Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies

Engel, Leeya; Gaietta, Guido; Dow, Liam P.; Swift, Mark F.; Pardon, Gaspard; Volkmann, Niels; Weis, William I.; Hanein, Dorit; Pruitt, Beth L.

doi:10.1101/657072

Cited by 2 publications

(2 citation statements)

References 49 publications

(43 reference statements)

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“…EM grids (UltrAuFoil R2/2 Au 200 mesh Q250AR2A and Quantifoil R2/2 Au 200 mesh Q2100AR2) were micropatterned using the Alveole PRIMO micropatterning system [20][21][22] . Unpatterned grids were placed holey film up on silicone sheeting (Specialty Manufacturing Inc.) on glass coverslips to prevent them from moving during exposure to plasma and incubation.…”

Section: Micropatterning Of Em Gridsmentioning

confidence: 99%

Cryo-electron tomography reveals the structural diversity of cardiac proteins in their cellular context

Woldeyes,

Nishiga,

Vander Roest

et al. 2023

Preprint

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Cardiovascular diseases are a leading cause of death worldwide, but our understanding of the underlying mechanisms is limited, in part because of the complexity of the cellular machinery that controls the heart muscle contraction cycle. Cryogenic electron tomography (cryo-ET) provides a way to visualize diverse cellular machinery while preserving contextual information like subcellular localization and transient complex formation, but this approach has not been widely applied to the study of heart muscle cells (cardiomyocytes). Here, we deploy a platform for studying cardiovascular disease by combining cryo-ET with human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). After developing a cryoET workflow for visualizing macromolecules in hiPSC-CMs, we reconstructed sub-nanometer resolution structures of the human thin filament, a central component of the contractile machinery. We also visualized a previously unobserved organization of a regulatory complex that connects muscle contraction to calcium signaling (the troponin complex), highlighting the value of our approach for interrogating the structures of cardiac proteins in their cellular context.

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Section: Micropatterning Of Em Gridsmentioning

confidence: 99%

Cryo-electron tomography reveals the structural diversity of cardiac proteins in their cellular context

Woldeyes,

Nishiga,

Vander Roest

et al. 2023

Preprint

Self Cite

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“…to combine the multiple Airyscan 32-detector array images into deconvolved final images with high SNR and resolution. 12 Widefield microscopy imaging Epifluorescence images ( Fig. 2d inset, Supplementary Fig.…”

Section: Comparison Between the Two Patterning Approachesmentioning

confidence: 99%

Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies

Toro-Nahuelpan

Zagoriy

Senger

et al. 2019

Preprint

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Spatially-controlled cell adhesion on electron microscopy (EM) supports remains a bottleneck in specimen preparation for cellular cryo-electron tomography. Here, we describe contactless and maskfree photo-micropatterning of EM grids for site-specific deposition of extracellular matrix-related proteins. We attained refined cell positioning for micromachining by cryo-focused ion beam milling.Complex patterns generated predictable intracellular organization, allowing direct correlation between cell architecture and in-cell 3D-structural characterization of the underlying molecular machinery. MainIn parallel to the ongoing resolution revolution in cryo-electron microscopy for macromolecular structure determination 1 , cryo-electron tomography (ET) has matured to reveal the molecular sociology in situ sensu stricto 2-4 . Yet, cryo-ET of adherent mammalian cells can only be directly performed on their thin peripheries (< 300 nm). To reveal function-related structural variation, conformational states and function-related assemblies of macromolecules at the cell interior, thinning by advanced cryo-focused ion beam (FIB) has proved an optimal, artifact-free preparation method 2,5,6 .Specimen preparation for cellular cryo-ET, whether performed directly on thin cellular peripheries or following cryo-FIB micromachining, involves seeding of adherent cells directly on EM grids made of a biocompatible metal, e.g. gold. Standard EM grids are 3 mm diameter metal meshes overlaid with a delicate perforated thin film. Cells are typically allowed to spread, subjected to genetic or molecular perturbation to represent different physiological settings to be examined in molecular detail, that are then arrested by vitrification 7 . For cells to be thinned by cryo-FIB, they must be positioned roughly at the center of an individual grid square (Fig. 1a) 6 , within a few squares away from the grid center. Whilst the first requirement is necessary to allow access to cellular material for ablation by the FIB, the latter is posed by the subsequent requirement of stage tilt in the transmission electron microscope (TEM) for

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Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies

Cited by 2 publications

References 49 publications

Cryo-electron tomography reveals the structural diversity of cardiac proteins in their cellular context

Cryo-electron tomography reveals the structural diversity of cardiac proteins in their cellular context

Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies

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