Christian Petzelt

Latrunculin inhibits the microfilament-mediated processes during fertilization, cleavage and early development in sea urchins and mice.

Latrunculin A, a marine toxin from a Red Sea sponge, is a potent inhibitor of the microfilament-mediated processes of fertilization and early development in sea urchins and in mice. Sperm from sea urchins, but not those from Limulus or mice, were affected by latrunculin, and fertilization in both sea urchins and in mice was arrested but at different stages. Sea urchin sperm treated with 2.6 microM latrunculin are unable to assemble acrosomal processes and their ability to fertilize eggs is impaired. The unwinding of the Limulus sperm acrosomal process occurs in the presence of latrunculin. Treated mouse sperm are able to fertilize mouse oocytes in vitro, suggesting that microfilaments may not be required in this mammalian sperm. In sea urchin eggs, sperm incorporation, microvillar elongation and cytokinesis are inhibited. Microtubule-mediated motility occurs normally. 20 nM latrunculin prevents the morphogenetic movements during gastrulation. It reduces the viscosity of actin gels from sea urchin egg homogenates. In unfertilized mouse oocytes, it prevents the colcemid-induced dispersion of the meiotic chromosomes; accumulations of cortical actin are noted adjacent to the scattered chromosomes. Sperm incorporation during mouse fertilization in vitro is unaffected suggesting that sperm entry may occur independent of microfilament activity in mammals. However, the apposition of the pronuclei at the center of the egg cytoplasm does not occur, providing evidence that cytoplasmic microfilaments may be required for the motions leading to pronuclear union during mouse fertilization. It inhibits the second polar body formation and cytokinesis. These results indicate that latrunculin is a potent inhibitor of microfilament-mediated processes in sperm, eggs and embryos, and that it may prove to be a powerful new drug for exploring the cellular behavior of microfilaments in the maintenance of cell shape and during motility.

Biochemistry of the Mitotic Spindle

Publisher Summary The mitotic spindle consists of the chromatic and the achromatic figure. The chromatic figure consists of the chromosomes and includes their attachment points to the spindle, the kinetochores. The achromatic figure is more difficult to define. It consists of the transient spindle-like structure, and, where presents, the asters and the centrioles. This chapter discusses the biochemistry of the achromatic figure, the evolution of the mitotic spindle, the ultrastructure of the mitotic spindle, the molecules in the isolated mitotic apparatus, the characteristics of the various isolation procedures, the criteria used for evaluating them, and the composition of the isolated spindle. The chapter discusses the repetition of the mitotic process in vitro , using spindle proteins in spindle models. Microtubules are one of the prominent structures found in the spindle. They are composed of one major protein that was named “tubulin.” The two protomeric subunits are termed “α-tubulin” and “β-tubulin.”

Rhythmic oscillations of cytosolic free calcium m rat C-cells

The relationship between stimulation of single C-cells (rMTC-6-23 cell line) with extracellular calcium, glucagon or 8-bromo-cAMP and fluctuations of intracellular free calcium concentration was studied. After pretreatment of rMTC cells with either 1 microM glucagon (30-60 min) or 1 mM 8-bromo-cAMP (5 min) [Ca2+]i started to oscillate when extracellular calcium was raised to 3 mM. These fluctuations in [Ca2+]i could be stopped by chelating the external calcium with EGTA or by adding calcium channel blockers. The voltage-dependent calcium channels in the plasma membrane seem to play a major role in maintaining the oscillations of [Ca2+]i.

Ca2+-stimulated ATpase during the early development of parthenogenetically activated eggs of the sea urchin Paracentrotus lividus

Abstract A Ca 2+ -stimulated ATPase shows fluctuations in the activity in parthenogenetically activated sea urchin eggs very similar to those described earlier for fertilized eggs. Besides activity peaks in the first part of a cell cycle the enzyme activity increases when the mitotic apparatus (MA) or MA-like structures like monasters or cytasters are formed. A possible function of the enzyme in the assembly of the MA is discussed.

NH3-treatment of unfertilized sea urchin eggs turns on the Ca2+-ATPase cycle

Abstract Cyclic fluctuations of a Ca 2+ -ATPase activity are turned on in NH 3 -activated unfertilized sea urchin eggs, although no mitotic apparatus is formed. Subsequent fertilization of the activated eggs and the concomitant formation of spindle-like structures does not change drastically the course of the enzymatic fluctuations.

UV-irradiation of sea urchin eggs changes the course of the Ca2+-activated ATPase activity☆

Abstract UV-irradiation of sea urchin eggs extends the time of increased Ca2+-ATPase activity of the first peak in the cell cycle if the eggs are irradiated before fertilization and the time where in controls the activity of the first peak declines. Irradiation after this time causes delay of the second cell cycle; it extends also the duration of increased enzyme activity of the first peak in the second cycle. The enzyme itself does not seem to be UV-sensitive as UV-irradiation of homogenates does not alter the enzymatic activity.

FUNCTIONAL ANALYSIS OF THE INTRACELLULAR CA2+-REGULATING SYSTEM

Publisher Summary This chapter discusses the functional analysis of fast intracellular Ca 2+ regulating system. This trasnport capacity is vital for the cell to maintain required low Ca 2+ concentration and to exert local control of these ions in intracellular microdomains. The isolated Ca 2+ -transport system in chicken fibroblasts and after subsequent transformation with Rous Sarcoma Virus have been studied. To obtain more information on the essential components of the Ca 2+ -transport system, in vitro mouse lymphocytes were activated with the isolated membranes and those were subsequently fused with myeloma cells. The results showed that for the maintenance of the mitotic apparatus, especially its microtubules, an intact intracellular Ca 2+ transport system was required. Its breakdown, induced by the injection of the antibody, creates presumably a massive disturbance of the Ca 2+ homoeostasis, and causes depolymerization of microtubules and arrest of the cell in its cycle. A transformed cell changes its InsP 3 sensitivity allows a closer look on Tyr-phosphorylated components with regard to their capacity as InsP 3 receptors. The injection of antibodies specific for components of the intracellular Ca 2+ transport system allows a direct interference with Ca 2+ regulated processes in the living cell.