Development of cryogenic correlated light electron microscopy methods to study mechanisms of intracellular trafficking and their relationships to the secretory pathway

The application of cryogenic electron microscopy (cryo‐EM) to the study of cellular ultrastructure provides a resolution several orders of magnitude better than light microscopy. Although this approach is increasingly applied in situ, it suffers from limitations in our ability to target imaging to specific intracellular features including the subcellular localization of specific events of interest. Cryogenic correlated light and electron microscopy (cryo‐CLEM) helps to overcome this problem by spatially locating areas of interest inside cells using fluorescence from genetically tagged or stained cellular molecules and allows for the visualization of localized fluorescently‐tagged proteins down to the level of individual organelles. Here, we attempted to study the secretory pathway in a specialized mammalian cell line, insulin‐secreting INS‐1E cells, expressing genetically‐encoded fluorophores as a model system to develop a cryo‐CLEM methodology. We discovered that there are many bright sources of autofluorescence in frozen cells. Based on our initial observations and the current understanding in the field, we hypothesized that autofluorescence from endogenous cellular substrates exhibits a broader spectrum of fluorescence than the fluorescence range of our expressed fluorescent proteins. To test this, we developed a quantitative approach to discriminate between autofluorescence and the fluorescent signal from genetically‐encoded fluorophores by measuring fluorescent intensities across different bandwidths. To validate this new methodology, we visualized multiple fluorophore‐tagged organelle markers in our experimental cell system. We found that DsRed2‐cytochrome c oxidase and chromogranin A‐GFP proteins were targeted in INS‐1E cells to mitochondria and secretory granules by cryo‐CLEM, consistent with their respective well‐established intracellular localizations. Moreover, these fluorescent signals were clearly distinguishable from autofluorescence emanating from endogenous structures including insulin crystals and multilamellar bodies. Overall, our novel cryo‐CLEM methods open the door to the study of cellular phenomena and structures with a new degree of specificity

Three-dimensional analysis of mitochondrial crista ultrastructure in a patient with Leigh syndrome by in situ cryoelectron tomography

Mitochondrial diseases produce profound neurological dysfunction via mutations affecting mitochondrial energy production, including the relatively common Leigh syndrome (LS). We recently described an LS case caused by a pathogenic mutation in USMG5, encoding a small supernumerary subunit of mitochondrial ATP synthase. This protein is integral for ATP synthase dimerization, and patient fibroblasts revealed an almost total loss of ATP synthase dimers. Here, we utilize in situ cryoelectron tomography (cryo-ET) in a clinical case-control study of mitochondrial disease to directly study mitochondria within cultured fibroblasts from a patient with LS and a healthy human control subject. Through tomographic analysis of patient and control mitochondria, we find that loss of ATP synthase dimerization due to the pathogenic mutation causes profound disturbances of mitochondrial crista ultrastructure. Overall, this work supports the crucial role of ATP synthase in regulating crista architecture in the context of human disease

A YAC-based physical map of the mouse genome

A physical map of the mouse genome is an essential tool for both positional cloning and genomic sequencing in this key model system for biomedical research. Indeed, the construction of a mouse physical map with markers spaced at an average interval of 300 kb is one of the stated goals of the Human Genome Project. Here we report the results of a project at the Whitehead Institute/MIT Center for Genome Research to construct such a physical map of the mouse. We built the map by screening sequenced-tagged sites (STSs) against a large-insert yeast artificial chromosome (YAC) library and then integrating the STS-content information with a dense genetic map. The integrated map shows the location of 9,787 loci, providing landmarks with an average spacing of approximately 300 kb and affording YAC coverage of approximately 92% of the mouse genome. We also report the results of a project at the MRC UK Mouse Genome Centre targeted at chromosome X. The project produced a YAC-based map containing 619 loci (with 121 loci in common with the Whitehead map and 498 additional loci), providing especially dense coverage of this sex chromosome. The YAC-based physical map directly facilitates positional cloning of mouse mutations by providing ready access to most of the genome. More generally, use of this map in addition to a newly constructed radiation hybrid (RH) map provides a comprehensive framework for mouse genomic studies