LOCALIZATION OF POLYMERIZED ACTIN AND CYCLIC NUCLEOTIDE MEASUREMENTS DURING SEA URCHIN FERTILIZATION (MICROFILAMENTS, LATRUNCULIN).

摘要

Filamentous actin is localized in cells using rhodaminyl-phalloidin (RPHD) after p-formaldehyde fixation and lysolecithin permeabilization. The surface of unfertilized Arbacia punctulata and Lytechinus variegatus eggs have numerous punctate fluorescence sites, indicating the presence of actin oligomers or polymers. Within thirty seconds of insemination a fertilization cone is detected as an erect structure on the surface of the egg. The fertilization cone grows to a maximum size by 8 to 9 minutes and is resorbed by 16 minutes after insemination. Fluorescent fibers are found on the surface of eggs by 10 minutes after insemination as a result of a burst in actin polymerization. Elongated microfilaments persist through cytokinesis. At cytokinesis a narrow bundle of microfilaments, the contractile ring, is found in the cleavage furrow.;Latrunculin-A (Lat-A), a recently identified inhibitor of microfilaments, reduces the viscosity of actin gels formed from sea urchin egg homogenates. An acrosomal process is not formed in sea urchin sperm treated with Lat-A, although, the acrosomal vesicle contents are released. Unpacking of the Limulus (horseshoe crab) sperm pre-assembled acrosomal process is not affected by Lat-A, indicating filaments not undergoing subunit exchange are unaffected by Lat-A.;To determine if cyclic nucleotides regulate cytoskeletal activity, radioimmune assays were performed to measure cyclic nucleotide levels. cAMP and cGMP were found to fluctuate in cycles similar to cytoskeletal activity in sea urchin eggs. Treatment with isobutylmethyl xanthine, a phosphodiesterase inhibitor, resulted in elevated levels of cAMP and cGMP and division did not occur. However, treatments with another phosphodiesterase inhibitor, Ro 20 1724/1, or cholera toxin, or cyclic nucleotide analogues, resulted in elevated cAMP and cGMP levels, but did not affect normal development. Therefore, in these experiments the levels of cyclic nucleotides in eggs do not appear to regulate motion directly.