Project description:The fibroblast growth factor FGF21 was labeled with molecularly defined gold nanoparticles (AuNPs), applied to human adipocytes, and imaged by cryo-electron tomography (cryo-ET). Most AuNPs were in pairs about 80 Å apart, on the outer cell surface. Pairs of AuNPs were also abundant inside the cells in clathrin-coated vesicles and endosomes. AuNPs were present but no longer paired in multivesicular bodies. FGF21 could thus be tracked along the endocytotic pathway. The methods developed here to visualize signaling coupled to endocytosis can be applied to a wide variety of cargo and may be extended to studies of other intracellular transactions.
Project description:Cryo-electron tomography (cryo-ET) provides three-dimensional (3D) structural information of bacteria preserved in a native, frozen-hydrated state. The typical low contrast of tilt-series images, a result of both the need for a low electron dose and the use of conventional defocus phase-contrast imaging, is a challenge for high-quality tomograms. We show that Zernike phase-contrast imaging allows the electron dose to be reduced. This limits movement of gold fiducials during the tilt series, which leads to better alignment and a higher-resolution reconstruction. Contrast is also enhanced, improving visibility of weak features. The reduced electron dose also means that more images at more tilt angles could be recorded, further increasing resolution.
Project description:Cryo-electron tomography (cryo-ET) has enabled high resolution three-dimensional(3D) structural analysis of virus and host cell interactions and many cell signaling events; these studies, however, have largely been limited to very thin, peripheral regions of eukaryotic cells or to small prokaryotic cells. Recent efforts to make thin, vitreous sections using cryo-ultramicrotomy have been successful, however,this method is technically very challenging and with many artifacts. Here, we report a simple and robust method for creating in situ, frozen-hydrated cell lamellas using a focused ion beam at cryogenic temperature (cryo-FIB), allowing access to any interior cellular regions of interest. We demonstrate the utility of cryo-FIB with high resolution 3D cellular structures from both bacterial cells and large mammalian cells. The method will not only facilitate high-throughput 3D structural analysis of biological specimens, but is also broadly applicable to sample preparation of thin films and surface materials without the need for FIB "lift-out".
Project description:Cryo-electron tomography (CET) is a well-established technique for imaging cellular and molecular structures at sub-nanometer resolution. As the method is limited to samples that are thinner than 500 nm, suitable sample preparation is required to attain CET data from larger cell volumes. Recently, cryo-focused ion beam (cryo-FIB) milling of plunge-frozen biological material has been shown to reproducibly yield large, homogeneously thin, distortion-free vitreous cross-sections for state-of-the-art CET. All eukaryotic and prokaryotic cells that can be plunge-frozen can be thinned with the cryo-FIB technique. Together with advances in low-dose microscopy, this has shifted the frontiers of in situ structural biology. In this protocol we describe the typical steps of the cryo-FIB technique, starting with fully grown cell cultures. Three recently investigated biological samples are given as examples.
Project description:The combination of cryo-microscopy and electron tomographic reconstruction has allowed us to determine the structure of one of the more complex viruses, intracellular mature vaccinia virus, at a resolution of 4-6 nm. The tomographic reconstruction allows us to dissect the different structural components of the viral particle, avoiding projection artifacts derived from previous microscopic observations. A surface-rendering representation revealed brick-shaped viral particles with slightly rounded edges and dimensions of approximately 360 x 270 x 250 nm. The outer layer was consistent with a lipid membrane (5-6 nm thick), below which usually two lateral bodies were found, built up by a heterogeneous material without apparent ordering or repetitive features. The internal core presented an inner cavity with electron dense coils of presumptive DNA-protein complexes, together with areas of very low density. The core was surrounded by two layers comprising an overall thickness of approximately 18-19 nm; the inner layer was consistent with a lipid membrane. The outer layer was discontinuous, formed by a periodic palisade built by the side interaction of T-shaped protein spikes that were anchored in the lower membrane and were arranged into small hexagonal crystallites. It was possible to detect a few pore-like structures that communicated the inner side of the core with the region outside the layer built by the T-shaped spike palisade.
Project description:Subcellular neighborhoods, comprising specific ratios of organelles and proteins, serve a multitude of biological functions and are of particular importance in secretory cells. However, the role of subcellular neighborhoods in insulin vesicle maturation is poorly understood. Here, we present single-cell multiple distinct tomogram acquisitions of β cells for in situ visualization of distinct subcellular neighborhoods that are involved in the insulin vesicle secretory pathway. We propose that these neighborhoods play an essential role in the specific function of cellular material. In the regions where we observed insulin vesicles, a measurable increase in both the fraction of cellular volume occupied by vesicles and the average size (diameter) of the vesicles was apparent as sampling moved from the area near the nucleus toward the plasma membrane. These findings describe the important role of the nanometer-scale organization of subcellular neighborhoods on insulin vesicle maturation.
Project description:The vault complex is the largest cellular ribonucleoprotein complex ever characterized and is present across diverse Eukarya. Despite significant information regarding the structure, composition and evolutionary conservation of the vault, little is know about the complex's actual biological function. To determine if intracellular vaults are morphologically similar to previously studied purified and recombinant vaults, we have used electron cryo-tomography to characterize the vault complexes found in the thin edges of primary human cells growing in tissue culture. Our studies confirm that intracellular vaults are similar in overall size and shape to purified and recombinant vaults previously analyzed. Results from subtomogram averaging indicate that densities within the vault lumen are not ordered, but randomly distributed. We also observe that vaults located in the extreme periphery of the cytoplasm predominately associate with granule-like structures and actin. Our ultrastructure studies augment existing biochemical, structural and genetic information on the vault, and provide important intracellular context for the ongoing efforts to understand the biological function of the native cytoplasmic vault.
Project description:Cilia play essential roles in normal human development and health; cilia dysfunction results in diseases such as primary ciliary dyskinesia (PCD). Despite their importance, the native structure of human cilia is unknown, and structural defects in the cilia of patients are often undetectable or remain elusive because of heterogeneity. Here we develop an approach that enables visualization of human (patient) cilia at high-resolution using cryo-electron tomography of samples obtained noninvasively by nasal scrape biopsy. We present the native 3D structures of normal and PCD-causing RSPH1-mutant human respiratory cilia in unprecedented detail; this allows comparisons of cilia structure across evolutionarily distant species and reveals the previously unknown primary defect and the heterogeneous secondary defects in RSPH1-mutant cilia. Our data provide evidence for structural and functional heterogeneity in radial spokes, suggest a mechanism for the milder RSPH1 PCD phenotype and demonstrate that cryo-electron tomography can be applied to human disease by directly imaging patient samples.
Project description:Cryo-electron tomography (cryoET) provides sub-nanometer protein structure within the dense cellular environment. Existing sample preparation methods are insufficient at accessing the plasma membrane and its associated proteins. Here, we present a correlative cryo-electron tomography pipeline optimally suited to image large ultra-thin areas of isolated basal and apical plasma membranes. The pipeline allows for angstrom-scale structure determination with sub-tomogram averaging and employs a genetically-encodable rapid chemically-induced electron microscopy visible tag for marking specific proteins within the complex cell environment. The pipeline provides fast, efficient, distributable, low-cost sample preparation and enables targeted structural studies of identified proteins at the plasma membrane of cells.
Project description:Cryo-electron tomography (cryoET) provides sub-nanometer protein structure within the dense cellular environment. Existing sample preparation methods are insufficient at accessing the plasma membrane and its associated proteins. Here, we present a correlative cryo-electron tomography pipeline optimally suited to image large ultra-thin areas of isolated basal and apical plasma membranes. The pipeline allows for angstrom-scale structure determination with subtomogram averaging and employs a genetically encodable rapid chemically-induced electron microscopy visible tag for marking specific proteins within the complex cellular environment. The pipeline provides efficient, distributable, low-cost sample preparation and enables targeted structural studies of identified proteins at the plasma membrane of mammalian cells.