Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies

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

doi:10.1088/1361-6439/ab419a

Cited by 32 publications

(34 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recently, photo‐micropatterning of EM grids enabled controlled positioning of cells with predictable actin organization 83,84 . By applying cryo‐FIB milling to a RP1E cell grown on a crossbow micropattern, actin transverse arcs and internal stress fibers could be targeted by cryo‐ET 83 .…”

Section: The Architecture Of the Actin Cytoskeletonmentioning

confidence: 99%

Three‐dimensional organization of the cytoskeleton: A cryo‐electron tomography perspective

2020

View full text Add to dashboard Cite

Traditionally, structures of cytoskeletal components have been studied ex situ, that is, with biochemically purified materials. There are compelling reasons to develop approaches to study them in situ in their native functional context. In recent years, cryo-electron tomography emerged as a powerful method for visualizing the molecular organization of unperturbed cellular landscapes with the potential to attain near-atomic resolution. Here, we review recent works on the cytoskeleton using cryo-electron tomography, demonstrating the power of in situ studies. We also highlight the potential of this method in addressing important questions pertinent to the field of cytoskeletal biomechanics. K E Y W O R D S actin filaments, cryo-electron tomography, in situ architecture, intermediate filaments, microtubules Abbreviations: 3D, three-dimensional; ATP, adenosine triphosphate;

show abstract

Section: The Architecture Of the Actin Cytoskeletonmentioning

confidence: 99%

Three‐dimensional organization of the cytoskeleton: A cryo‐electron tomography perspective

2020

View full text Add to dashboard Cite

show abstract

“…Since EM grid micropatterning shows great promise for further simplified and more efficient cell seeding (Engel et al, 2019;Toro-nahuelpan et al, 2019) we designed grid holders that are also compatible with micropatterning approaches using a standard, noncommercial setup. Specifically, high-throughput patterning on EM grids requires grid holders to provide a low, stable and even support of grids for controlled ablation of the Polyethylene glycol(PEG)-brush layer, which is applied to EM grids to render them nonadherent.…”

Section: Micropatterning-compatible Grid Holdersmentioning

confidence: 99%

“…Hence, a significant level of user experience and a steady hand are required to achieve sufficient throughput, an appropriate cell density and to ensure minimal damage to the grids during specimen preparation. A notable improvement for controllable cell seeding conditions on grids was recently introduced by EM grid compatible substrate micropatterning approaches (Engel et al, 2019;Toro-nahuelpan et al, 2019). Here, only defined areas on grids are made adherent or are specifically functionalized with extracellular matrix (ECM) proteins, allowing one to precisely control the positioning, shape, and size of adherent cells.…”

Section: Introductionmentioning

confidence: 99%

3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy

Fäßler

Zens

Hauschild

et al. 2020

Preprint

View full text Add to dashboard Cite

Cryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micropatterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications. Design and 3D-printing of grid holdersGrid holders were designed in Autodesk Fusion 360 (free education license) and printed with either an Original Prusa MINI (Prusa Research) or BLV mgn Cube (open source 3D printer project). Individual grid holders were printed in a matter of minutes using 100% infill. For printing, either PLA (Prusa Research) or PETG (Filament PM) filaments were used. Grid holders were sterilized with perform® classic alcohol EP and UV irradiation prior to use in cell culture experiments. Grid holders have been re-used up to 15 times without any signs of negative effects on sample preparation. Plans for the presented grid holders are available online at https://schurlab.ist.ac.at/downloads with a creative commons CC BY-NC-SA 4.0 license. Cell Culture and cell seedingWildtype B16-F1 melanoma and wildtype NIH 3T3 cells, as well as HeLa cells expressing cytosolic mCherry were kindly provided by Klemens Rottner (Technical University Braunschweig, Helmholtz Centre for Infection Research) and Michael Sixt (IST Austria), respectively. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM GlutaMAX, ThermoFischer Scientific, #31966047), supplemented with 10% (v/v) fetal bovine serum (ThermoFischer Scientific, #10270106) and 1% (v/v) penicillin-streptomycin (ThermoFischer Scientific, #15070063). Cells were incubated at 37°C and 5% CO2. Either 200 mesh gold holey carbon grids (R2/2; Quantifoil Micro Tools) or 200 mesh gold grids (Science Services, #G200-AU) coated with continuous Formvar film (0.75%) were used. Throughout all cell culture experiments Dumont tweezers, medical grade, style 5 and style 7 were used. Prior to seeding of cells, grids were glow discharged in an ELMO glow discharge unit (Cordouan Technologies) for 2min (holey carbon), or 30s (formvar), either directly in the specific grid holders or on Parafilm. Grids ...

show abstract

“…Optional: a silicon stencil can be used to keep the grid on place 11 . and cryo-ET please refer to 5,10 . o Grids should be stored in sealed boxes in liquid nitrogen until use.…”

Section: General Remarks and Guidelines For Micropatterningmentioning

confidence: 99%

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

Toro-Nahuelpan

Zagoriy

Senger

et al. 2019

Preprint

View full text Add to dashboard Cite

Micromachining by cryo-focused ion beam (FIB) milling coupled to cryo-electron tomography (ET) enables visualization of macromolecules directly inside cells. Yet, spatial control of cell adhesion on electron microscopy (EM) grids remains a bottleneck in the specimen preparation pipeline. This protocol describes a contactless and mask-free photo-micropatterning of EM grids for site-specific deposition of extracellular matrix-related proteins. We achieved accurate and reproducible cell positioning, leading to optimized preparations for cryo-FIB milling. We tested HeLa and RPE1 cell lines on various grid types (gold or titanium mesh coated with SiO2, gold or carbon films). Briefly, grids were passivated with an anti-fouling agent, followed by controlled ablation of the passivation layer, and further functionalization with fibronectin. The micropatterning procedure takes ~3 h. Employing micropatterning to produce complex shapes generated a predictable intracellular organization, allowing direct correlation between cellular architecture and in-cell 3D-structural characterization of the underlying machinery at molecular resolution.

show abstract

Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies

Abstract: Fig. S1: Epithelial cells are viable on continuous carbon films and EM grids. Left: 10x phase micrograph of MDCKII cells on untreated continuous carbon film. Right: Lifeact GFP MDCKII cells plated on EM grids treated with fibronectin, imaged at 10x GFP.

Cited by 32 publications

References 50 publications

Three‐dimensional organization of the cytoskeleton: A cryo‐electron tomography perspective

Three‐dimensional organization of the cytoskeleton: A cryo‐electron tomography perspective

3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy

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

Contact Info

Product

Resources

About