• PLCζ and calmodulin (CaM) co-localise in mature mammalian sperm • CaM binds the PLCζ XY-linker via cooperative engagement of both lobes • Ca 2+ /CaM inhibits PLCζ catalytic activity at elevated Ca 2+ levels • MARCKS effector-domain peptide competes with PLCζ for CaM binding • MARCKS-CaM interaction restores PLCζ activity and IP 3 generation • A PLCζ-CaM-MARCKS network model reproduces fertilisation-like Ca 2+ oscillations Precise regulation of intracellular calcium (Ca 2+ ) signalling is essential for successful egg activation and mammalian fertilisation, with sperm-specific phospholipase C zeta (PLCζ) acting as the physiological trigger of Ca 2+ oscillations following gamete fusion. Despite significant advances in understanding PLCζ function, the native regulatory partners and competitive interactions that modulate its activity during fertilisation remain poorly understood. In this study, we demonstrate that PLCζ and calmodulin (CaM) co-localise strongly in mature mammalian sperm and that direct binding between the two proteins requires cooperative engagement of both CaM lobes with the XY-linker region of PLCζ. Functional enzymatic assays reveal that at elevated Ca 2+ concentrations, Ca 2+ /CaM acts as a potent inhibitor of PLCζ activity, identifying a Ca 2+ -dependent negative regulatory mechanism that may contribute to tuning of Ca 2+ oscillations. Furthermore, molecular modelling and in vitro competition assays show that the MARCKS effector-domain peptide, an established CaM-binding protein enriched at cellular membranes, effectively competes with PLCζ for CaM binding. This competition disrupts the PLCζ-CaM complex and restores PLCζ activity, suggesting that MARCKS acts as a molecular switch regulating PLCζ access to CaM. These findings uncover a new regulatory axis in which CaM and MARCKS dynamically modulate PLCζ function, thereby fine-tuning the Ca 2+ signalling cascade that initiates embryo development. A dynamic mathematical model incorporating these interactions demonstrates that the PLCζ-CaM-MARCKS network is sufficient to generate sustained Ca 2+ oscillations with fertilisation-like periodicity. Together, our findings provide mechanistic insight into the regulation of PLCζ activity and support a model in which CaM-mediated inhibition in sperm, followed by competitive interaction with MARCKS in the oocyte, may control phasic IP 3 generation and Ca 2+ oscillations during fertilisation. This novel framework offers a basis for future studies aimed at understanding defects in human oocyte activation.
Thanassoulas et al. (Fri,) studied this question.