Genetically Encoded FRET Biosensor for Live-cell Visualization of Lamin A Phosphorylation at Serine 22

Abstract

Extensive phosphorylation at Serine 22 (pSer22) on lamin A is the hallmark of cell mitosis, which contributes to the breakdown of nuclear envelope. In the interphase, the pSer22 lamin A exists in low abundance and involves in mechanotransduction, virus infection and gene expression. Numerous evidence emerges to support lamin A regulation on cell function and fate by phosphorylation. However, live-cell imaging tools for visualizing the dynamics of pSer22 lamin A is yet to be established. Herein, we developed a novel lamin A phosphorylation sensor (LAPS) based on fluorescence resonance energy transfer (FRET) with high sensitivity and specificity. We observed the dynamic lamin A phosphorylation during cell cycle progression in single living cells: the increase of pSer22 modification when cells enter the mitosis and recovered upon the mitosis exit. Our biosensor also showed the gradual reduction of pSer22 modification during cell adhesion and in response to hypotonic environment. By applying LAPS, we captured the propagation of pSer22 modification from inside to outside of the inner nuclear membrane, which further led to the breakdown of nuclear envelope. Meanwhile, we found the synchronous phosphorylation of lamin A and H3S10 at mitosis entry. Inhibition of Aurora B, the responsible kinase for H3S10ph, could shorten the mitotic period without obvious effect on the pSer22 modification level of lamin A. Thus, LAPS allows the spatiotemporal visualization of lamin A phosphorylation at Serine 22 site, which will be useful for elucidating the molecular mechanisms underlying cell mitosis and mechanoresponsive processes.