Whole Cell Cryo-Electron Tomography Suggests Mitochondria Divide by Budding

2014 ◽  
Vol 20 (4) ◽  
pp. 1180-1187 ◽  
Author(s):  
Guo-Bin Hu
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Binary Fission ◽  
Related Protein ◽  
Mitochondrial Division ◽  
Whole Cell ◽  
Cryo Electron Tomography ◽  
Mitochondrial Networks ◽  
New Generation ◽  
Conventional Electron Microscopy

AbstractEukaryotes rely on mitochondrial division to guarantee that each new generation of cells acquires an adequate number of mitochondria. Mitochondrial division has long been thought to occur by binary fission and, more recently, evidence has supported the idea that binary fission is mediated by dynamin-related protein (Drp1) and the endoplasmic reticulum. However, studies to date have depended on fluorescence microscopy and conventional electron microscopy. Here, we utilize whole cell cryo-electron tomography to visualize mitochondrial division in frozen hydrated intact HeLa cells. We observe a large number of relatively small mitochondria protruding from and connected to large mitochondria or mitochondrial networks. Therefore, this study provides evidence that mitochondria divide by budding.

scholarly journals Cryo-electron tomography of cardiac myofibrils reveals a contraction-induced lattice twist in the Z-discs

2020 ◽  
Author(s):  
Toshiyuki Oda ◽  
Haruaki Yanagisawa
Keyword(s):  
Electron Microscopy ◽  
Muscle Contraction ◽  
Actin Filaments ◽  
Striated Muscle ◽  
Electron Tomography ◽  
3D Structure ◽  
Structural Flexibility ◽  
Cryo Electron Tomography ◽  
Contraction Cycle ◽  
Conventional Electron Microscopy

AbstractThe 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 been 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 actinactinin cross-bridging complexes within the Z-discs in relaxed and activated states. We found that the α-actinin showed a contraction-induced swing motion along with a global twist in the actin lattice. Our observation suggests that the elasticity and the integrity of the Z-disc during the muscle contraction cycle are maintained by the structural flexibility within the actin-actinin complex.

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

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Toshiyuki Oda ◽  
Haruaki Yanagisawa
Keyword(s):  
Electron Microscopy ◽  
Muscle Contraction ◽  
Muscle Tissue ◽  
Actin Filaments ◽  
Striated Muscle ◽  
Electron Tomography ◽  
3D Structure ◽  
Cryo Electron Tomography ◽  
Contraction Cycle ◽  
Conventional Electron Microscopy

Abstract 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.

scholarly journals Lattice Micropatterning of Electron Microscopy Grids for Improved Cellular Cryo-Electron Tomography Throughput

2021 ◽  
Vol 120 (3) ◽  
pp. 173a
Author(s):  
Leeya Engel ◽  
Claudia G. Vasquez ◽  
Elizabeth A. Montabana ◽  
Belle M. Sow ◽  
Marcin P. Walkiewicz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Cryo Electron Tomography

scholarly journals An Economical, Portable Manual Cryogenic Plunge Freezer for the Preparation of Vitrified Biological Samples for Cryogenic Electron Microscopy

2020 ◽  
Vol 26 (3) ◽  
pp. 413-418
Author(s):  
Jamie S. Depelteau ◽  
Gert Koning ◽  
Wen Yang ◽  
Ariane Briegel
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Particle Analysis ◽  
Bacterial Cells ◽  
Cellular Processes ◽  
Cryo Electron Tomography ◽  
Specialized Equipment ◽  
Improved Design ◽  
Commercial Equipment ◽  
Local Machine

AbstractVisualizing biological structures and cellular processes in their native state is a major goal of many scientific laboratories. In the past 20 years, the technique of preserving samples by vitrification has greatly expanded, specifically for use in cryogenic electron microscopy (cryo-EM). Here, we report on improvements in the design and use of a portable manual cryogenic plunge freezer that is intended for use in laboratories that are not equipped for the cryopreservation of samples. The construction of the instrument is economical, can be produced by a local machine shop without specialized equipment, and lowers the entry barriers for newcomers with a reliable alternative to costly commercial equipment. The improved design allows for successful freezing of isolated proteins for single particle analysis as well as bacterial cells for cryo-electron tomography. With this instrument, groups will be able to prepare vitreous samples whenever and wherever necessary, which can then be imaged at local or national cryo-EM facilities.

scholarly journals In situ structure of virus capsids within cell nuclei by correlative light and cryo-electron tomography

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Swetha Vijayakrishnan ◽  
Marion McElwee ◽  
Colin Loney ◽  
Frazer Rixon ◽  
David Bhella
Keyword(s):  
Electron Microscopy ◽  
Structure Determination ◽  
Electron Tomography ◽  
3D Structure ◽  
Three Dimensional ◽  
Cell Nuclei ◽  
Nuclear Egress ◽  
Virus Capsids ◽  
Cryo Electron Tomography

Abstract Cryo electron microscopy (cryo-EM), a key method for structure determination involves imaging purified material embedded in vitreous ice. Images are then computationally processed to obtain three-dimensional structures approaching atomic resolution. There is increasing interest in extending structural studies by cryo-EM into the cell, where biological structures and processes may be imaged in context. The limited penetrating power of electrons prevents imaging of thick specimens (> 500 nm) however. Cryo-sectioning methods employed to overcome this are technically challenging, subject to artefacts or involve specialised and costly equipment. Here we describe the first structure of herpesvirus capsids determined by sub-tomogram averaging from nuclei of eukaryotic cells, achieved by cryo-electron tomography (cryo-ET) of re-vitrified cell sections prepared using the Tokuyasu method. Our reconstructions confirm that the capsid associated tegument complex is present on capsids prior to nuclear egress. We demonstrate that this method is suited to both 3D structure determination and correlative light/electron microscopy, thus expanding the scope of cryogenic cellular imaging.

scholarly journals Whole Cell Cryo-Electron Tomography Reveals Distinct Disassembly Intermediates of Vaccinia Virus

PLoS ONE ◽  
2007 ◽  
Vol 2 (5) ◽  
pp. e420 ◽  
Author(s):  
Marek Cyrklaff ◽  
Alexandros Linaroudis ◽  
Marius Boicu ◽  
Petr Chlanda ◽  
Wolfgang Baumeister ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Electron Tomography ◽  
Whole Cell ◽  
Cryo Electron Tomography

scholarly journals Post-correlation on-lamella cryo-CLEM reveals the membrane architecture of lamellar bodies

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Steffen Klein ◽  
Benedikt H. Wimmer ◽  
Sophie L. Winter ◽  
Androniki Kolovou ◽  
Vibor Laketa ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Current Knowledge ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
A549 Cells ◽  
Airway Epithelial Cells ◽  
Lamellar Bodies ◽  
Membrane Structures ◽  
Alveolar Cells ◽  
Cryo Electron Tomography

AbstractLamellar bodies (LBs) are surfactant-rich organelles in alveolar cells. LBs disassemble into a lipid-protein network that reduces surface tension and facilitates gas exchange in the alveolar cavity. Current knowledge of LB architecture is predominantly based on electron microscopy studies using disruptive sample preparation methods. We established and validated a post-correlation on-lamella cryo-correlative light and electron microscopy approach for cryo-FIB milled cells to structurally characterize and validate the identity of LBs in their unperturbed state. Using deconvolution and 3D image registration, we were able to identify fluorescently labeled membrane structures analyzed by cryo-electron tomography. In situ cryo-electron tomography of A549 cells as well as primary Human Small Airway Epithelial Cells revealed that LBs are composed of membrane sheets frequently attached to the limiting membrane through “T”-junctions. We report a so far undescribed outer membrane dome protein complex (OMDP) on the limiting membrane of LBs. Our data suggest that LB biogenesis is driven by parallel membrane sheet import and by the curvature of the limiting membrane to maximize lipid storage capacity.

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