Biology:Cell unroofing
Cell unroofing is any of various methods to isolate and expose the cell membrane of cells. Differently from the more common membrane extraction protocols performed with multiple steps of centrifugation (which goal is to separate the membrane fraction from a cell lysate), in cell unroofing the aim is to tear and preserve patches of the plasma membrane in order to perform in situ experiments using (microscopy and biomedical spectroscopy).
History
The first observation the bi-layer cell membrane was made in 1959[1] on a section of a cell using the electron microscope. But the first micrograph of the internal side of a cell dates back to 1977[2] by M.V. Nermut. Professor John Heuser made substantial contributions in the field, imaging the detailed internal structure of the membrane and the cytoskeleton bound to it with extensive use of the electron microscope.
It was only after the development of the atomic force microscope operated in liquid that it was possible to image the cell membranes in almost-physiological conditions[3] and to test its mechanical properties.[4]
Methods
- Freeze-fracturing of monolayers[5]
- Quick-freeze deep-etch electron microscopy and cryofixation[6]
- Sonication for atomic force microscopy[7]
- Single-cell unroofing[4]
See also
References
- ↑ Robertson, J.D. (1959). "The ultrastructure of cell membranes and their derivatives". Biochem. Soc. Symp..
- ↑ Nermut, M. V.; Williams, Lynn D. (1977). "Freeze-fracturing of monolayers (capillary layers) of cells, membranes and viruses: some technical considerations" (in en). Journal of Microscopy 110 (2): 121–132. doi:10.1111/j.1365-2818.1977.tb00023.x. ISSN 1365-2818. PMID 335072.
- ↑ Cho, Sang-Joon; Ahn, Jeonghun; Youn, Daehwan; Minakata, Shiho; Yoshimura, Azumi; Usukura, Jiro (2012-10-01). "Use of the unroofing technique for atomic force microscopic imaging of the intra-cellular cytoskeleton under aqueous conditions" (in en). Journal of Electron Microscopy 61 (5): 321–326. doi:10.1093/jmicro/dfs055. ISSN 0022-0744. PMID 22872282.
- ↑ 4.0 4.1 Galvanetto, Nicola (2018-12-01). "Single-cell unroofing: probing topology and nanomechanics of native membranes" (in en). Biochimica et Biophysica Acta (BBA) - Biomembranes 1860 (12): 2532–2538. doi:10.1016/j.bbamem.2018.09.019. ISSN 0005-2736. PMID 30273580.
- ↑ Nermut, M. V.; Williams, Lynn D. (1977). "Freeze-fracturing of monolayers (capillary layers) of cells, membranes and viruses: some technical considerations" (in en). Journal of Microscopy 110 (2): 121–132. doi:10.1111/j.1365-2818.1977.tb00023.x. ISSN 1365-2818. PMID 335072.
- ↑ Heuser, John (2000). "The Production of 'Cell Cortices' for Light and Electron Microscopy" (in en). Traffic 1 (7): 545–552. doi:10.1034/j.1600-0854.2000.010704.x. ISSN 1600-0854. PMID 11208142.
- ↑ Usukura, Jiro; Ito, Shuichi; Yagi, Akira; Narita, Akihiro; Usukura, Eiji (2016-06-07). "An Unroofing Method to Observe the Cytoskeleton Directly at Molecular Resolution Using Atomic Force Microscopy" (in en). Scientific Reports 6: 27472. doi:10.1038/srep27472. ISSN 2045-2322. PMID 27273367.
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