The role of cytoplasmic calcium gradients, calmodulin, and actin filaments in the early development of the fucoid alga, Pelvetia compressa

The mechanisms by which the cellular polarity was established, maintained and regulated in the zygotes of the marine brown alga, Pelvetia compressa , were investigated. The foci of attention were the involvement of cytoplasmic calcium (Ca2+) gradients, calmodulin and actin cytoskeleton. To establish the long predicted existence of cytoplasmic Ca 2+ gradients during Pelvetia polarization, a long excitation wavelength Ca2+ indicator, Calcium Crimson dextran, and a Ca2+-insensitive reference dye, Rhodamine B dextran, were microinjected into living zygotes separately. Ratios representing relative Ca2+ concentrations were calculated by dividing the averaged fluorescent pixel values of Calcium Crimson images by the averaged fluorescent pixel values of corresponding Rhodamine B images. A cytoplasmic Ca 2+ gradient was found to form within one hour of the exposure of the zygotes to unilateral blue light during the photosensitive period. Similarly, zygotes polarizing in the dark formed cytoplasmic Ca2+ gradients prior to germination as well. The injection site was the preferred germination site for dark-grown zygotes, and injection-induced cytoplasmic Ca2+ gradients could be redirected elsewhere. The region of high Ca 2+ formed during polarization was localized to and predictive of the site of future germination. This indirect ratiometric approach was validated by its application to the growing rhizoid, where the known tip-focused Ca 2+ gradient was clearly revealed. Microinjection of zygotes with antibodies made against Dictyostelium calmodulin inhibited germination, and this inhibition was abolished when the calmodulin antibodies were coinjected with an excess of purified maize calmodulin. Likewise, the growth of the rhizoids was inhibited by calmodulin antibody injections. Those results indicate that calmodulin is the cellular mediator of cytoplasmic Ca2+ gradients during both germination and rhizoidal growth. Calmodulin may exert its effect through the activation of Ca2+/calmodulin-dependent protein kinase II or its homologue. Cytoplasmic Ca2+ gradients did not form during photopolarization in the presence of a specific actin filament inhibitor, but then formed normally upon the removal of the inhibitor. The dynamics of actin filaments in living zygotes imaged with Alexa 568-conjugated phalloidin revealed that actin cytoskeleton reorganization was rapid upon light exposure. Actin may be involved in actively transporting plasma membrane Ca2+ channels to the future germination site