scholarly journals Actin Organization in Cells Responding to a Perforated Surface, Revealed by Live Imaging and Cryo-Electron Tomography

Structure ◽  
2016 ◽  
Vol 24 (7) ◽  
pp. 1031-1043 ◽  
Author(s):  
Marion Jasnin ◽  
Mary Ecke ◽  
Wolfgang Baumeister ◽  
Günther Gerisch
Keyword(s):  
Electron Tomography ◽  
Live Imaging ◽  
Actin Organization ◽  
Cryo Electron Tomography ◽  
In Cells

scholarly journals Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Florian Fäßler ◽  
Georgi Dimchev ◽  
Victor-Valentin Hodirnau ◽  
William Wan ◽  
Florian K. M. Schur
Keyword(s):  
Actin Filament ◽  
Electron Tomography ◽  
Active Conformation ◽  
Structural Rearrangements ◽  
Cryo Electron Tomography ◽  
Complex Structural ◽  
Filament Networks ◽  
Subtomogram Averaging ◽  
Resolution Structure ◽  
In Cells

AbstractThe actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0 Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Notably, our model reveals a previously undescribed set of interactions of the Arp2/3 complex with the mother filament, significantly different to the previous branch junction model. Our structure also indicates a central role for the ArpC3 subunit in stabilizing the active conformation.

scholarly journals Novel cryo-electron tomography structure of Arp2/3 complex in cells reveals mechanisms of branch formation

2020 ◽  
Author(s):  
Florian Fäßler ◽  
Georgi Dimchev ◽  
Victor-Valentin Hodirnau ◽  
William Wan ◽  
Florian KM Schur
Keyword(s):  
Actin Filament ◽  
Electron Tomography ◽  
Cryo Electron Tomography ◽  
Branch Formation ◽  
Complex Structural ◽  
Filament Networks ◽  
Subtomogram Averaging ◽  
Resolution Structure ◽  
In Cells

AbstractThe actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Our structure indicates a central role for the ArpC3 subunit in stabilizing the active conformation and suggests that in the branch junction relocation of the ArpC5 N-terminus and the C-terminal tail of Arp3 is important to fix Arp2 and Arp3 in an actin dimer-like conformation. Notably, our model of the branch junction in cells significantly differs from the previous in vitro branch junction model.

scholarly journals Microtubule architecture in vitro and in cells revealed by cryo-electron tomography

2018 ◽  
Vol 74 (6) ◽  
pp. 572-584 ◽  
Author(s):  
Joseph Atherton ◽  
Melissa Stouffer ◽  
Fiona Francis ◽  
Carolyn A. Moores
Keyword(s):  
Molecular Motors ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Structural Heterogeneity ◽  
Dimensional Structure ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Cryo Electron Tomography ◽  
In Cells

The microtubule cytoskeleton is involved in many vital cellular processes. Microtubules act as tracks for molecular motors, and their polymerization and depolymerization can be harnessed to generate force. The structures of microtubules provide key information about the mechanisms by which their cellular roles are accomplished and the physiological context in which these roles are performed. Cryo-electron microscopy allows the visualization of in vitro-polymerized microtubules and has provided important insights into their overall morphology and the influence of a range of factors on their structure and dynamics. Cryo-electron tomography can be used to determine the unique three-dimensional structure of individual microtubules and their ends. Here, a previous cryo-electron tomography study of in vitro-polymerized GMPCPP-stabilized microtubules is revisited, the findings are compared with new tomograms of dynamic in vitro and cellular microtubules, and the information that can be extracted from such data is highlighted. The analysis shows the surprising structural heterogeneity of in vitro-polymerized microtubules. Lattice defects can be observed both in vitro and in cells. The shared ultrastructural properties in these different populations emphasize the relevance of three-dimensional structures of in vitro microtubules for understanding microtubule cellular functions.

scholarly journals In situ Microfluidic Cryofixation for Cryo Focused Ion Beam Milling and Cryo Electron Tomography

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marie Fuest ◽  
Miroslava Schaffer ◽  
Giovanni Marco Nocera ◽  
Rodrigo I. Galilea-Kleinsteuber ◽  
Jan-Erik Messling ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Light Microscope ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Live Imaging ◽  
Cryo Electron Tomography

AbstractWe present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5 µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.

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