and scientists studying multinucleate cells in diverse organisms, including fungi.

Multinucleate cells allow the proportion of nuclei that contribute to any specific function to be dynamically tuned according to cellular requirements. But such division of labor is impossible if nuclei that share cytoplasm also see identical proteins and environmental cues. So, for nuclei to behave autonomously, protoplasm must be organized to control communication between adjacent nuclei. This project will use computational imaging, genetics and mathematical modeling to study how nuclei form communities within multinucleate cells and to dissect cellular processes that control these emergent communities. The focus will be on digestion of complex carbohydrates by Neurospora crassa to: 1) analyze the adaptive costs/benefits of nuclear coordination and division of labor; 2) determine the length scales of nuclear cooperation and autonomous behaviors; and 3) develop a linked mathematical model for inter-nuclear communication and coordination, focused on identifying general mechanisms that enable nuclear communities to stably emerge from interactions of relatively few macromolecules whose abundances are dominated by Poisson noise. The project will help discover new rules of life for how coordinated behaviors emerge from autonomous nuclei in filamentous fungi, and in syncytial cells generally, within cellular compartments that range from microns to meters in size.

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.

Summary/abstract Cell shape is integral to function and can be described in terms of plasma membrane curvature. Many changes in cell curvature occur on the micrometer scale but proteins are nanometers in size, raising the question as to how cells can perceive, control and use micrometer-scale geometry. The septins are a conserved, filament-forming family of proteins that preferentially assemble at sites of micrometer-scale membrane curvature. Septins assemble on many curved surfaces including at the cytokinetic furrow, dendritic spines, membrane blebs, around intracellular bacteria and bases of cilia and flagella. Given these diverse cell contexts, malfunction of septins is linked to diverse human diseases including many cancers, neuropathies and infertility. At sites of micrometer-scale membrane curvature, septins can influence the diffusion of proteins in the membrane, act as scaffolds to bring together signaling proteins, and impact the rigidity of the cell cortex. How curved septin assemblies form and recruit signaling proteins to the local membrane is critical to understand how septins link cell geometry to responses. Septin filament assembly occurs through annealing of short (~24-32nm) oligomeric rods on lipid bilayers or other cytoskeletal networks. We hypothesize that cells modulate the membrane affinity, length, density, and geometrical arrangement of septins in a curvature-dependent manner. The goal of this proposal is to identify the mechanisms directing assembly of septins on curved surfaces and to measure how curved assemblies regulate signaling networks. We will combine a variety of imaging approaches including high-resolution fluorescence, SEM and high-speed atomic force microscopy (HS-AFM), modeling, proteomics, and molecular genetics. Based on preliminary data, we hypothesize that curvature-dependent septin assembly involves mechanisms at work on several length scales. This work will be directed by three aims: (1) Analyze septin membrane interaction in curvature sensing; (2) Determine the biophysical properties of septin filaments that enable curvature sensing; (3) Identify how curved septin assemblies recruit specific signaling proteins. From the proposed experiments, we will learn how nanometer length scale mechanisms contribute to the emergent mesoscale process of sensing micron- scale curvature. These studies will also reveal how septin scaffolding may change as a function of local curvature. The long-term goal of this proposed study is to identify how septins recognize micrometer-scale curvature and then use shape information to modulate cellular functions.

Project Summary/Abstract (PI Gladfelter, AS) Cells must compartmentalize biochemistry in time and space. A newly appreciated mechanism of organization is biomolecular condensation. In many cases, condensates form via weak, multivalent interactions among disordered proteins and nucleic acids. These interactions determine the material states of condensates such as viscosity, surface tension and porosity, which in turn impact the concentrations, reaction and transport rates in, out and within condensates of key constituents. There are major gaps in understanding how cells control where condensates form, which molecules coassemble, and how condensate material state contributes to function. We discovered a physiological function for condensates in controlling nuclear division and cell polarity in the filamentous fungus, Ashbya gossypii. These condensates can control translation and are formed by an RNA-binding protein called Whi3 binding to target RNAs important for nuclear division (cyclins) and cell polarity (formins). The power of this cell system is that we can link physical properties and locations of condensates to functional outputs of protein translation, cell shape and nuclear division. The goals of the proposed work are to determine how structured elements in proteins, RNAs and cell membranes control the material state, location and function of condensates in the cell. We will determine how nanometer scale features of protein and RNA sequences promote mesoscale physical states of condensates to spatially pattern protein translation. We use an interdisciplinary suite of advanced imaging, genetic, biophysical and modeling approaches to tackle these fundamental open problems that not yet understood for any phase-separating system. Specifically, we will: Aim 1: Determine roles of hidden structured domains of proteins. We hypothesize that transiently ordered states promote specific protein-protein interactions and condensate material properties. Aim 2. Establish the architecture and function of RNA-based scaffolds. We hypothesize that mRNA forms a higher-order network using base-pairing that determines condensate properties. Aim 3: Delineate how membrane platforms control condensate assemblies. We hypothesize that endomembranes provide sites of assembly to specify the location of condensates. The proposed work will define how protein structure, RNA scaffolds and cell membranes are harnessed to control the properties, functions and locations of condensates in cells. The importance of condesates is underscored by numerous findings that link aberrant formation of condensates to multiple human diseases, including cancer and neurodegenerative diseases. While it is clear condensates undoubtably impact biochemistry, we do not yet understand how condensates actually contribute to normal cell function which is critical to understand how their malfunction leads to human pathologies.

Fungi are integral to the productivity of all terrestrial ecosystems as they play critical roles in nutrient cycling, soil structure and as parasites and symbionts. The estimates for the number of fungal species on the planet range from 1.5 to over 5 million and it is thought that likely over 90% of fungi remain to be identified. To date, a relatively small percentage of the identified fungi are associated with marine environments. However, fungi have been found from the surface of the ocean to depths of many kilometers in ocean sediment, and these organisms have potential key roles in carbon cycling and degradation of anthropogenic materials. Fungi that survive in the marine environment have remarkable abilities to respond to the myriad of stresses, including UV exposure, limited nutrients and high salinity that are features of the ocean. This work examines how marine fungi cope with environmental stresses by changing cell shapes and controlling how the cells divide. Fundamental understanding of growth, division and stress response is a missing link to understanding how marine fungi can contribute to the function of oceans. The work will involve the training of undergraduate and graduate student researchers along with several community outreach efforts.

Cell function is intimately tied to cell morphogenesis across the biosphere. This is especially clear in fungi where combinations of spherical, ovoid and hyphal-shaped cells build diverse structures across many scales. From micrometer-sized spores, centimeter-sized mushrooms and kilometer-spanning hyphae, a conserved family of cytoskeletal proteins called septins is integral to creating this morphological diversity. Despite their ubiquity, the basic biophysical properties and regulation of septin polymers are only beginning to be understood. This project links biophysical properties of septin polymers at the nanometer scale to their function in cell morphology at the micron scale. Septin form and function will be analyzed across highly morphologically diverse fungal systems called ?black yeasts? that were isolated from the ocean. Fungi in this group are considered to be amongst the most stress tolerant eukaryotes. Comparative analysis of protein sequence variation, biophysics and cell biology of septins in these diverse black yeast species will reveal how plasticity in the septin cytoskeleton supports diversity in cell shape, function and stress tolerance. The research will analyze this problem from three scales: 1. Polymer properties: How do biophysical properties of septin filaments control higher-order structures? 2. Dynamics: How are septin dynamics used to sculpt assemblies and enable stress responses? 3. Morphogenesis: How do different septin assemblies promote distinct cell morphologies. These questions will be addressed using interdisciplinary approaches including biochemical reconstitution, advanced live cell imaging, molecular genetics and quantitative modeling. Integral to this research plan is the training of undergraduate and graduate student scientists, along with several community outreach activities.

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.