Calcium Nanodomains at Spindles

Abstract

The spindle apparatus is arguably the most important organelle in cell division. Despite the comprehensive characterization of microtubule regulatory proteins and protein complexes in the past decades, we are still far from being able to reconstitute a functional spindle using purified components. One important question remaining unanswered is whether calcium signaling participates in spindle assembly. Calcium signaling is involved in cell division but is thought to be dispensable for spindle assembly, mainly because excessive EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) is present in the popular in vitro spindle assembly system – Xenopus egg extracts. However, a recent report showed that injection of the fast calcium chelator 1,2-bis(o-aminopheyoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA), but not the slow chelator EGTA, causes rapid depolymerization of spindle microtubules in Xenopus oocytes. Differential sensitivity to BAPTA is the defining characteristic of highly restricted, or nanodomain Ca²⁺ signaling, best known to function in presynaptic neurotransmitter release. However, no local Ca²⁺ signals have been identified to be functionally linked to spindle assembly. In this project, a novel microtubule-binding calcium sensor, TubeCamp1 (TC1), was designed to test the hypothesis that spindles are associated with calcium nanodomains. Consistent with the hypothesis, confocal imaging with TC1 revealed microtubule-proximal calcium increase at the spindle poles in Xenopus oocytes and HeLa cells. Calcium nanodomains also formed in spindles assembled in vitro and at the center of monoastral spindles, suggesting that they are a general feature of spindle assembly. Disruption of nanodomain calcium signaling, via rapid chelation of calcium or perturbation of inositol-1,4,5-trisphosphate pathway, resulted in rapid spindle disassembly in intact oocytes and in oocyte extracts. These results demonstrate the existence of spindle-associated calcium nanodomains and suggest that such domains are an important and common feature of spindles in vertebrates. In addition, a Ca²⁺ transient was identified during polar body emission in Xenopus oocytes by using a novel ratiometric probe Tubecamp4 (TC4) and mobile probes Oregon Green-2 and -6f. A close examination of its spatiotemporal pattern with membrane-targeted probes revealed that the Ca²⁺ transient was associated with polar body abscission whereby the polar body is separated from the oocyte, suggesting a role of Ca²⁺ in ensuring the correct number of chromosomes and proper division of cytoplasm. Collectively, these results expand our knowledge in highly localized Ca²⁺ control of spindle assembly and cell cycles, providing new angles for understanding disease pathology and drug intervention.