Elizabeth Villa, Ph.D. - US grants

Project Summary One of the most remarkable aspects of biological systems is their ability to operate on many length and time scales to perform their functions. Thus, methods to study those functions need to cover, and preferably connect, such scales. When studying the architecture of cells and their molecular components, imaging modalities range from high-resolution X-ray crystallography and single-particle cryo-EM, where molecules are isolated from their context, to light microscopy and conventional EM, where the localization within the cell is precise, but the structural details are lost. Thus, the grand challenge is to bring together structural and cellular studies. Cryo-electron tomography (CET) holds great promise to bridge together current structural, biochemical, and biophysical approaches. CET allows us to obtain 3D reconstructions of pleiomorphic structures, such as organelles and cells at molecular resolution, providing snapshots of molecular landscapes inside cells captured in action. However, limitations in sample thickness had hindered the application of cryo- EM to eukaryotic samples. We use cryo-FIB milling to micromachine intact cryo-fixed cells into thin-enough regions to study with CET. The resulting data shows readily identifiable features of cells such as individual molecular complexes, proteins embedded in membranes, and filamentous networks, at unprecedented detail. Our data show that we can use this technology to observe the structure of macromolecular complexes deeply embedded and entangled inside the cell, e.g., in the nucleus. Under the right sampling conditions, we can derive the structural dynamics of these macromolecular complexes by statistically comparing individual molecules. Just like super-resolution light microscopy has extended the realm of questions than can be asked in cell biology, I believe that CET will have a transformative impact through its ability to look not only at the location and context, but also at the structure of molecules in their natural environment. The goal of this project is to unleash the full potential of CET by integrating new and available tools around CET, notably, we will: (1) extend the experimental set up to create devices that resemble experimental conditions better than EM grids in order to perturb samples under controlled conditions, (2) use these devices to correlate CET data with light microscopy and powerful tags used in conventional EM, and (3) create computational and modeling tools to analyze such data quantitatively and to integrate data from other sources to reveal the structural dynamics of these complexes and the molecular architectures they form in the cell. Putting in place these techniques will allow us to study structurally uncharted territory: the nuclear periphery. Our first target is the study of the organization of the networks that connect the cytoskeleton and nucleoskeleton formed by LINC complexes, nuclear lamina, and cytoskeletal filaments, in health and disease.

The phagocytosis-like process of engulfment is the hallmark of endospore formation in bacteria from the genera Bacillus and Clostridium, which produce unusually durable endospores that are the infectious agent of Anthrax and Botulism. Engulfment mediates a dramatic change in cellular architecture, rearranging the sporangium from two cells that lie side by side, to an endospore in which one cell (the forespore) lies within the cytoplasm of another (the mother cell). The studies supported by this grant have provided new insights into the mechanisms by which coordinated peptidoglycan synthesis and degradation mediate this dramatic example of the architectural plasticity of bacterial cells, and they are providing insight into the role essential proteins play in this process. We here propose to visualize the dramatic morphological rearrangements of sporulation and the protein complexes that mediate them, by using a new implementation of cryo-electron tomography that uses a focused ion beam to produce thin lamella of bacterial cells, producing images that reveal structures within cells at nanometer resolution. We also propose to pursue preliminary data that has revealed that sporulation entails a dramatic and previously unrecognized metabolic differentiation of the two cells, after which the future spore is completely dependent on the mother cell for the precursors for biosynthesis. This process, which appears to be mediated by a massive forespore-specific proteolysis event, effectively converts the two cells into synthrophic partners, providing an accessible model for studying coupled metabolism, which is prevalent in microbial communities and biofilms. We here propose to study these processes, using genetics, metabolomics, fluorescence and cryo- electron microscopy, integrating these experimental efforts with computational analysis and modeling. These studies will reveal the cellular and metabolic landscape of sporulation in unprecedented detail, capitalizing on the dispensability and streamlined machinery of sporulation to provide insight into conserved processes that are often essential for growth.

We recently described the discovery of a nucleus like structure assembled by phage 201?2-1 after infection of Pseudomonas chlororaphis. The phage nucleus is composed of a protein shell (GP105) that segregates phage and host bacterial proteins according to function, with metabolic enzymes and ribosomes in the cytoplasm and proteins related to DNA and RNA processing inside the phage nucleus. This compartment is centered by a bipolar spindle composed of the phage-encoded tubulin PhuZ. Capsids assemble on the bacterial membrane and then migrate to and dock on the surface of the phage nucleus where phage DNA is packaged. Ultimately, capsids assemble with tails to create mature particles and the cell lyses. The GP105 shell appears in our preliminary cryoEM as an irregularly shaped 5 nm wide border that encloses phage DNA. Remarkably, this shell allows selective entry or retention of specific proteins. This work raises a number of questions such as: Is the GP105 shell essential for phage replication? Are other proteins required for shell assembly? What is the structural organization of GP105 within the shell and how does it assemble and incorporate new subunits as it grows? Does it contain pores that allow diffusion of mRNA, proteins and small molecules in and out of the structure? How is the PhuZ spindle organized over time as it pushes the growing GP105 to midcell? Does the spindle participate in other aspects of phage development such as capsid movement? Here, we propose to use a combination of genetics, biochemistry, structural biology, and cell biology to study the nucleus like structure assembled by GP105 and the spindle assembled by PhuZ and determine how these two structures participate in viral lytic growth.

An award is made to the University of California San Diego to support the acquisition of a cryo-dual beam microscope to enable the three dimensional visualization of cells at the level of the structure of individual biomolecules. This technology will enable the development of completely novel hypothesis on how molecules perform their function within the molecular landscapes that they operate in. This project will realize this potential for laboratories at UC San Diego, and will extend the training opportunities from technically trained scientists to non-expert users, effectively democratizing this technology and training the next generation of instrumentalists from diverse backgrounds. Furthermore, the molecular landscapes that this project will reveal are great teaching and dissemination tools for general audiences, including the creation and circulation of virtual reality and videos that allow users to navigate and explore cellular territories from within.

The acquisition of this instrument will support research projects that aim to observe intricate molecular networks in their natural environment. The scientific projects it will support span from technical projects that aim to develop new methodologies that will leverage the use of cryo-FIB milling for structural cell biology, to biologically driven projects with goals of unveiling the molecular mechanisms that result from specialized molecular networks in various model systems in cell biology. Method-related projects include development of novel computational algorithms to identify proteins within the molecular panoramas and enabling simulations of entire cells to predict cellular behavior under a variety of perturbations. Biologically driven projects include: unveiling unexplored mechanisms in bacterial cell biology, such as how bacterial cells divide and differentiate, how they respond to viral infection or keep track of time; the study of fundamental processes in the nucleus of eukaryotic cells such as how the genome organizes in 3-D and how this architecture determines the state of the cell. Together with future projects that will be enabled by this technology, the awarded project has the potential to make transformative discoveries that will unleash yet-unimagined hypothesis of cellular mechanisms in a wide range of biological scenarios.

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.