Structural Cell Biology
The Wright lab explores a range of topics in bacteriology, cell biology, and virology. Simultaneously, we develop cryo-electron microscopy, cryo-electron tomography, and correlative imaging technologies. Our approach provides a basis for defining the architecture of complex cellular and host-pathogen systems so that this information may be used to develop novel antimicrobials, therapeutics, and vaccines.
Bacterial Motility and Adherence. The flagella and pili of many bacteria are essential for motility, adherence, biofilm formation, and are often crucial virulence factors for pathogenic species. For many species, the two appendages are temporally and spatially regulated and organized to work either in synchrony or alternately in order to coordinate motility and surface colonization. Our long-term goal is to understand 1) the structural implications of the incorporation of multiple flagellins into the flagellar filament and 2) the structural variation between pili at the macromolecular level. We use a number of model systems to study bacterial pathogenesis and virulence, bacterial appendage structure and function, and bacteria-bacteriophage interactions.
Helical reconstruction of Caulobacter crescentus flagellum. 3D map at ~2 Å resolution of a functional Caulobacter crescentus flagellum composed of the single flagellin protein FljM. Data: Mr. Juan Sanchez.
Eukaryotic Cells. We are interested in how eukaryotic cells (Drosophila and mouse neurons, human and mouse blood cells, and a number of cell lines) regulate their function and what causes cellular dysfunction. The dynamic three-dimensional landscape within cells drives how macromolecules, complexes, and organelles interact to maintain cellular processes such as those associated with metabolism, cell division, and trafficking. To further define concepts of cell development and structural plasticity, we are investigating a number of neuron cell types. We use cryo-microscopy methods to study platelet cells to determine relationships that are present between known patient genetics, platelet function, and platelet structure.
Virus Entry and Assembly. Enveloped viruses, such as HIV, respiratory syncytial virus (RSV), and measles virus (MeV), are human pathogens. Due to the significant variation in virus morphology, cryo-CLEM, cryo-FIB milling, and cryo-ET are the best technologies to use for targeting structures of interest, revealing them in intact cells, and for determining the structures at macromolecular to high-resolution. Our long-term goals are to understand the structural bases for 1) glycoprotein-mediated viral fusion, 2) virus replication, and 3) virus assembly and egress.
RSV Matrix (M) protein lattice. Sub-tomogram average at ~4.6 Å resolution of the RSV M protein helical-like lattice from native RSV particles. M protein crystal dimers (cyan and green) modeled into the 3D average. Data: Dr. Bryan Sibert.
Neurons, neurodegeneration, and amyloid diseases. Simultaneous with our cryo-ET structural studies of primary neurons, we are investigating the role of alpha-synuclein (a-Syn) in the pathogenesis of Parkinson Disease (PD). We collaborate with Chad Rienstra (UW-Madison) and Paul Kotzbauer (Wash U.), experts in ssNMR and synucleinopathies. PD is defined pathologically by the accumulation of a-Syn fibrils in neuronal cytoplasmic and neuritic inclusions known as Lewy bodies (LBs) and Lewy neurites (LNs). We are using cryo-EM and helical reconstruction techniques to solve structures of in vitro assembled a-Syn fibrils. We have generated 2 Å resolution structures of these fibrils. We are using in-situ cryo-ET methods to investigate a-Syn structures in intact neurons and brain tissue.
Cryo-EM helical reconstruction of alpha-synuclein fibril. 3D map at ~2 Å resolution of full-length a-Syn fibril depicting two protofilaments (one in red and one in grey). Data: Mr. Juan Sanchez.
Technology Development. In addition to the biological projects, we develop methods and tools to push the limits of cryo-EM and its correlation with other imaging modalities.
Correlative light and electron microscopy (CLEM). We develop novel equipment and molecular approaches for bridging the information gap between cryo-EM and fluorescence microscopy. This includes the design and use of cryo-stages for fluorescence microscopy and the software for correlation between microscope systems. By rapidly freezing cells cultured on EM substrates, we are able to directly correlate fluorescence microscopy images to images collected in the electron microscope. This technology is being applied to fundamental questions in all areas of structural cell biology.
Affinity capture. We develop methods for targeting macromolecules, cells, and viruses to EM substrates. Many of the enveloped viruses we study are pleiomorphic, grow to low titers, are cell-associated, and require the use of purification strategies that may alter the native structure of the virus. We adapt and use affinity technologies to address challenges associated with structural studies of enveloped viruses.
Micropatterning. Substrates used for imaging and other biophysical measurements poorly replicate the native physiological environments where cells exist. We engineer strategies to mimic cellular environments so that we are better able to define structure-function relationships within and between cells.
Grid Micropatterning Pipeline. (Left) Curved or straight pattern design to support seeding of Drosophila neurons on EM grids. (Left, middle) Patterned grids ready for neuron seeding. (Right, middle and Right) Fluorescence microscopy images of Drosophila neurons cultured on EM grids. Neurons remain within the boundaries of the pattern. Data: Mr. Joe Kim.