Expression and distribution of vimentin and keratin filaments in heterokaryons of human fibroblasts and amnion epithelial cells.

The expression of intermediate filaments of the keratin- and the vimentin-type was studied in heterokaryons of human fibroblasts and amnion epithelial cells by immunofluorescence microscopy. Fibroblasts and their homokaryons showed a fibrillar, vimentin-specific fluorescence throughout the cytoplasm but were negative when stained for keratin. Amnion epithelial cells and their homokaryons, on the other hand, showed a keratin-specific fibrillar staining, and only some of them contained also detectable vimentin. When suspended epithelial cells were fused with adherent fibroblasts, keratin fibrils spread within 3 h into the fibroblasts, intermixing with the vimentin fibrils. 1-3 d after fusion, both vimentin and keratin filaments were expressed as typical fibrillar cytoplasmic arrays, and the distribution of keratin in heterokaryons resembled closely that of vimentin. A typical cell-to-cell arrangement of keratin fibrils, seen in cultures of amnion epithelial cells, could also be found between heterokaryons. Treatment of the cultures with vinblastine sulphate induced coiling of the vimentin filaments in both homo- and heterokaryons, whereas the keratin organization was only slightly affected. Our results show that both vimentin and keratin filaments are incorporated into the cytoskeleton of heterokaryons formed between fibroblasts and epithelial cells, and that they behave in the same way as in their parental cells. Both epithelial and fibroblastic characteristics thus appear to the coexpressed in such heterokaryons.

Regulation of insulin secretion from islets of Langerhans rendered permeable by electric discharge

1. High-voltage electric discharge has been used to increase the permeability of B-cells of isolated islets of Langerhans to facilitate studies of the effects of normally impermeable substances on insulin secretion. 2. The application of an intense electric field increased the [14C]sucrose space of the islets from 37.8±3.1% to 86.2±5.2% of their total volume as assessed by 3H2O content. The cells remained permeable for at least 40min. 3. Ultrastructural studies showed no deleterious changes in the structure of the B-cells after discharge. 4. Insulin secretion from normal islets was unaffected by increasing the medium [Ca2+] from 10nm to 10μm. In the islets that had been rendered permeable by discharge, insulin secretion was significantly increased under these conditions, without any alteration in the release of lactate dehydrogenase, a cytoplasmic marker enzyme. 5. Studies of the dynamics of insulin release during perifusion showed that the response to increased (10μm) Ca2+ concentration was rapid and sustained over a period of at least 13min. 6. Secretion responses to Ca2+ in perifusion established that maximum release in permeabilized islets occurs at approx. 1μm-Ca2+ and half-maximum release occurs at approx. 0.6μm-Ca2+. 7. The study of the effect of agents that interfere with the microtubular microfilamentous system in B-cells using a perifusion system revealed that cytochalasin B caused a considerable increase, whereas vinblastine sulphate caused a significant inhibition, in insulin release in response to 1μm-Ca2+. 8. This technique should facilitate the study of the role of normally impermeable ions and metabolic intermediates in the regulation of insulin secretion.

The role of the cytoskeleton in the motility of coccidian sporozoites

The sporozoites of Eimeria tenella and Eimeria acervulina show bending, pivoting and gliding motility. All these types of motility occur intermittently and with decreasing frequency during the life of a sporozoite. Gliding is the only locomotive action expressed by these sporozoites and is only seen when the sporozoites are in contact with the substratum. All gliding sporozoites adopt a set pattern of body ‘attitudes’, which suggests that locomotion involves a fixed body shape. The microtubule inhibitors, colchicine, griseofulvin, vinblastine sulphate and nocodazole, have no effect on sporozoite motility. Ultrastructural examination reveals, in addition, that they have no effect on the subpellicular microtubules. The microfilament inhibitor, cytochalasin B, completely, and reversibly, inhibits pivoting and gliding but bending is only slightly depressed by the drug. High magnesium ion concentration inhibits all motility completely. The cell membrane was readily labelled with fluorescein isothiocyanate-conjugated cationized ferritin, the label was rapidly capped and shed from the posterior of the sporozoite. This capping reaction takes place only during sporozoite locomotion. The membrane label was seen to ‘move’ backwards realtive to the sporozoite at the same rate as the sporozoite moved forwards relative to the substratum. The substratum and the leading edge of the cap remained static relative to each other. Both capping and locomotion are sensitive to low temperature and cytochalasin B. From these results a theory of sporozoite motility is postulated. The sporozoites adhere to the substratum by surface ligands. This ligand/substratum complex is then capped along the fixed spiral of the sporozoite body by a microfilament-based contractile system. This proposed model for motility of coccidia sporozoites is consistent with all current observations on cell invasion by the sporozoa and therefore suggests that locomotion is an integral component of host cell invasion in this group of parasites.