GPCR-induced calcium transients trigger nuclear actin assembly for chromatin dynamics

Although the properties of the actin cytoskeleton in the cytoplasm are well characterized, the regulation and function of nuclear actin filaments are only recently emerging. We previously demonstrated serum-induced, transient assembly of filamentous actin within somatic cell nuclei. However, the extracellular cues, cell surface receptors as well as underlying signaling mechanisms have been unclear. Here we demonstrate that physiological ligands for G protein-coupled receptors (GPCRs) promote nuclear F-actin assembly via heterotrimeric Gαq proteins. Signal-induced nuclear actin responses require calcium release from the endoplasmic reticulum (ER) targeting the ER-associated formin INF2 at the inner nuclear membrane (INM). Notably, calcium signaling promotes the polymerization of linear actin filaments emanating from the INM towards the nuclear interior. We show that GPCR and calcium elevations trigger nuclear actin-dependent alterations in chromatin organization, uncovering a general cellular mechanism by which physiological ligands and calcium promote nuclear F-actin assembly for rapid responses towards chromatin dynamics.


Statistics
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Software and code
Policy information about availability of computer code Data collection Quantitative PCR data were collected with Bio-Rad CFX system. Live cell imaging data were collected using Zeiss LSM 800 system or Visitron systems. FLIM data were collected on Leica TCS SP8 system. EM data were acquired on FEI Tecnai 12 TEM.

Data analysis
Statistical data analysis was performed using GraphPad Prism 7 or Microsoft Office Excel. Two-sided Student's t-test was performed when there were only 2 experimental conditions. One-way ANOVA was performed for experiments with more than two conditions. Stars in the figures indicate P values: *: p≤0.05; **: p≤0.01; ***: p≤0.001; ****: p≤0.0001. Images were analyzed with FIJI/Image J (NIH), Zen software (Zeiss) or Metamorph software (Molecular Devices). FLIM data were fitted using the FLIMfit (Imperial College London). For quantification of heterochromatin, nuclei and nucleoli were manually segmented. A FIJI machine learning plugin (Trainable Weka Segmentation) and a macro were used to quantify the region classed as heterochromatin, including the proportion of the nucleus occupied by heterochromatin. Calcium images by GCaMP6f and nAC-mCherry were analyzed by MATLAB. Calcium elevation in the whole cell or nucleus was measured by quantifying the fluorescence intensity of GCaMP6f, and the nuclear actin polymerization was measured by calculating the maximal spatial signal heterogeneity of nAC-mCherry.
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