Zellbiologische Untersuchungen zur Augenlinse der Säugetiere : Zellkultivierung, Interaktion mit transformierten Zellen und strukturelle Eigenschaften des Cytoskeletts
This thesis covers three interrelated topics: 1. The spontaneous differentiation of lens epithelial cells to elongated, thin spindle-shaped fiber-like cells in primary cell culture. 2. The interaction of primary cultured lens epithelial cells with malignant cell lines in co-culture, with regard to the establishment of a distinct proliferation border. 3. Structural reorganization processes of the microfilament and microtubule cytoskeleton during differentiation of lens epithelial to lens fiber cells. In primary cell culture, using a standard culture medium with fetal calf serum as single additionally supplement, lens epithelial cells reproducibly started a spontaneous differentiation to fiber-like cells. Accompanied by significant changes of morphology the formation of fiber-like cells appeared only in spots of cells regularly arranged in a compact monolayer. From co-culture of lens epithelial cells with non-adherent growing malignant leukemia cells, no interactions by chemo-repulsive effects could be observed. In contrast, studying a co-culture of lens epithelial cells with also adherent growing micro vascular endothelial cells, it was found that these two cell types formed a distinct proliferation border which was stable for more than two weeks. The co-culture experiments indicate that the contribution of lens epithelial cells to create a barrier for invasive growing tissues is in all probability the maintenance of a tight, compact and impenetrable cell layer. If repulsive effects (chemically or physically by contact) play a critical role at all, than probably only over very short distances within direct vicinity of the lens epithelium. The development of transparency in the mammalian eye lens includes complex structural reorganization processes during differentiation of lens epithelial to lens fiber cells. Whereas apoptotic processes cause an extensive organelle degradation to eliminate light-scattering macromolecular structures, the lens fiber cell cytoskeleton is not degraded but basically reorganized. Regarding the microfilament and microtubule cytoskeleton during lens cell differentiation dramatic reorganizations could be observed in vitro and in vivo. In lens fiber cells, alterations in the microfilament (f-actin) network resulted in an actin free cytoplasm with compact stress fibers parallel aligned to the cell membrane of these highly elongated cells. By visualizing the microtubule system in lens epithelial and cortical fiber cells the restructuring of the microtubule network from a monocentric array in epithelial to a decentralized organization in fiber cells was shown. Also the microtubule system became aligned to the central axis (from pole to pole) of the differentiating fiber cells. These effects were found to initiate already in primary cell culture during differentiation of lens epithelial to fiber-like cells. By immunofluorescence techniques using anti-gamma-tubulin antibodies, it was observed that the microtubule-organizing centre (MTOC) was separated from the cell nucleus both in primary cultured lens epithelial cells and also in isolated cortical lens fiber cells. After degradation of the cell nucleus, cortical lens fiber cells still contained gamma-tubulin complexes. Microtubules were never found to be associated with these gamma-tubulin complexes and did never radiate from one main focus. Instead, they formed a decentralized network with several crossing points, where few microtubules interlinked. The immunofluorescence and immunoblot results indicate that the reorganization of the cytoskeleton of differentiating lens fiber cells includes the separation of the MTOC from the nucleus, its immobilization at the cell membrane and subsequent disintegration. As a result the organization of the microtubule system by one main MTOC becomes lost. Disintegration of the MTOC could eliminate a light scattering center and therefore contribute to the improvement of the optical properties of the eye lens. By contrast, the cytoskeleton of cortical lens fiber cells is not degraded and might therefore be necessary for the final differentiation to a cell type which remains stable until death of the organism.
In der vorliegenden zellbiologischen Arbeit zur Augenlinse der Säugetiere wird zum einen das Verhalten von Linsenepithelzellen bezüglich der Induktion einer Teildifferenzierung zu Linsenfaserzellen und zum anderen die Interaktion von Linsenepithelzellen mit transformierten Zelllinien in Co-Kultur hinsichtlich der Ausbildung einer Wachstumsgrenze für invasiv wachsende Gewebe beschrieben. Weiterhin werden strukturelle und biochemische Eigenschaften des Cytoskeletts von kultivierten undifferenzierten und teildifferenzierten Linsenepithelzellen (Lentoiden) sowie von sekundären Linsenfaserzellen im Kontext einer Umstrukturierung während der limitierten Apoptose untersucht.