Defining the Dynamics and Regulation of CaMKIIy in Egg Activation

About This Grant

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a critical serine/threonine kinase that translates cal- cium signals into long-term cellular responses across diverse systems, including the brain, heart, and reproduc- tive cells. This project focuses on CaMKIIγ, the variant essential for egg activation in mammals, seeking to define how its activation and autophosphorylation are regulated during oocyte maturation, fertilization, and early em- bryonic development. Our long-term goal is to unravel how divalent cations homeostasis and their targets, in- cluding CaMKII, are regulated and function in oocytes, eggs, and during embryo development. The overall ob- jective here is to monitor CaMKII kinase activity in real-time using FRET sensors during maturation, fertilization, and parthenogenetic stimulation and elucidate its regulation by autophosphorylation. Our central hypothesis is that the enhanced sensors will accurately report CaMKII activity, enabling us to monitor its activity and dynamics in real-time during oocyte maturation, throughout fertilization, and early cleavages. We further hypothesize that CaMKIIγ T287 phosphorylation is essential to maintaining kinase activity during the extended intervals between Ca2+ rises of fertilization. These hypotheses are supported by preliminary data showing that: 1) the origi- nal CaMKII biosensor FRESCA-1 only partially tracked CaMKII activity in cells in real-time, 2) newer FRESCA versions (FRESCA-2 and FRESCA-3) offer increased sensitivity and dynamic range, and signal stability for more than 6 hours, 3) eggs of Camk2g knockout (KO) mice have no detectable CaMKII activity, and 4) CaMKII activity and Ca²⁺-dependent egg activation can be restored in KO eggs by expressing CaMKII variants. The rationale for our study is that using the newly developed, robust, and sensitive FRESCA biosensors will enable the detection of CaMKII activity in oocytes and embryonic stages, where this has not been possible. This ap- proach will define the functional significance of CaMKIIγ, the sole isoform expressed in eggs, its regulation, and its role during fertilization and development. These insights will clarify the Ca²⁺ signals and CaMKII activity thresholds required for egg activation and their influence on developmental potential and early embryogenesis. Ultimately, it may inform novel fertility treatments and advance our understanding of CaMKII regulation. We will test these questions using the following specific aims: 1) Define the timing and activity of CaMKII during matu- ration, egg activation, and preimplantation embryo development. 2) Determine the role of CaMKIIγ autophos- phorylation in egg activation. This project investigates how CaMKII, a single kinase, can decode vastly different calcium signals—from rapid neural firing to the slow calcium waves of fertilization—by sustaining its activity through autophosphorylation. While this mechanism is well-established in neurons for memory formation, its role in slow-timescale events, such as in mammalian fertilization, remains unknown. By directly visualizing CaMKII activation in real-time, this work addresses a fundamental gap in our understanding of how a conserved regula- tory switch enables signal decoding across diverse physiological contexts.