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.

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

2020 ◽  
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

Lamellar 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.

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.

scholarly journals A cryo–electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane

2021 ◽  
Vol 7 (13) ◽  
pp. eabc6345
Author(s):  
Shrawan Kumar Mageswaran ◽  
Wei Yuan Yang ◽  
Yogaditya Chakrabarty ◽  
Catherine M. Oikonomou ◽  
Grant J. Jensen
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Electron Tomography ◽  
Membrane Damage ◽  
Light And Electron Microscopy ◽  
Actin Dynamics ◽  
Endosomal Sorting ◽  
Plasma Membrane Damage ◽  
Cryo Electron Tomography ◽  
Vacuolar Protein

Cryo–electron tomography (cryo-ET) provides structural context to molecular mechanisms underlying biological processes. Although straightforward to implement for studying stable macromolecular complexes, using it to locate short-lived structures and events can be impractical. A combination of live-cell microscopy, correlative light and electron microscopy, and cryo-ET will alleviate this issue. We developed a workflow combining the three to study the ubiquitous and dynamic process of shedding in response to plasma membrane damage in HeLa cells. We found filopodia-like protrusions enriched at damage sites and acting as scaffolds for shedding, which involves F-actin dynamics, myosin-1a, and vacuolar protein sorting 4B (a component of the ‘endosomal sorting complex required for transport’ machinery). Overall, shedding is more complex than current models of vesiculation from flat membranes. Its similarities to constitutive shedding in enterocytes argue for a conserved mechanism. Our workflow can also be adapted to study other damage response pathways and dynamic cellular events.

scholarly journals Effects of the cationic protein poly-L-arginine on airway epithelial cellsin vitro

2002 ◽  
Vol 11 (3) ◽  
pp. 141-148 ◽  
Author(s):  
Shahida Shahana ◽  
Caroline Kampf ◽  
Godfried M. Roomans
Keyword(s):  
Electron Microscopy ◽  
Epithelial Cells ◽  
Cell Membrane ◽  
Cell Damage ◽  
Membrane Damage ◽  
Light And Electron Microscopy ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Cationic Protein ◽  
Induced Apoptosis

Background: Allergic asthma is associated with an increased number of eosinophils in the airway wall. Eosinophils secrete cationic proteins, particularly major basic protein (MBP).Aim: To investigate the effect of synthetic cationic polypeptides such as poly-L-arginine, which can mimic the effect of MBP, on airway epithelial cells.Methods: Cultured airway epithelial cells were exposed to poly-L-arginine, and effects were determined by light and electron microscopy.Results: Poly-L-arginine induced apoptosis and necrosis. Transmission electron microscopy showed mitochondrial damage and changes in the nucleus. The tight junctions were damaged, as evidenced by penetration of lanthanum. Scanning electron microscopy showed a damaged cell membrane with many pores. Microanalysis showed a significant decrease in the cellular content of magnesium, phosphorus, sodium, potassium and chlorine, and an increase in calcium. Plakoglobin immunoreactivity in the cell membrane was decreased, indicating a decrease in the number of desmosomes.Conclusions: The results point to poly-L-arginine induced membrane damage, resulting in increased permeability, loss of cell-cell contacts and generalized cell damage.

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

Correlated 3D Light and Electron Microscopy of Large, Complex Structures: Analysis of Transverse Tubules in Heart Failure

1998 ◽  
Vol 4 (S2) ◽  
pp. 440-441
Author(s):  
Maryann E. Martone ◽  
Andrea Thor ◽  
Stephen J. Young ◽  
Mark H. Ellisman.
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
Structural Features ◽  
Electron Microscopic Level ◽  
Specimen Preparation ◽  
Large Sample Size ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron

Light microscopic imaging has experienced a renaissance in the past decade or so, as new techniques for high resolution 3D light microscopy have become readily available. Light microscopic (LM) analysis of cellular details is desirable in many cases because of the flexibility of staining protocols, the ease of specimen preparation and the relatively large sample size that can be obtained compared to electron microscopic (EM) analysis. Despite these advantages, many light microscopic investigations require additional analysis at the electron microscopic level to resolve fine structural features.High voltage electron microscopy allows the use of relatively thick sections compared to conventional EM and provides the basis for excellent new methods to bridge the gap between microanatomical details revealed by LM and EM methods. When combined with electron tomography, investigators can derive accurate 3D data from these thicker specimens. Through the use of correlated light and electron microscopy, 3D reconstructions of large cellular or subcellular structures can be obtained with the confocal microscope,

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.

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