Visualization of proteins in intact cells with a clonable tag for electron microscopy

Diestra, Elia; Fontana, Juan; Guichard, Paul; Marco, Sergio; Risco, Cristina

doi:10.1016/j.jsb.2008.11.009

Cited by 90 publications

(93 citation statements)

References 49 publications

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“…AmiA-GFP remains dispersed in the periplasm throughout the cell cycle, and AmiC-GFP remains dispersed only in nondividing cells. In contrast, AmiC-GFP was concentrated mostly at the septal ring in dividing cells, as confirmed by an electron microscopy analysis of the metallothionein-tagged protein (55).…”

Section: Amia Amib and Amic N-acetylmuramoyl-l-alanine Amidasesmentioning

confidence: 57%

“…Upstream of the hemF gene at centisome 55, an open reading frame codes for a 32-kDa protein homologous to the CwlB amidase from B. subtilis (262). The designation amiA is now used for the encoding gene (91) and must not be confused with the same previously proposed designation (261) for the 39-kDa amidase (197,283).…”

Section: Amia Amib and Amic N-acetylmuramoyl-l-alanine Amidasesmentioning

confidence: 99%

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Peptidoglycan Hydrolases of Escherichia coli

Heijenoort

2011

Microbiol Mol Biol Rev

187

233

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SUMMARY The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway.

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Section: Amia Amib and Amic N-acetylmuramoyl-l-alanine Amidasesmentioning

confidence: 57%

Section: Amia Amib and Amic N-acetylmuramoyl-l-alanine Amidasesmentioning

confidence: 99%

Peptidoglycan Hydrolases of Escherichia coli

Heijenoort

2011

Microbiol Mol Biol Rev

187

233

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“…Seventh, it will be necessary to use electron microscopy to improve the resolution of the information obtained using confocal light immunofluorescence microscopy of GFP-labeled proteins. A recent and important development in this area is the introduction of a new tag that can be detected using electron microscopy based on the use of the metal-binding protein metallothionein (Diestra et al 2009). Finally, the various freeze-fracture techniques, especially in association with immunocytochemistry, will continue to be a powerful tool in the analysis of the structure of the cytoskeleton and various membranes.…”

Section: Perspectivesmentioning

confidence: 99%

Structural organization of Trypanosoma cruzi

Souza

2009

Mem. Inst. Oswaldo Cruz

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“…GFP is 27 kDa), but many of them are too small for direct in situ visualization using cryo-ET. Therefore, for EM visualization, small clonable tags are often specifically linked to additional electron-dense probes, such as metal ion binding by metallothionein (7 kDa) (16,18), or tags such as MiniSOG that catalyze the photooxidation of diaminobenzidine into a localized precipitate that can be visualized by OsO 4 treatment (19,20). Despite these recent advances, the techniques used are still limited and/or are not compatible with cryo-EM, because of low contrast, toxicity of metal ion solutions, and/or the requirement to chemically fix and post-stain the cells.…”

mentioning

confidence: 99%

In Situ Localization of N and C Termini of Subunits of the Flagellar Nexin-Dynein Regulatory Complex (N-DRC) Using SNAP Tag and Cryo-electron Tomography

Song

Awata

Tritschler

et al. 2015

Journal of Biological Chemistry

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Background: Techniques to localize proteins in situ at high resolution are important but limited. Results: Combining SNAP tag technology with cryo-electron tomography, we precisely localized proteins within the N-DRC that are important for ciliary motility. Conclusion: The developed method was applied to localize proteins with ϳ3 nm resolution without interfering with the complex function. Significance: The method is a powerful tool for studies of proteins in situ.

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Visualization of proteins in intact cells with a clonable tag for electron microscopy

Cited by 90 publications

References 49 publications

Peptidoglycan Hydrolases of Escherichia coli

Peptidoglycan Hydrolases of Escherichia coli

Structural organization of Trypanosoma cruzi

In Situ Localization of N and C Termini of Subunits of the Flagellar Nexin-Dynein Regulatory Complex (N-DRC) Using SNAP Tag and Cryo-electron Tomography

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