scholarly journals Structural Cell Biology: Preparing Specimens for Cryo-Electron Tomography Using Focused-Ion-Beam Milling

2014 ◽  
Vol 20 (S3) ◽  
pp. 1222-1223
Author(s):  
Elizabeth Villa ◽  
Miroslava Schaffer ◽  
Ben Engel ◽  
Jürgen Plitzko ◽  
Wolfgang Baumeister
Author(s):  
Tobias Zachs ◽  
Andreas Schertel ◽  
João Medeiros ◽  
Gregor L Weiss ◽  
Jannik Hugener ◽  
...  

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Kanika Khanna ◽  
Javier Lopez-Garrido ◽  
Ziyi Zhao ◽  
Reika Watanabe ◽  
Yuan Yuan ◽  
...  

The study of bacterial cell biology is limited by difficulties in visualizing cellular structures at high spatial resolution within their native milieu. Here, we visualize Bacillus subtilis sporulation using cryo-electron tomography coupled with cryo-focused ion beam milling, allowing the reconstruction of native-state cellular sections at molecular resolution. During sporulation, an asymmetrically-positioned septum generates a larger mother cell and a smaller forespore. Subsequently, the mother cell engulfs the forespore. We show that the septal peptidoglycan is not completely degraded at the onset of engulfment. Instead, the septum is uniformly and only slightly thinned as it curves towards the mother cell. Then, the mother cell membrane migrates around the forespore in tiny finger-like projections, whose formation requires the mother cell SpoIIDMP protein complex. We propose that a limited number of SpoIIDMP complexes tether to and degrade the peptidoglycan ahead of the engulfing membrane, generating an irregular membrane front.


2014 ◽  
Vol 106 (2) ◽  
pp. 600a ◽  
Author(s):  
Elizabeth Villa ◽  
Miroslava Schaffer ◽  
Jürgen M. Plitzko ◽  
Wolfgang Baumeister

2018 ◽  
Author(s):  
Kanika Khanna ◽  
Javier Lopez-Garrido ◽  
Ziyi Zhao ◽  
Reika Watanabe ◽  
Yuan Yuan ◽  
...  

AbstractThe study of cell biology is limited by the difficulty in visualizing cellular structures at high spatial resolution within their native milieu. Here, we have visualized sporulation inBacillus subtilisusing cryo-electron tomography coupled with cryo-focused ion beam milling, a technique that allows the 3D reconstruction of cellular structures in near-native state at molecular resolution. During sporulation, an asymmetrically-positioned septum divides the cell into a larger mother cell and a smaller forespore. Subsequently, the mother cell phagocytoses the forespore in a process called engulfment, which entails a dramatic rearrangement of the peptidoglycan (PG) cell wall around the forespore. By imaging wild-type sporangia, engulfment mutants, and sporangia treated with PG synthesis inhibitors, we show that the initiation of engulfment does not entail the complete dissolution of the septal PG by the mother cell SpoIIDMP complex, as was previously thought. Instead, DMP is required to maintain a flexible septum that is uniformly and only slightly thinned at the onset of engulfment. Then, the mother cell membrane migrates around the forespore by forming tiny finger-like projections, the formation of which requires both SpoIIDMP and new PG synthesized ahead of the leading edge of the engulfing membrane. We propose a molecular model for engulfment membrane migration in which a limited number of SpoIIDMP complexes tether the membrane to and degrade the new PG ahead of the leading edge, thereby generating an irregular engulfing membrane front. Our data also reveal other structures that will provide a valuable resource for future mechanistic studies of endospore formation.


2019 ◽  
Author(s):  
Tobias Zachs ◽  
João M. Medeiros ◽  
Andreas Schertel ◽  
Gregor L. Weiss ◽  
Jannik Hugener ◽  
...  

AbstractCryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, with the unique ability to determine structures of macromolecular complexes in their cellular context. A major limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.


2020 ◽  
Vol 15 (6) ◽  
pp. 2041-2070 ◽  
Author(s):  
Felix R. Wagner ◽  
Reika Watanabe ◽  
Ruud Schampers ◽  
Digvijay Singh ◽  
Hans Persoon ◽  
...  

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Tobias Zachs ◽  
Andreas Schertel ◽  
João Medeiros ◽  
Gregor L Weiss ◽  
Jannik Hugener ◽  
...  

Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and determining structures of macromolecular complexes in their cellular context. A limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here, we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.


2013 ◽  
Vol 23 (5) ◽  
pp. 771-777 ◽  
Author(s):  
Elizabeth Villa ◽  
Miroslava Schaffer ◽  
Jürgen M Plitzko ◽  
Wolfgang Baumeister

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