Novel freeze-substitution electron microscopy provides new aspects of virulent Mycobacterium tuberculosis with visualization of the outer membrane and satisfying biosafety requirements

Yamada, Hiroyuki; Mitarai, Satoshi; Chikamatsu, Kinuyo; Mizuno, Kazue; Yamaguchi, Masashi

doi:10.1016/j.mimet.2009.09.022

Cited by 14 publications

(14 citation statements)

References 16 publications

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“…We previously reported the excellent preservation of the ultrastructure of M . tuberculosis cells provided by CRF-RFS [ 5 , 7 , 8 ]. CRF-RFS preserves not only the cell envelope with OM but also ribosomes within the cytoplasm.…”

Section: Resultsmentioning

confidence: 99%

“…Briefly, a portion (<1 μl) of the highly concentrated bacterial pellet prepared as described above was applied to a glow-discharge-treated single-hole cupper grid (Veco; hole size, 0.1-mm diameter) and then sandwiched with another glow-discharge-treated single-hole grid. The grids were then picked up with tweezers and frozen by plunging them into melting propane (propane cooled with liquid nitrogen in a cooling device) for 20 seconds, as described previously [ 5 ]. The pair of grids was transferred, detached in liquid nitrogen, and immersed quickly into 2% osmium tetroxide/acetone solution and then placed in the device described above and cooled.…”

Section: Methodsmentioning

confidence: 99%

“…determined the number and volume of organelles, including the mitochondria, endoplasmic reticulum, Golgi apparatus, and ribosomes using serial ultrathin sections of yeast cells. They proposed the term “structome” for this technique, which is defined as the ‘quantitative and three-dimensional structural information of a whole cell at the electron microscopic level’ [ 3 , 5 ].…”

Section: Introductionmentioning

confidence: 99%

“…The ribosome density (number per 0.1 fl of cytoplasm) for M . tuberculosis was calculated and compared to that determined for yeast cells [ 2 , 5 ] as well as previously published estimations [ 10 – 14 ]. This is the first report of a structome analysis of individual M .…”

Section: Introductionmentioning

confidence: 99%

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Structome Analysis of Virulent Mycobacterium tuberculosis, Which Survives with Only 700 Ribosomes per 0.1 fl of Cytoplasm

et al. 2015

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We previously reported the exquisite preservation of the ultrastructures of virulent Mycobacterium tuberculosis cells processed through cryofixation and rapid freeze substitution. Here, we report the “structome” analysis (i.e., the quantitative three-dimensional structural analysis of a whole cell at the electron microscopic level) of virulent M. tuberculosis using serial ultrathin sections prepared after cryofixation and rapid freeze substitution and analyzed by transmission electron microscopy. Five M. tuberculosis cells, which were contained in the serial ultrathin cross sections encompassing from one end to the other, were cut into 24, 36, 69, 55, and 63 serial ultrathin sections, respectively. On average, the cells were 2.71 ± 1.05 μm in length, and the average diameter of the cell was 0.345 ± 0.029 μm. The outer membrane and plasma membrane surface areas were 3.04 ± 1.33 μm2 and 2.67 ± 1.19 μm2, respectively. The cell, outer membrane, periplasm, plasma membrane, and cytoplasm volumes were 0.293 ± 0.113 fl (= μm3), 0.006 ± 0.003 fl, 0.060 ± 0.021 fl, 0.019 ± 0.008 fl, and 0.210 ± 0.091 fl, respectively. The average total ribosome number was 1,672 ± 568, and the ribosome density was 716.5 ± 171.4/0.1 fl. This is the first report of a structome analysis of M. tuberculosis cells prepared as serial ultrathin sections following cryofixation and rapid freeze substitution and examined by transmission electron microscopy. These data are based on the direct measurement and enumeration of exquisitely preserved single-cell structures in transmission electron microscopy images rather than calculations or assumptions from indirect biochemical or molecular biological data. In addition, these data may explain the slow growth of M. tuberculosis and enhance understanding of the structural properties related to the expression of antigenicity, acid-fastness, and the mechanism of drug resistance, particularly in regard to the ratio of target to drug concentrations.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Structome Analysis of Virulent Mycobacterium tuberculosis, Which Survives with Only 700 Ribosomes per 0.1 fl of Cytoplasm

et al. 2015

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“…To determine whether a cytoskeletal filament is a route of ibNoTS, the filament should be visualized. For the visualization, rapid-freeze substitution [31][32][33] is an effective method which applied to visualization of the fine structures of surface and released vesicle in V. cholerae [34][35][36]. Further studies of a route of ibNoTS should be performed using rapid-freeze substitution and/or immunoelectron microscopy double staining [30].…”

Section: Discussionmentioning

confidence: 99%

ATP-association to intrabacterial nanotransportation system in Vibrio cholerae

Matsuzaki

Nakano

et al. 2015

Med Mol Morphol

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Vibrio cholerae colonizes the lumen of the proximal small intestine, which has an alkaline environment, and secretes cholera toxin (CT) through a type II secretion machinery. V. cholerae possesses the intrabacterial nanotransportation system (ibNoTS) for transporting CT from the inner portion toward the peripheral portion of the cytoplasm, and this system is controlled by extrabacterial pH. Association of ATP with ibNoTS has not yet been examined in detail. In this study, we demonstrated by immunoelectron microscopy that ibNoTS of V. cholerae under the extrabacterial alkaline condition was inhibited by ATP inhibitors, 2,4-dinitrophenol (DNP), a protonophore, or 8-amino-adenosine which produces inactive form of ATP. The inhibition of CT transport can be reversed by neutralization of DNP. Those inhibitions were associated with decrease of CT secretion by which ibNoTS followed. We propose that ATP closely associates with V. cholerae ibNoTS for transporting CT.

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Pre-fixation of virulent Mycobacterium tuberculosis with glutaraldehyde preserves exquisite ultrastructure on transmission electron microscopy through cryofixation and freeze-substitution with osmium-acetone at ultralow temperature

Yamada

Chikamatsu

Ando

et al. 2014

Journal of Microbiological Methods

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Novel freeze-substitution electron microscopy provides new aspects of virulent Mycobacterium tuberculosis with visualization of the outer membrane and satisfying biosafety requirements

Cited by 14 publications

References 16 publications

Structome Analysis of Virulent Mycobacterium tuberculosis, Which Survives with Only 700 Ribosomes per 0.1 fl of Cytoplasm

Structome Analysis of Virulent Mycobacterium tuberculosis, Which Survives with Only 700 Ribosomes per 0.1 fl of Cytoplasm

ATP-association to intrabacterial nanotransportation system in Vibrio cholerae

Pre-fixation of virulent Mycobacterium tuberculosis with glutaraldehyde preserves exquisite ultrastructure on transmission electron microscopy through cryofixation and freeze-substitution with osmium-acetone at ultralow temperature

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