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Three-dimensional reconstruction of cytoplasmic membrane networks in parietal cells

Joseph G. Duman1, Nimesh J. Pathak1, Mark S. Ladinsky3, Kent L. McDonald2 and John G. Forte1,*

1 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
2 Electron Microscope Laboratory, University of California, Berkeley, CA 94720, USA
3 Boulder Laboratory for 3-D Fine Structure, Department of MCD Biology, University of Colorado, Boulder, CO 80309, USA



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  		Fig. 1. Low-power electron micrograph of a thin section through a rabbit gastric gland preparation fixed by high pressure freezing. Three parietal cells are clearly evident, including numerous dark staining mitochondria (m), secretory canaliculi (c) and a nucleus (n) within one of the cells. Bar, 5 µm.

 


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  		Fig. 2. High power electron micrograph through a typical parietal cell. Microvilli project into the lumina of the canaliculi (can). Endocytic activity is commonly seen at the canalicular surface (arrows). Numerous membrane profiles of H,K-ATPase-rich structures (arrowheads), most of which appear tubular and vesicular in thin section, are located in spaces between the mitochondria (m). Bar, 1 µm.

 


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  		Fig. 3. Three steps in the modeling process applied to the secretory canaliculi of a parietal cell. (A) A single thin section from a series of 30 consecutive serial sections from a specific intracellular region. In this section two canaliculi are outlined (blue) to form one set of contours defining the canalicular surface (exclusive of microvilli, which project into the lumens of the canaliculi). Bar marker is 1 µm. (B) A series of consecutive canalicular contours traced from 29 serial sections as shown in (A) (one contour was not included in the data set owing to a fold in the section). (C) Meshed model of three canaliculi from contours shown in (A) and (B). Interconnections can be seen between two of the canaliculi in some planes. Morphological complexity made it difficult to follow the exact outline of the entire canalicular surface including each microvillar extension, so this model was constructed using only the surface that outlined the canaliculus at the base of the microvilli.

 


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  		Fig. 4. Meshed model of a canaliculus showing the placement of microvilli within the lumen. Contours were separately drawn for the canaliculus and each of the microvilli, and the two sets of images were merged together in the meshed model of the entire apical surface. For clarity, a lighter blue was used for the microvilli. (A) Transverse section through a canaliculus with part of a surface removed. (B) Rotation of the model 90° to provide a transsectional view of microvilli projecting into the lumen of the canaliculus. Bar, 0.5 µm.

 


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  		Fig. 5. Meshed model of parietal cell mitochondria reconstructed from a parietal cell cytoplasmic space of about 7 µmx4 µmx2 µm (lxwxh). Note that virtually every mitochondrion appears to be interconnected with an adjacent mitochondrion, either by major branching or via more diminished tight annular interconnections. Bar, 1 µm.

 


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  		Fig. 6. 3D modeling of H,K-ATPase-rich membranes in parietal cell.

(A) Contours were drawn around membranes in a cell volume of about 6 µm3. Each membrane unit was defined as an object and assigned a separate color. Three crude mitochondrial profiles are inserted in red for size comparison and orientation. Note that some membranes appear as tubules but the majority are flattened cisternal discs, often arranged in small stacks. Bar, 0.5 µm.

(B) Reconstruction of tubulocisternae from a larger area surrounding a canalicular lumen (large blue structure). Bar, 1 µm.

 


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  		Fig. 7. Modeling of H,K-ATPase-rich membranes from a 0.4 µm thick section reconstructed by tomography. (A) A section is depicted from which membrane profiles were accumulated and stacked into objects of individual color. A partial mitochondrion (red contours) is included for size comparison and orientation. The insert shows an actual tomographic section, near the middle of the stack, outlining the membrane structures that were included in the model. (B) The model of tubulocisternae has been meshed and rotated to provide another view. The mitochondrial object is still represented as contours.

 

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© The Company of Biologists Ltd 2002