Cytoplasmic calcium gradients and calmodulin in the early development of the fucoid alga Pelvetia compressa

The predicted existence of cytoplasmic Ca2+ gradients during the photopolar ization of the zygotes of the brown algae, Pelvetia and Fucus, has proved t o be difficult to establish, and the downstream targets of the putative gra dients are not known. We have used quantitative microinjection of the long excitation wavelength Ca2+ indicator, Calcium Crimson, and of antibodies ag ainst calmodulin to investigate these matters in the zygotes and early embr yos of Pelvetia, We found that there is a window of cytoplasmic Calcium Crimson concentratio n that gives an adequate signal above autofluorescence yet allows normal de velopment of the zygotes, As Calcium Crimson is not a ratiometric indicator , we injected other zygotes with a Ca2+-insensitive dye, rhodamine B, and i maged the cells at the same time that Calcium Crimson-injected cells were i maged. Ratios were calculated by dividing the averaged pixel values of Calc ium Crimson images by the averaged pixel values of corresponding rhodamine B images. By this method, we observed the formation of a cytoplasmic Ca2+ g radient within one hour of the exposure of the cells to unilateral blue lig ht during the photosensitive period. The region of high Ca2+ was localized to and predictive of the site of future rhizoid formation. We validated thi s somewhat indirect method by applying it to the growing rhizoid, where the existence of a tip-localized Ca2+ gradient is well established. The method clearly revealed the known gradient, The injection of ungerminated zygotes with antibodies made against Dictyost elium calmodulin inhibited germination, and this inhibition was abolished i f the calmodulin antibodies were coinjected with an excess of purified maiz e calmodulin, Likewise, the growth of the rhizoids was inhibited by calmodu lin antibody injections. The fungus-derived calmodulin antagonist, ophiobol in A, which has previously been shown to be a potent inhibitor of germinati on, also inhibited rhizoidal growth. Our results provide evidence that a cy toplasmic Ca2+ gradient is present during photopolarization and that calmod ulin acts as a mediator of Ca2+ gradients throughout the early developmenta l processes of germination and rhizoidal growth in Pelvetia compressa.