Structural analysis of multicellular organisms with cryo-electron tomography

Abstract Cryo-focused ion beam milling of frozen hydrated cells for the production of thin lamellas in combination with cryo-electron tomography (cryo-ET) has yielded unprecedented insights into the cell interior. This method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, it is only suitable for cells that can be vitrified by plunge freezing (<10 μm). Multicellular organisms and tissues are considerably thicker and high-pressure freezing is required to ensure optimal preservation. Here, we describe a preparation method for extracting lamellas from high pressure frozen samples with a new cryo-gripper tool. This in situ lift-out technique at cryo-temperatures enables cryo-ET to be performed on multicellular organisms and tissue, extending the range of applications for in situ structural biology.

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Structural analysis of Mycoplasma pneumoniae by cryo-electron tomography

Journal of Structural Biology ◽

10.1016/j.jsb.2006.04.010 ◽

2006 ◽

Vol 156 (2) ◽

pp. 342-354 ◽

Cited By ~ 73

Author(s):

Anja Seybert ◽

Richard Herrmann ◽

Achilleas S. Frangakis

Keyword(s):

Structural Analysis ◽

Mycoplasma Pneumoniae ◽

Electron Tomography ◽

Cryo Electron Tomography

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Structural Analysis of Hemagglutinin-Induced Hemifusion by Volta Phase-Plate Cryo-Electron Tomography

Biophysical Journal ◽

10.1016/j.bpj.2015.11.1377 ◽

2016 ◽

Vol 110 (3) ◽

pp. 250a-251a

Author(s):

Petr Chlanda ◽

Elena Mekhedov ◽

Hang Waters ◽

Cindi L. Schwartz ◽

Elizabeth R. Fischer ◽

...

Keyword(s):

Structural Analysis ◽

Electron Tomography ◽

Phase Plate ◽

Cryo Electron Tomography

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Developments in cryo-electron tomography for in situ structural analysis

Archives of Biochemistry and Biophysics ◽

10.1016/j.abb.2015.04.006 ◽

2015 ◽

Vol 581 ◽

pp. 78-85 ◽

Cited By ~ 18

Author(s):

Anna Dubrovsky ◽

Simona Sorrentino ◽

Jan Harapin ◽

K. Tanuj Sapra ◽

Ohad Medalia

Keyword(s):

Structural Analysis ◽

Electron Tomography ◽

Cryo Electron Tomography

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A lamin A/C variant causing striated muscle disease provides insights into filament organization

10.1101/2020.10.20.347088 ◽

2020 ◽

Author(s):

Rafael Kronenberg-Tenga ◽

Meltem Tatli ◽

Matthias Eibauer ◽

Wei Wu ◽

Ji-Yeon Shin ◽

...

Keyword(s):

Structural Analysis ◽

Striated Muscle ◽

Electron Tomography ◽

Nuclear Lamina ◽

Muscle Disease ◽

Relative Reduction ◽

Embryonic Fibroblasts ◽

Thick Filaments ◽

Cryo Electron Tomography

AbstractThe LMNA gene encodes the A-type lamins that polymerize into ~3.5 nm thick filaments, and together with B-type lamins and lamin binding proteins form the nuclear lamina. Mutations in LMNA are associated with a wide variety of pathologies. In this study, we analyzed the nuclear lamina of embryonic fibroblasts from LmnaH222P/H222P mice, which develop cardiomyopathy and muscular dystrophy. Although the organization of the lamina appeared unaltered, there were changes in chromatin and B-type lamin expression. An increase in nuclear size and consequently a relative reduction in heterochromatin near the lamina allowed for a higher resolution structural analysis of lamin filaments using cryo-electron tomography. This was most apparent when visualizing lamin filaments in situ, and using a nuclear extraction protocol. Averaging of individual segments of filaments in LmnaH222P/H222P mouse fibroblasts resolved two-polymers that constitute the mature filaments. Our findings provide better views of the organization of lamin filaments and the effect of a striated muscle disease-causing mutation on nuclear structure.

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