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The aim of this study was to establish a retinal organ culture and to follow the cellular and cytoskeletal changes. For this purpose the authors detached porcine neuroretinas from the underlying pigment epithelium, and incubated them for 2 weeks under standardized conditions. After 3, 6, 10, and 13 days in culture the retinal tissues were fixed, embedded in LR-White resin or paraffin wax and processed for electron-, light-, immunofluorescence- and confocal laser scanning microscopy. Antibodies directed against alpha-tubulin, actin, glial fibrillary acidic protein (GFAP), vimentin, neurofilament(200) and beta-catenin were used to investigate the cytoskeletal changes over a certain period of time. After experimental detachment Müller cells quickly started to hypertrophy showing increased levels of intermediate filaments (i.e. vimentin and GFAP). The actin labelling of photoreceptor cells decreased concomitantly with a rapid degeneration of the outer segments. After 1 week of detachment the Müller cell cytoplasm revealed increasing amounts of actin and tubulin staining. Actin filaments appeared frequently organized in thick bundles across the full width of the retina, whereas increasing levels of tubulin shifted into the outer nuclear region especially concentrated near the outer limiting membrane. A prolonged time of explant culturing resulted in a discontinuous staining of beta-catenin along the adherent junctions of the outer limiting membrane, followed by an outgrowth of Müller cell extensions into the subretinal space. Double staining of tubulin and cones showed that this outgrowth predominantly occurred between cone inner segments. The outer limiting membrane was penetrated by stalk-like structures, highly enriched with tubulin and associated with swollen tips, reaching into the subretina. Electron microscopy demonstrated in detail the focal disruption of the outer limiting membrane by Müller cell extensions and subsequent subretinal gliosis. The cytoskeletal reactions described here were compared with degenerative changes observed after induced retinal detachments.

The microfilament protein α-actinin exists as a dimer. The N-terminal regions of both polypeptides, arranged in antiparallel orientation, comprise the actin-binding regions, while the C-terminal, larger parts consist of four spectrin-like repeats that interact to form a rod-like structure. To elucidate the fine structure of smooth-muscle α-actinin, we used energy-filtered transmission electron microscopy in conjunction with negative staining. Survey pictures of the protein purified from chicken gizzard revealed discrete, elongated particles whose length and width varied with the ionic strength of the buffer. It was determined to to 29.3 nm×4.8 nm in 0.05 M KCI and 32.6 nm×4.4 nm in 0.15 M KCI. Both ends of the molecule displayed hook-like structures consisting of globular domains, which were highly variable in their orientation with respect to the long axis of the molecule. Their location at the ends of the molecule, and the finding that these hooks were missing from particles obtained by thermolysin digestion indicated that they probably correspond to the N-terminal actin-binding regions. The rod-like center of the molecule revealed discrete globular masses which probably comprise the spectrin-like repeats. Their arrangement was compatible with the interpretation that three spectrin repeats of each polypeptide chain can form pairs with the respective sequences of the other chain. The rod-like 53-kDa fragment obtained after thermolysin digestion largely retained this structural organization but appeared wider (22.5 nm×5.9 nm). Our results help to clarify previous discrepancies on the quatenary organization of α-actinin and suggest that effective actin-binding and cross-linking of α-actinin is based on the high flexibility of the terminal hooks. [ABSTRACT FROM AUTHOR]

Talin is a multidomain cytoskeletal protein containing discrete binding sites for acidic phospholipids, β-integrin, actin and vinculin. Hence, it is thought to link microfilaments to the cytoplasmic membrane in cell-matrix adhesion sites, and this should critically depend on talin structure. To obtain more information on the latter; we used energy-filtered transmission electron microscopy of negatively stained talin purified from chicken smooth muscle. We show that in buffers of physiological ionic strength, talin adopts an elongated shape (56 ± 7 nm in length), consisting of a series of globular masses, While these compact elements, arranged like beads on a string, were of rather uniform dimensions (3.8 nm in diameter), their center-to-center spacings varied, indicating tile flexibility of the connecting strands. The ends of the elongated molecules frequently formed loops. The images obtained are consistent with the assumption that, under the conditions used, the majority of the talin molecules are monomeric. A minor fraction appeared as dimers, composed of two chains only partially intertwined, thus giving rise to Y-shaped particles. Electron micrographs revealed that the biochemically defined 50-kDa N-terminal talin head domain is composed of two globular subunits, while chemical cross-linking provided evidence that the Cterminal 220-kDa fragment is solely responsible for dimerization. These results imply that in the dimeric molecules, the polypeptide chains are arranged in parallel, in contrast to what has been described for human-platelet talin. In buffers of low ionic strength (0.02 M instead of 0.15 M KCl), the molecules collapsed into a compact shape. By showing the high flexibility and versatility of its morphology, our data favour the concept of talin as an important resilient link in microfilament-plasma-membrane attachment. [ABSTRACT FROM AUTHOR]