Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography

Wagner, Felix; Watanabe, Reika; Schampers, Ruud; Singh, Digvijay; Persoon, Hans; Schaffer, Miroslava; Fruhstorfer, Peter; Plitzko, Jürgen M.; Villa, Elizabeth

doi:10.1038/s41596-020-0320-x

Cited by 145 publications

(112 citation statements)

References 112 publications

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“…Cryo-ET image quality is highly sensitive to sample thickness and limited to specimens which are less than ~400 nm in depth (Rice et al, 2018). It is possible to overcome this by thinning the sample using FIB-milling but this multi-step procedure reduces the throughput for data collection (Wagner et al, 2020). We therefore decided to directly image the thin parts of neurites, first targeting Dm neurons as they are known to contain many narrow regions (Egger et al, 1997).…”

Section: Resultsmentioning

confidence: 99%

A cryo-ET study of microtubules in axons

Foster

2021

Preprint

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The microtubule cytoskeleton in axons plays key roles in intracellular transport and in defining cell shape. Despite many years of study of microtubules, many questions regarding their native architecture remain unanswered. Here, we performed cryo-electron tomography of mouse dorsal root ganglion (DRG) and Drosophila melanogaster (Dm) neurons and examined their microtubule ultrastructure in situ. We found that the microtubule minus and plus ends in DRG axons are structurally similar and frequently contact nearby components. The microtubules in DRG axons maintained a 13 protofilament (pf) architecture, even close to lattice break sites. In contrast, microtubules in Dm neurons had 12 or 13 pfs and we detected sites of pf number transition. The microtubule lumen in DRG axons is filled with globular microtubule inner proteins (MIPs). Our data suggest these have a defined structure, which is surprising given they are thought to contain the disordered protein MAP6. In summary, we reveal novel morphological and structural features of microtubules in their native environment.

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Section: Resultsmentioning

confidence: 99%

A cryo-ET study of microtubules in axons

Foster

2021

Preprint

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“…It is possible that our "diamond-shaped" form corresponds to, or related to the "diagonal square" form, which is thought to be different from the small square form. It would be necessary to conduct a cryo-FIB tomography 29,30 of native muscle fibers to make a clear distinction among these forms. Given that the basket-weave form is observed in the relaxed state, the diamond-shaped form is thought to be under mechanical stress imposed by the sliding between the thin and thick filaments.…”

Section: Discussionmentioning

confidence: 99%

“…It is possible that our “diamond-shaped” form corresponds to, or related to the “diagonal square” form, which is thought to be different from the small square form. It would be necessary to conduct a cryo-FIB tomography 29 , 30 of native muscle fibers to make a clear distinction among these forms.…”

Section: Discussionmentioning

confidence: 99%

Cryo-electron tomography of cardiac myofibrils reveals a 3D lattice spring within the Z-discs

Oda

Yanagisawa

2020

Commun Biol

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The Z-disc forms a boundary between sarcomeres, which constitute structural and functional units of striated muscle tissue. Actin filaments from adjacent sarcomeres are cross-bridged by α-actinin in the Z-disc, allowing transmission of tension across the myofibril. Despite decades of studies, the 3D structure of Z-disc has remained elusive due to the limited resolution of conventional electron microscopy. Here, we observed porcine cardiac myofibrils using cryo-electron tomography and reconstructed the 3D structures of the actin-actinin cross-bridging complexes within the Z-discs in relaxed and activated states. We found that the α-actinin dimers showed contraction-dependent swinging and sliding motions in response to a global twist in the F-actin lattice. Our observation suggests that the actin-actinin complex constitutes a molecular lattice spring, which maintains the integrity of the Z-disc during the muscle contraction cycle.

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“…Grids with Caulobacter were prepared using cryo-FIB milling as previously described using an Aquilos (Thermo Fisher Scientific) dual-beam SEM equipped 65,66 . Briefly, areas covered with a monolayer of cells were targeted first for coarse milling with an ion beam current of 0.10-0.50 nA, followed by fine milling using 10-50 pA. Lamella width was typically 10-12 um.…”

Section: Methodsmentioning

confidence: 99%

A modular platform for engineering function of natural and synthetic biomolecular condensates

Lasker

Boeynaems

et al. 2021

Preprint

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Phase separation is emerging as a universal principle for how cells use dynamic subcompartmentalization to organize biochemical reactions in time and space. Yet, whether the emergent physical properties of these biomolecular condensates are important for their biological function remains unclear. The intrinsically disordered protein PopZ forms membraneless condensates at the poles of the bacterium Caulobacter crescentus and selectively sequesters kinase-signaling cascades to regulate asymmetric cell division. By dissecting the molecular grammar underlying PopZ phase separation, we find that unlike many eukaryotic examples, where unstructured regions drive condensation, a structured domain of PopZ drives condensation, while conserved repulsive features of the disordered region modulate material properties. By generating rationally designed PopZ mutants, we find that the exact material properties of PopZ condensates directly determine cellular fitness, providing direct evidence for the physiological importance of the emergent properties of biomolecular condensates. Our work codifies a clear set of design principles illuminating how sequence variation in a disordered domain alters the function of a widely conserved bacterial condensate. We used these insights to repurpose PopZ as a modular platform for generating synthetic condensates of tunable function in human cells.

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Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography

Cited by 145 publications

References 112 publications

A cryo-ET study of microtubules in axons

A cryo-ET study of microtubules in axons

Cryo-electron tomography of cardiac myofibrils reveals a 3D lattice spring within the Z-discs

A modular platform for engineering function of natural and synthetic biomolecular condensates

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