Measuring nuclear calcium and actin assembly in living cells.

Abstract

Nuclear calcium signalling has emerged as a critical mechanism regulating processes like chromatin organization and gene expression. Recently, we have shown that nuclear calcium elevation triggers rapid and transient actin filament assembly inside the nucleus. Here, we constructed and employed a nuclear-specific calcium sensor based upon the new generation of genetically encoded probes jGCaMP7f. By fusing a nuclear localization signal to jGCaMP7f, we achieved highly efficient nuclear-specific targeting. Comparing the jGCaMP7f-NLS probe with the previous GCaMP6f-NLS calcium sensor showed clearly that jGCaMP7f-NLS is more sensitive and reverses significantly quicker thereby reflecting rapid nuclear calcium transients in a closely physiological manner. We further confirm that nuclear calcium transients precede nuclear actin polymerization by several seconds. Our data show that calcium-triggered nuclear actin assembly in fibroblasts is independent of the actin nucleating Arp2/3 complex. Together, jGCaMP7f-NLS represents an easy to use, reliable and highly sensitive nuclear calcium sensor that allows to tightly interrogate real-time, spatiotemporal calcium signalling and calcium-elicited effects in the nucleus of living cells.