Fluorenone-azomethines, a novel class of microtubule inhibitors that specifically affect cell proliferation.

The effects of various azomethine derivatives on microtubule (MT) assembly in vitro as well as on cell proliferation, cell shape, and the cytoskeleton of some cultured murine cell lines were studied. 3 of them, the alpha-diphenylene-N-(p-[bis-(beta-hydroxyethyl)-amino]-phenyl)-nit rone (DHPN), alpha-diphenylene-N-(p-[N-(hydroxyethyl)-N-(gamma-hydroxypropyl)- amino]-phenyl)-nitrone, and alpha-diphenylene-N-(p-diethylaminophenyl)-nitrone, strongly inhibit the assembly of microtubules (MTs) in vitro (50% inhibition at 4 to 7 mumol/l). The same compounds are also able to disrupt preformed microtubules. Moreover, they were found to inhibit proliferation of leukaemia L 1210, melanoma B16 K, fibroblast L 929, and embryo fibroblast cells down to 1 to 10 mumol/l, completely. Immunofluorescence microscopy revealed that DHPN, used as a representative of the active azomethines, causes a reversible destruction of the microtubule part of the cytoskeleton. Apparently resulting from microtubule disruption, the intermediate filament system collapsed whereas the microfilament system remained unaffected. The results indicate that the antiproliferative action of the azomethines is based, at least partially, on their ability to attack microtubules.

The microtubule system and the reduplication of microtubule organizing centres in Dictyostelium discoideum.

The microtubule system of normal and microtubule-poisoned amoebae of Dictyostelium discoideum has been investigated both by indirect immunofluorescence with antibodies to microtubule proteins and electron microscopy. Nocodazole, like some other microtubule poisons, destroys most of the microtubules in both interphase and dividing cells resulting in an inhibition of nuclear and cell division. The microtubule organizing centres, however, continue to duplicate once or twice. The daughter organizing centres segregate, they seem to be connected with nuclear material, that splits partly, too, forming more or less extended nuclear clefts. This segregation, at least over short distances, takes place without intranuclear microtubules. Duplication of microtubule organizing centres is not strictly correlated with nuclear division and cytokinesis. Microtubule poisons are able to uncouple these events. Different levels of regulation should be responsible for microtubule organizing centre, nuclear, and cell division.

DMSO induces the formation of microfilament bundles in nuclei of zoospores of Phytophthora infestans (Mont.) De Bary (Oomycetes, Pythiaceae).

Cytoskeletons of zoospores of the fungus Phytophthora infestans (Mont.) De Bary treated with dimethylsulfoxide (DMSO) were studied by electron microscopy. High concentrations of DMSO (greater than or equal to 5%) resulted in lysis of the cells and disturbances of the microtubule pattern of flagellar axonemes. Treatment with low concentrations of DMSO (1%-0.1%) induced the formation of bundled intranuclear filaments. It is assumed that these filament bundles should consist of F-actin.

Septum pattern in ts mutants of Schizosaccharomyces pombe defective in genes cdc3, cdc4, cdc8 and cdc12.

Septum-defective mutants of Schizosaccharomyces pombe impaired in cdc genes 3, 4, 8 and 12 were compared by fluorescence microscopy, freeze-etching and ultrathin sectioning. This approach made it possible to recognize the internal organization of defective phenotypes under restrictive conditions. Of special interest in this study was the pattern of unusual septum malformations found to be regular features of the terminal phenotypes of the mutants. Their overall topology was visualized at the cellular level by primulin fluorescence. The subcellular location of septum defects was found to be identical in origin to the compartment where normal septum was assembled in the wild type. Delocalized septation involved both microfibrillar and matrix components, which participated in the final assembly of malformations. Unique contour views of delocalized septa were exposed by freeze-fracturing. Cytoplasmic microtubules and microfilaments were detected in ultrathin sections of the cytoplasm of mutant cells. The internal organization of malformation-accumulating phenotypes suggested a disruption of the directional mechanism that steers septum material to the periplasm at the cell equator.

Photomicrography of weakly fluorescent objects--employment of p-phenylene diamine as a blocker of fading.

By adding p-phenylene diamine (PPD) to the embedding medium, the fading of fluorescent objects labeled with FITC or mithramycin is substantially reduced. Thus, a multiple quantity of light, as compared to without additive, may be obtained from the objects and so photomicrography be improved or made possible at all. For microfluorometry as well as for subjective fluorescence microscopy the employment of PPD is not very helpful.