Anne Bushnell

Studies on intercellular LETS glycoprotein matrices.

Intercellular matrices secreted by chick embryo fibroblasts in culture were studied by scanning electron microscopy. Cell-cell contact is a prerequisite for the expression of such matrices. The smallest fiber detected by transmission electron microscopy is 5--10 nm in diameter. These matrix fibers tend to cluster to form bundles. Immunofluorescence and immunoferritin procedures reveal that LETS protein is one of the components of the matrices. The matrices are isolated from other cellular organelles by detergent treatment. More than 90% of the proteins in cell-free matrices are LETS protein, suggesting that the matrices are probably made of only one component--LETS protein. Since the solubilization of matrices requires beta-mercaptoethanol, LETS protein matrices may be the first known polymer system in nature to use disulfide linkage as an intermolecular polymerization vehicle. Collagen does not appear to be involved in such matrices. The LETS protein matrix supports the morphological conversion of rounded cells into spindle-shaped, and also promotes myoblast fusion. It does not, however, exert an effect upon cell growth, the rate of glucose uptake or protease production.

Characterization of the intermediate (10 nm) filaments of cultured cells using an autoimmune rabbit antiserum.

An antiserum has been found in a nonimmunized rabbit which reacts strongly with a system of filaments in various fibroblasts, epithelial cells, macrophages and neuroblastoma. These filaments are distinct from the actin microfilament bundles visualized by an antibody against actin, and they are not affected by brief treatment with cytochalasin B. The pattern of these filaments somewhat resembles that described for microtubules, but the filaments could be clearly distinguished from microtubules by a comparison of their respective immunofluorescent patterns during cell division. In response to the drugs colcemid and vinblastine, the filaments reacting with this preimmune serum condense to form a compact perinuclear coil of fibers, a distribution and behavior in agreement with that previously described for the 10 nm or intermediate filaments studied by electron microscopy. Further evidence supporting our conclusion that this antiserum reacts with intermediate filaments is provided by a comparison of electron micrographs and the immunofluorescent patterns from parallel cell cultures. To identify the antigens reacting with this antiserum we have used the new technique of immuno-autoradiography on SDS gels of whole cell extracts. Two reactive polypeptide chains have been identified with apparent molecular weights of 56,000 and 30,000 daltons.

The orientation of centrioles in migrating 3T3 cells.

Abstract Based on electron microscopic examination of 20 migrating 3T3 cells, the paper suggests that one centriole of a pair is preferentially oriented perpendicular, the other parallel to the substrate. The finding is interpreted as further support for the possibility that centrioles are involved in the control of migration in 3T3 cells.

The ultrastructure of primary cilia in quiescent 3T3 cells

Abstract Electron microscopy was used to investigate primary cilia in quiescent 3T3 cells. As in the case of primary cilia of other cell types, their basal centriole was found to be a focal point of numerous cytoplasmic microtubules which terminate at the basal feet. There are also intermediate filaments which appear to converge at the basal centriole. Cross-striated fibers of microtubular diameter, reminiscent of striated rootlets of ordinary cilia, appear associated with the proximal end of the basal centriole. Usually less than nine cross-banded basal feet surround the basal centriole in a well-defined plane perpendicular to the centriolar axis. The ciliary shaft was found to be entirely enclosed in the cytoplasm of fully flattened cells. In rounded cells, it could be found extending to the outside of the cell. Periodic striations along the entire shaft were observed after preparing the cells in a special way. The tip of the shaft showed an electron-dense specialization. Several unusual forms of primary cilia were observed which were reminiscent of olfactory flagella or retinal rods. Using tubulin antibody for indirect immunofluorescence, a fluorescent rod is visible in the cells [18] which we demonstrate is identical with the primary cilium.

Immunofluorescent and ultrastructural studies of polygonal microfilament networks in respreading non-muscle cells

Abstract Respreading gerbil fibroma cells (CCL146) have been found to display cytoplasmic actin-based polygonal fiber networks 10 h after replating (stage III of respreading according to Vasiliev & Gelfand, [1]). The networks have been analyzed by immunofluorescence and electron microscopy. The foci, sites of actin, α-actinin and filamin distribution, are condensed meshworks of microfilaments attached to the inner surface of the plasma membrane. The interconnecting fibers, sites of uniform actin distribution and complementary periodicities of α-actinin and myosin, are bundles of parallel microfilaments with periodic dense bodies. Heavy meromyosin (HMM) labelling of the microfilaments in the foci and interconnecting bundles confirm that they contain actin. In addition, approx. 70% of the microfilaments associated with an individual focus have a uniform polarity relative to it (arrowheads pointing away) suggesting that they have their origin there. Our results support earlier conclusions [2] that polygonal networks are structural intermediates responsible for organizing contractile proteins of the cortical microfilament layer into stress fibers.

Observations on the vimentin-10-nm filaments during mitosis in BHK21 cells

Mitotic BHK21 cells were examined by immunofluorescence optical sectioning after staining with anti-vimentin. Throughout all stages of mitosis, intact 10-nm filaments were observed to form a cage around the mitotic spindle. These observations were confirmed by serial section electron microscopy which demonstrated assembled 10-nm filaments in the cytoplasm surrounding the spindle. Therefore, in contrast to previous reports, mitotic BHK21 cells show a similar cage-like 10-nm filament arrangement, as found in a variety of other cell types.

Reversible compression of cytoplasm

Abstract Incubation for 3 min in 1 M sorbitol causes animal cells to lose 50–75% of their water content. Upon return to normal medium, the ultrastructure and morphology of the cells return to normal within 3 min. Mitoses continue normally; migration patterns and growth curves also return to normal. An intriguing aspect of irreversibility was observed when blebbing microplasts switched to ruffling. They would have continued to bleb without the prior episode of dehydration in sorbitol. During incubation with 1 M sorbitol the shrunken cells appear to store excess surface area in numerous, rapidly forming microvilli. The cytoplasm is also altered. Bundles of individual microfilaments and intermediate filaments condense into homogeneous streaks of materials which retain their birefringence. In contrast, microtubules display a clearly visible exclusion zone around each individual tubule. Sharply outlined patches of tiny granules appear in electron micrographs. The results are discussed with respect to the morphological compartmentalization and the viscoelasticity of the cytoplasm.

Properties of enucleated cells. III. Changes in cytoplasmic architecture of enucleated BHK21 cells following trypsinization and replating.

Abstract BHK21 cells enucleated with cytochalasin B were trypsinized and replated. Subsequent spreading and shape formation were monitored with light and transmission electron microscopy. Assembly and changes in distribution of microtubules, microfilaments and 100 A filaments were related to these processes. These results indicate that cytoplasm contains the information necessary for the normal assembly and distribution of cytoplasmic fibers.