Alternate header for print version


Reconstruction
Display image description
Single computed slice through a tomographic reconstruction 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
Tomographic reconstruction in IMOD format.
Volume_dimension
3072, 3072, 76
Volume scale
0.0012, 0.0012, 0.0012
Animation description
Animation through the computed slices of a tomographic reconstruction 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 at zero degress of tilt 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 are 20 nm gold particles applied to the surface of the section to serve as fiducial marks for subsequent alignment.
Full resolution image description
Tar file containing the raw and aligned tilt images, along with all supporting files from IMOD (beyers1a_*). Note that some of the tilt images were discarded.
Animation description
Animation through an aligned 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. Dark particles are 20 nm gold particles applied to the surface of the section to serve as fiducial marks for subsequent alignment.

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

CCDB:6347*
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
6347
Instrument
JEOL4000EX IVEM
Microscopy type
IVEM
Product type
SINGLE TILT
Image basename
beyers1a
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
0.15 µm
Specimen description
Organ
lens
Structure
Multi-lamellar structure
Cell type
HeLa
Imaging parameters
Type
Electron microscopy product
Recording medium
Spectral Instruments 1100 series
Magification
15000
Accelerating voltage
400 kV
Notes
Used second largest objective apeture in the JEOL 4000EX.
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 acquisition of the tilt series.
Imaging product type
Type
Single tilt
Description
singlet_desc
Min range
-60 degrees
Max range
40 degrees
Tilt increment
2 degrees
Notes
Collected tilts -60 to +56 degrees, but discarded tilts over 40 degrees. (in IMOD views skipped 51-58)