Rs. Pu et Kr. Robinson, Cytoplasmic calcium gradients and calmodulin in the early development of the fucoid alga Pelvetia compressa, J CELL SCI, 111, 1998, pp. 3197-3207
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.