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3D Analysis of HCMV Induced-Nuclear Membrane Structures by FIB/SEM Tomography: Insight into an Unprecedented Membrane Morphology.

Villinger C, Neusser G, Kranz C, Walther P, Mertens T - Viruses (2015)

Bottom Line: We found that the previously described infoldings of the inner nuclear membrane, which are unique among its kind, form an extremely complex network of membrane structures not predictable by previous two-dimensional studies.Only 0.8% proved to be enveloped capsids which were exclusively detected in 1st order infoldings (perinuclear space).Distribution of the capsids between 1st, 2nd and 3rd order infoldings is in complete agreement with the envelopment/de-envelopment model for egress of HCMV capsids from the nucleus and we confirm that capsid budding does occur at the large infoldings.

View Article: PubMed Central - PubMed

Affiliation: Electron Microscopy Facility, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany. thomas.mertens@uniklinik-ulm.de.

ABSTRACT
We show that focused ion beam/scanning electron microscopy (FIB/SEM) tomography is an excellent method to analyze the three-dimensional structure of a fibroblast nucleus infected with human cytomegalovirus (HCMV). We found that the previously described infoldings of the inner nuclear membrane, which are unique among its kind, form an extremely complex network of membrane structures not predictable by previous two-dimensional studies. In all cases they contained further invaginations (2nd and 3rd order infoldings). Quantification revealed 5498HCMV capsids within two nuclear segments, allowing an estimate of 15,000 to 30,000 capsids in the entire nucleus five days post infection. Only 0.8% proved to be enveloped capsids which were exclusively detected in 1st order infoldings (perinuclear space). Distribution of the capsids between 1st, 2nd and 3rd order infoldings is in complete agreement with the envelopment/de-envelopment model for egress of HCMV capsids from the nucleus and we confirm that capsid budding does occur at the large infoldings. Based on our results we propose the pushing membrane model: HCMV infection induces local disruption of the nuclear lamina and synthesis of new membrane material which is pushed into the nucleoplasm, forming complex membrane infoldings in a highly abundant manner, which then may be also used by nucleocapsids for budding.

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Sample manipulation by the focused ion beam (FIB) and image acquisition with the scanning electron microscope (SEM) beam. Sample preparation is described in [39]. Embedded HCMV infected fibroblasts are located directly under the surface of the approximately 1 mm high Epon block. The sample is coated with a thin platinum layer prior to mounting into the dual beam microscope. The area of interest is chosen at 10 kV acceleration voltage. (A) The sample is then tilted to 52° and a trench is FIB-milled into the Epon block, generating a new surface (block face); (B) Removal of the next thin layer (“slice”) of the sample generates a new block face. After every “slice” step the generated block face is imaged by the scanning electron beam (“view”). The “slice and view” cycle is repeated to gain a three-dimensional dataset of the volume of interest. Reprinted from [38] Figure 1 with kind permission from Springer Science+Business Media.
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viruses-07-02900-f001: Sample manipulation by the focused ion beam (FIB) and image acquisition with the scanning electron microscope (SEM) beam. Sample preparation is described in [39]. Embedded HCMV infected fibroblasts are located directly under the surface of the approximately 1 mm high Epon block. The sample is coated with a thin platinum layer prior to mounting into the dual beam microscope. The area of interest is chosen at 10 kV acceleration voltage. (A) The sample is then tilted to 52° and a trench is FIB-milled into the Epon block, generating a new surface (block face); (B) Removal of the next thin layer (“slice”) of the sample generates a new block face. After every “slice” step the generated block face is imaged by the scanning electron beam (“view”). The “slice and view” cycle is repeated to gain a three-dimensional dataset of the volume of interest. Reprinted from [38] Figure 1 with kind permission from Springer Science+Business Media.

Mentions: First, we sawed the Epon block into two halves. One half was objected to serial ultrathin sectioning (see 2.5.). As described in [38,39], we reduced the size of the second half with a jigsaw to a height of ~1 mm. The produced Epon disc was then mounted onto a SEM specimen stub (Figure 1). The sample was then coated with 5 nm of platinum using an electron beam evaporator (Baltec, Balzers, Liechtenstein) to enhance electrical conductivity. FIB/SEM tomography was conducted with a Helios Nanolab 600 FIB/SEM (FEI, Eindhoven, The Netherlands). The sample surface was imaged at a high acceleration voltage of 10 kV to visualize the contours of the embedded cells. In order to protect the cellular structures in the chosen area from beam damage during FIB-milling, the surface was covered with an additional platinum layer using ion beam-induced deposition based on the fragmentation of methylcyclopentadienyl(trimethyl)platinum (C9H16Pt).


3D Analysis of HCMV Induced-Nuclear Membrane Structures by FIB/SEM Tomography: Insight into an Unprecedented Membrane Morphology.

Villinger C, Neusser G, Kranz C, Walther P, Mertens T - Viruses (2015)

Sample manipulation by the focused ion beam (FIB) and image acquisition with the scanning electron microscope (SEM) beam. Sample preparation is described in [39]. Embedded HCMV infected fibroblasts are located directly under the surface of the approximately 1 mm high Epon block. The sample is coated with a thin platinum layer prior to mounting into the dual beam microscope. The area of interest is chosen at 10 kV acceleration voltage. (A) The sample is then tilted to 52° and a trench is FIB-milled into the Epon block, generating a new surface (block face); (B) Removal of the next thin layer (“slice”) of the sample generates a new block face. After every “slice” step the generated block face is imaged by the scanning electron beam (“view”). The “slice and view” cycle is repeated to gain a three-dimensional dataset of the volume of interest. Reprinted from [38] Figure 1 with kind permission from Springer Science+Business Media.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664973&req=5

viruses-07-02900-f001: Sample manipulation by the focused ion beam (FIB) and image acquisition with the scanning electron microscope (SEM) beam. Sample preparation is described in [39]. Embedded HCMV infected fibroblasts are located directly under the surface of the approximately 1 mm high Epon block. The sample is coated with a thin platinum layer prior to mounting into the dual beam microscope. The area of interest is chosen at 10 kV acceleration voltage. (A) The sample is then tilted to 52° and a trench is FIB-milled into the Epon block, generating a new surface (block face); (B) Removal of the next thin layer (“slice”) of the sample generates a new block face. After every “slice” step the generated block face is imaged by the scanning electron beam (“view”). The “slice and view” cycle is repeated to gain a three-dimensional dataset of the volume of interest. Reprinted from [38] Figure 1 with kind permission from Springer Science+Business Media.
Mentions: First, we sawed the Epon block into two halves. One half was objected to serial ultrathin sectioning (see 2.5.). As described in [38,39], we reduced the size of the second half with a jigsaw to a height of ~1 mm. The produced Epon disc was then mounted onto a SEM specimen stub (Figure 1). The sample was then coated with 5 nm of platinum using an electron beam evaporator (Baltec, Balzers, Liechtenstein) to enhance electrical conductivity. FIB/SEM tomography was conducted with a Helios Nanolab 600 FIB/SEM (FEI, Eindhoven, The Netherlands). The sample surface was imaged at a high acceleration voltage of 10 kV to visualize the contours of the embedded cells. In order to protect the cellular structures in the chosen area from beam damage during FIB-milling, the surface was covered with an additional platinum layer using ion beam-induced deposition based on the fragmentation of methylcyclopentadienyl(trimethyl)platinum (C9H16Pt).

Bottom Line: We found that the previously described infoldings of the inner nuclear membrane, which are unique among its kind, form an extremely complex network of membrane structures not predictable by previous two-dimensional studies.Only 0.8% proved to be enveloped capsids which were exclusively detected in 1st order infoldings (perinuclear space).Distribution of the capsids between 1st, 2nd and 3rd order infoldings is in complete agreement with the envelopment/de-envelopment model for egress of HCMV capsids from the nucleus and we confirm that capsid budding does occur at the large infoldings.

View Article: PubMed Central - PubMed

Affiliation: Electron Microscopy Facility, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany. thomas.mertens@uniklinik-ulm.de.

ABSTRACT
We show that focused ion beam/scanning electron microscopy (FIB/SEM) tomography is an excellent method to analyze the three-dimensional structure of a fibroblast nucleus infected with human cytomegalovirus (HCMV). We found that the previously described infoldings of the inner nuclear membrane, which are unique among its kind, form an extremely complex network of membrane structures not predictable by previous two-dimensional studies. In all cases they contained further invaginations (2nd and 3rd order infoldings). Quantification revealed 5498HCMV capsids within two nuclear segments, allowing an estimate of 15,000 to 30,000 capsids in the entire nucleus five days post infection. Only 0.8% proved to be enveloped capsids which were exclusively detected in 1st order infoldings (perinuclear space). Distribution of the capsids between 1st, 2nd and 3rd order infoldings is in complete agreement with the envelopment/de-envelopment model for egress of HCMV capsids from the nucleus and we confirm that capsid budding does occur at the large infoldings. Based on our results we propose the pushing membrane model: HCMV infection induces local disruption of the nuclear lamina and synthesis of new membrane material which is pushed into the nucleoplasm, forming complex membrane infoldings in a highly abundant manner, which then may be also used by nucleocapsids for budding.

Show MeSH
Related in: MedlinePlus