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Reconstruction
Display image description
Single computed slice through a double tilt electron tomogram of a lamellar structure observed in HeLa cells transfected with mutant connexin 50, imaged using intermediate voltage electron microscopy, and labeled using the tetracysteine-ReHAsH system and fluorescence photoooxidation. The concentric double membrane layers of the aggregate appear to form "onion skin" sheets that do not form a closed structure along the long axis. The differential staining between the outside and inside is most likely due to impeded diffusion of DAB into the interior of these accumulations.
Full resolution image description
Double tilt tomographic volume file in IMOD (MRC) format.
Volume_dimension
3228, 2986, 211
Volume scale
0.00097, 0.00097, 0.00097
Animation description
Animation through the computed slices of a double tilt electron tomogram through a Single computed slice through a double tilt electron tomogram of a lamellar structure observed in HeLa cells transfected with mutant connexin 50, imaged using intermediate voltage electron microscopy, and labeled using the tetracysteine-ReHAsH system and fluorescence photoooxidation. The concentric double membrane layers of the aggregate appear to form "onion skin" sheets that do not form a closed structure along the long axis. The differential staining between the outside and inside is most likely due to impeded diffusion of DAB into the interior of these accumulations.

Image 2D
Display image description
Single tilt image taken at zero degrees tilt of a lamellar structure observed in HeLa cells transfected with mutant connexin 50, imaged using intermediate voltage electron microscopy, and labeled using the tetracysteine-ReHAsH system and fluorescence photoooxidation. The concentric double membrane layers of the aggregate appear to form "onion skin" sheets that do not form a closed structure along the long axis. The differential staining between the outside and inside is most likely due to impeded diffusion of DAB into the interior of these accumulations. Dark particles represent 20 nm gold particles applied to the surface of the section to serve as fiducial marks for subsequent alignment.
Full resolution image description
Zipped archive containing the tilt images in IMOD format along with all supporting IMOD files for alignment (beyers2a_*).
Animation description
Animation through the aligned sections of a tilt series through a lamellar structure observed in HeLa cells transfected with mutant connexin 50, imaged using intermediate voltage electron microscopy, and labeled using the tetracysteine-ReHAsH system and fluorescence photoooxidation. The concentric double membrane layers of the aggregate appear to form "onion skin" sheets that do not form a closed structure along the long axis. The differential staining between the outside and inside is most likely due to impeded diffusion of DAB into the interior of these accumulations. Dark particles represent 20 nm gold particles applied to the surface of the section to serve as fiducial marks for subsequent alignment.

Segmentation
Display image description
Manual segmentation of CX50 labeled lamellae, rough endoplasmic reticulum and vesicles using IMOD. Segmentations were surface rendered using Amira.
Segmentation file description
Gzipped, tar file which contains: Volume used to produce model file(s), IMOD model file containing three objects (each with many contours), model files of each contour in IMOD and VRML format, Amira network used to create surfaced models and movies.

License
Attribution Only: This image is licensed under a Creative Commons Attribution License. View License Deed | View Legal Code

CCDB:6348*
Project: P2020
Project name
Trafficking of cataract-associated mutant connexins
Description
Mutant connexins have been linked to hereditary congenital cataracts. One such mutant causes a proline-to-serine substitution at position 88 in human connexin 50 (CX50P88S). In transfected cells, CX50P88S does not form gap junctions, but localizes in cytoplasmic multilamellar structures. We studied the dynamics of formation and the stability of these structures in HeLa cells stably transfected with CX50P88S containing a tetracysteine motif appended to its C-terminus (HeLa-CX50P88S(Cys)4 cells) using a combination of light and electron microscopy.
Funding agency
National Eye Institute, National Institutes of Health
Leader(s)
Eric Beyer
Collaborator(s)
Gina Sosinsky
John Crum
Viviana Berthoud
Alexandra Lichtensetin
Guido Geietta
Start date
unspecified
End date
unspecified
 
Experiment
Experiment ID
6340
Title
Electron tomography of connexin trafficking
Purpose
In order to clarify the origin of CX50P88S accumulations, ReAsH-labeled structures in HeLa-CX50P88S(Cys)4 cells were used to drive the photoconversion of diaminobenzidine at labeled structures which were then studied by transmission electron microscopy
Experimenter(s)
John Crum
Microscopy product
Microscopy product ID
6348
Instrument
JEOL4000EX IVEM
Microscopy type
IVEM
Product type
DOUBLE TILT
Image basename
beyers2a
Spatial Axis Image Size Pixel Size
X 4000px 0.0012 um/pixels
Y 4000px 0.0012 um/pixels
Subject
Species
human
Scientific name
Homo sapiens
Strain
sapiens
Group by
Transfection with mutant connexin
Treatment
Transfection with CX50 bearing a mutation of proline 88 to a serine (CX50P88S) and a tetracysteine motif appended to its carboxyl terminus (CX50P88S(Cys)4)
Age class
adult
Tissue section
Microtome
Ultramicrotome
Thickness
0.25 µm
Specimen description
Structure
Multi-lamellar structure
Cell type
HeLa
Imaging parameters
Type
Electron microscopy product
Recording medium
Spectral Instruments 1100 series
Magification
15000
Energy filter type
n/a
Energy filter dispersion
n/a
Energy filter slit
n/a
Energy filter type
n/a
Accelerating voltage
400 kV
Notes
Applied 20nm colloidal gold (for fiducial tracking) over glow-discharged evaporated carbon film.
Specimen preparation
Protocol used
ReAsH-labeled cells were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4 at 4C. After 20 min, the cells were rinsed in 0.1 M cacodylate pH 7.4 and incubated for 30-45 min with blocking buffer (10 mM KCN, 10 mM aminotriazole, 0.01% hydrogen peroxide/50 mM glycine in 0.1 M cacodylate pH 7.4). This buffer was then replaced with 1 mg/ml diaminobenzidine (DAB) in blocking buffer, and photoconversion was performed using intense illumination (75 W xenon arc lamp without neutral density filters) focused through the microscope objective. Rinsing and further preparation of the sample for transmission electron microscopy were performed as described in Gaietta et al. (2002). Sections were stained with uranyl acetate prior to acquiring tilt images.
Imaging product type
Type
Double tilt
X min range
-60 degrees
X max range
60 degrees
X tilt increment
2 degrees
Y min range
-60 degrees
Y max range
60 degrees
Y tilt increment
2 degrees
Description
Rotated specimen approximately 90 degrees