Nuclear Ca2+ concentration measured with specifically targeted recombinant aequorin

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Nuclear events like transcription factor activation and apoptosis are potentially regulated by nucleoplasmic calcium (Ca2+) concentration ([Ca2+]n).
• Direct measurement of [Ca2+]n in living cells has been technically challenging, limiting understanding of its role.

Purpose of the Study:

• To develop and validate a novel method for monitoring Ca2+ concentrations specifically within the nucleus of living cells.
• To investigate the dynamics and characteristics of nuclear Ca2+ signaling in response to cellular stimuli.

Main Methods:

• Construction of a chimeric cDNA encoding a fusion protein (nuAEQ) of photoprotein aequorin and a nuclear translocation signal.
• Stable expression of nuAEQ in HeLa cells for selective nucleoplasmic localization and Ca2+ monitoring.
• Simultaneous measurement of nuclear ([Ca2+]n) and cytosolic ([Ca2+]i) Ca2+ concentrations under resting and stimulated conditions.

Main Results:

• The modified aequorin (nuAEQ) was successfully targeted to the nucleoplasm, enabling reliable [Ca2+]n measurements.
• Resting [Ca2+]n and [Ca2+]i showed no significant differences.
• Upon stimulation, the kinetics of [Ca2+]i and [Ca2+]n increases were similar, but the amplitude of nuclear Ca2+ increase was greater when evoked by internal Ca2+ store mobilization compared to plasma membrane influx.

Conclusions:

• The developed nuAEQ system provides a robust tool for real-time monitoring of nuclear Ca2+ in living cells.
• Nucleus-cytosol Ca2+ gradients exist, with distinct amplitudes depending on the Ca2+ signaling pathway.
• These findings highlight the importance of localized Ca2+ signaling within the nucleus for cellular processes.