Limits...
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.

Show MeSH

Related in: MedlinePlus

Methodology and resolution of FIB/SEM tomography. (A) The Epon embedded cells are first visualized by SEM at an acceleration voltage of 10 kV. A region of interest can be easily chosen for the subsequent “slice and view” process. The boxes show the positions of the volumes chosen for FIB/SEM tomography; (B) Images of human cytomegalovirus (HCMV) particles acquired from an ultrathin section by transmission electron microscopy (TEM) and from the block face by FIB/SEM. The high resolution of the FIB/SEM image allows clear visibility of the two leaflets of the lipid bilayer. The secondary electron SEM image was acquired with an acceleration voltage of 5 kV.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664973&req=5

viruses-07-02900-f002: Methodology and resolution of FIB/SEM tomography. (A) The Epon embedded cells are first visualized by SEM at an acceleration voltage of 10 kV. A region of interest can be easily chosen for the subsequent “slice and view” process. The boxes show the positions of the volumes chosen for FIB/SEM tomography; (B) Images of human cytomegalovirus (HCMV) particles acquired from an ultrathin section by transmission electron microscopy (TEM) and from the block face by FIB/SEM. The high resolution of the FIB/SEM image allows clear visibility of the two leaflets of the lipid bilayer. The secondary electron SEM image was acquired with an acceleration voltage of 5 kV.

Mentions: We analyzed two segments of one infected fibroblast nucleus at five days post infection (Figure 2A). FIB/SEM proved to be ideal for gaining high quality structural information of adherent cells and allowed the selection of an optimum region before starting the “slice and view” process (Figure 2A). Both analyzed volumes had the same dimensions (6.4 µm × 5.5 µm × 5 µm) and each dataset consisted of 250 subsequent images from the slice and view processes with an increment of 20 nm. The three-dimensional reconstruction was based on stacking the obtained images to a tomogram. With our advanced specimen preparation and FIB/SEM imaging approach using the secondary electron signal at 5 kV acceleration voltage, we gained a resolution comparable to standard TEM (Figure 2B).


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)

Methodology and resolution of FIB/SEM tomography. (A) The Epon embedded cells are first visualized by SEM at an acceleration voltage of 10 kV. A region of interest can be easily chosen for the subsequent “slice and view” process. The boxes show the positions of the volumes chosen for FIB/SEM tomography; (B) Images of human cytomegalovirus (HCMV) particles acquired from an ultrathin section by transmission electron microscopy (TEM) and from the block face by FIB/SEM. The high resolution of the FIB/SEM image allows clear visibility of the two leaflets of the lipid bilayer. The secondary electron SEM image was acquired with an acceleration voltage of 5 kV.
© Copyright Policy
Related In: Results  -  Collection

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

viruses-07-02900-f002: Methodology and resolution of FIB/SEM tomography. (A) The Epon embedded cells are first visualized by SEM at an acceleration voltage of 10 kV. A region of interest can be easily chosen for the subsequent “slice and view” process. The boxes show the positions of the volumes chosen for FIB/SEM tomography; (B) Images of human cytomegalovirus (HCMV) particles acquired from an ultrathin section by transmission electron microscopy (TEM) and from the block face by FIB/SEM. The high resolution of the FIB/SEM image allows clear visibility of the two leaflets of the lipid bilayer. The secondary electron SEM image was acquired with an acceleration voltage of 5 kV.
Mentions: We analyzed two segments of one infected fibroblast nucleus at five days post infection (Figure 2A). FIB/SEM proved to be ideal for gaining high quality structural information of adherent cells and allowed the selection of an optimum region before starting the “slice and view” process (Figure 2A). Both analyzed volumes had the same dimensions (6.4 µm × 5.5 µm × 5 µm) and each dataset consisted of 250 subsequent images from the slice and view processes with an increment of 20 nm. The three-dimensional reconstruction was based on stacking the obtained images to a tomogram. With our advanced specimen preparation and FIB/SEM imaging approach using the secondary electron signal at 5 kV acceleration voltage, we gained a resolution comparable to standard TEM (Figure 2B).

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