Spindle function in Xenopus oocytes involves possible nanodomain calcium signaling

Intracellular calcium transients are a universal phenomenon at fertilization and are required for egg activation, but the exact role of Ca in second polar body emission remains unknown. On the other hand, similar calcium transients have not been demonstrated during oocyte maturation and, yet, manipulating intracellular calcium levels interferes with first polar body emission, in mice and frogs. To determine the precise role of calcium signaling in polar body formation, we employed live cell imaging coupled with temporally precise intracellular calcium buffering. We found that BAPTA-based calcium chelators caused immediate depolymerization of spindle microtubules in meiosis I and meiosis II. Surprisingly, EGTA, at similar or higher intracellular concentrations, had no effect on spindle function or polar body emission. Using two calcium probes containing permutated GFP and the calcium sensor calmodulin (Lck-GCaMP3 and GCaMP3), we demonstrated enrichment of the probes at the spindle but failed to detect calcium increase during oocyte maturation, at the spindle or elsewhere. Finally, endogenous calmodulin was found to co-localize with spindle microtubules throughout all stages of meiosis. Our results, most importantly the different sensitivity of the spindle to BAPTA and EGTA, suggest that meiotic spindle function in frog oocytes requires highly localized, or nanodomain, calcium