iMEM: Isolation of Plasma Membrane for Cryoelectron Microscopy

Peitsch, Camille Françoise; Beckmann, Sven; Zuber, Benoît

doi:10.1016/j.str.2016.09.016

Cited by 5 publications

(3 citation statements)

References 33 publications

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“…To overcome this limitation, we used cryo-ET to observe the molecular-scale interactions between single triskelia in flat lattices on frozen cell membranes (Peitsch et al, 2016). Flat, domed, and spherical structures were observed, but precise segmentation of curved lattices was not possible due to the missing wedge feature of cryo-ET and the fixed geometry of our samples.…”

Section: Flat Lattices Are Flexibly Assembled With Loosely Connected ...mentioning

confidence: 99%

The structure and spontaneous curvature of clathrin lattices at the plasma membrane

et al. 2021

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Section: Flat Lattices Are Flexibly Assembled With Loosely Connected ...mentioning

confidence: 99%

The structure and spontaneous curvature of clathrin lattices at the plasma membrane

et al. 2021

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“…The second is fracturing: it consists on sandwiching the cells, freeze and separate the sandwich [6,7]; this allowed to achieve a more natural, life-like appearance of the samples. A variant of the fracturing method is the recently developed iMEM [8] that consist of isolating the cell membranes during the blotting step of a Cryo-EM grid preparation. The third uses sonic waves to break the body of the cells: in this way only the layer of membrane in contact with the substrate remains [9].…”

Section: Introductionmentioning

confidence: 99%

Single-cell unroofing: probing topology and nanomechanics of native membranes

Galvanetto

2018

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Cell membranes separate the cell interior from the external environment. They are constituted by a variety of lipids; their composition determines the dynamics of membrane proteins and affects the ability of the cells to adapt. Even though the study of model membranes allows to understand the interactions among lipids and the overall mechanics, little is known about these properties in native membranes. To combine topology and nanomechanics analysis of native membranes, I designed a method to investigate the plasma membranes isolated from a variety of single cells. Five cell types were chosen and tested, revealing 20% variation in membrane thickness. I probed the resistance of the isolated membranes to indent, finding their line tension and spreading pressure. These results show that membranes isolated from neurons are stiffer and less diffusive than brain cancer cell membranes. This method gives direct quantitative insights on the mechanics of native cell membranes.

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“…Thus, it would be more authentic if the pure plasma membranes were observed in situ by cryo-EM. Although traditional methods, for example, sucrose gradient centrifugation and membrane patch unroofing procedures, have been utilised for plasma membrane preparation( 13, 14 ), neither of them can mostly eliminate cellular content and fulfil the requirements of vitrification for cryo-ET, such as suitable thickness and homogenous fractions.…”

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confidence: 99%

Revealing the structure of somatic cell membranes by in situ cryo-electron tomography

Liu

Zhou

Zou

et al. 2022

Preprint

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The cell membrane, which separates the cell from the environment, plays a key role in signal transduction, energy conversion and substance transport. Although previous membrane models have successfully interpreted some functions of the cell membrane, no consensus has been reached for the lack of direct and in situ evidence. Here, we characterized the high-resolution 3D structure of 293T cell membranes in situ for the first time by cryo-electron tomography. Due to the excellent thickness of our cryo-samples, we could clearly observe membrane proteins with an average molecular weight of 100 kD. By analysing tomograms, we found that the total thickness of a 293T plasma membrane is approximately 20 nm and that there is a 4-nm lipid bilayer structure within the membrane. We observed that membrane proteins and protein complexes with a similar height (7-11 nm) are densely embedded in the ectoplasmic side of 293T plasma membranes, whereas membrane proteins aggregate to form islands with heights reaching dozens of nanometres on the cytoplasmic side. Additionally, we measured the average sizes of membrane proteins on the cytoplasmic side of 293T plasma membranes and found them to be approximately 7 nm in length and 4 nm in width. Moreover, if more precise structural information is obtained in future studies, we will identify the molecular interactions and detailed structures of membrane protein clusters that can be easily distinguished on a 293T cell membrane. Our work represents the first in situ structural characterization of a native somatic cell membrane with cryo-electron tomography and advances cell membrane structural studies from the model prediction stage to the real structure observation stage.

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iMEM: Isolation of Plasma Membrane for Cryoelectron Microscopy

Cited by 5 publications

References 33 publications

The structure and spontaneous curvature of clathrin lattices at the plasma membrane

The structure and spontaneous curvature of clathrin lattices at the plasma membrane

Single-cell unroofing: probing topology and nanomechanics of native membranes

Revealing the structure of somatic cell membranes by in situ cryo-electron tomography

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