Session M68: Visualizing the Physics Behind Cell Biology Through Cryo-Electron Tomography

Three-dimensional imaging of force-generating molecular networks inside of cells using cryo-electron tomography

Dynamic networks of cytoskeletal polymers commonly take part in generating
fundamental cellular forces. In order to understand the mechanical role of these cytoskeletal
networks in development and disease, multimodal information across multiple dimensions and
scales need to be integrated. In this presentation, I will briefly overview the protein
components that make and regulate these cytoskeletal networks, and talk about how my lab is
using live-cell imaging and cryo-electron tomography to study these molecules in the context of
neuronal outgrowth.   

Opening a new window into the cell with super-resolution imaging and in situ cryo-electron tomography

Super-resolution light and electron microscopy have revolutionized our ability to visualize cell machinery. We use live-cell super-resolution imaging including stimulated emission depletion (STED) microscopy together with highly inclined thin illumination (HiLo) and high-speed three-dimensional widefield imaging to visualize organelle dynamics in real time. We have integrated these approaches with in situ cryo-electron tomography (cryo-ET), and cryo-correlative light and electron microscopy (cryo-CLEM) to visualize the endoplasmic reticulum (ER) and its relationships with other intracellular organelles in near-native states. The combination of these methods has revealed a novel ER-derived compartment that is mobile, vesicular, and associated with mammalian 80S cytoplasmic ribosomes. Moreover, with cryo-focused-ion-beam (FIB) milling and cryo-ET, we show that these vesicles exist as discrete structures separate from the intact reticular ER architecture. We call these organelles Ribosome-Associated Vesicles (RAVs). Detailed characterization of the RAVs revealed that these structures are conserved across multiple cell types and species using both conventional transmission electron microscopy (TEM) and cryo-ET.   

Automatic analysis of cryo-electron tomography using computer vision and machine learning

Cryo-electron tomography (cryo-ET) is an emerging technology for the 3D visualization of structural organizations and interactions of subcellular components at near-native state and sub-molecular resolution. Tomograms captured by cryo-ET contain heterogeneous structures representing the complex and dynamic subcellular environment. Since the structures are not purified or fluorescently labeled, the spatial organization and interaction between both the known and unknown structures can be studied in their native environment. The rapid advances of cryo-electron tomography (cryo-ET) have generated abundant 3D cellular imaging data. However, the systematic localization, identification, segmentation, and structural recovery of the subcellular components require efficient and accurate large-scale image analysis methods. We developed and adapted a suite of computer vision and machine learning methods for such analysis.   

Visualizing mitochondrial division machinery in situ

Mitochondria are dynamic organelles that serve a variety of metabolic roles for eukaryotic cells, including ATP generation and cell signaling. Unlike other organelles, mitochondria cannot be produced “de novo”, and instead rely on a tightly regulated division process called “fission”, which functions as a quality control mechanism to maintain a healthy population of mitochondria. However, several cellular stress pathways promote hyperactivation of the mitochondrial fission pathway that fragment the mitochondrial network and induce cell death (apoptosis). Despite increasing evidence that mitochondrial fragmentation is a hallmark feature of many neurodegenerative diseases, the molecular mechanisms that contribute to the mitochondrial fission process remain poorly defined. We recently developed a three-dimensional imaging approach using a combination of cryo-focused ion beam milling and cryo-electron tomography to visualize snapshots of mitochondrial constriction events in mammalian cells. Our three-dimensional reconstructions represent the highest resolution structures of the ultrastructural interactions between mitochondria and other subcellular components to date, enabling unprecedented analysis of these associations. Further analyses reveal that the endoplasmic reticulum and the cytoskeleton preferentially associate with mitochondrial membrane constrictions. Interestingly, we also observe the presence of a previously unidentified filamentous structure in association with dividing mitochondria, which we propose is a member of the septin family of intermediate cytoskeletal filaments. By mapping out the precise interactions of these components relative to mitochondrial membranes, our work describes the complete ultrastructural architecture of the mitochondrial fission machinery required for membrane constriction, and establishes cellular tomography as a valuable approach for studying snapshots of mitochondrial dynamics in situ.