Abstract
Chromophore-assisted light inactivation (CALI) enables us to inactivate proteins in specific cells or induce target cell death based on light-inducible and tissue-selective cell ablation. KillerRed can produce at least 1000 times as much oxygen as EGFP. CALI using proteins fused with KillerRed is a useful optogenetic method that has revealed the spatiotemporal function of proteins in vivo. Cohesion is essential for the identification of sister chromatids and for the biorientation of chromosomes until their segregation. An RNA-binding motif protein encoded on the X chromosome (RBMX) was identified as a chromosomal protein responsible for chromosome cohesion. Depletion of RBMX by RNA interference (RNAi) causes the loss of cohesin from the centromeric regions before anaphase, resulting in premature chromatid separation. In order to confirm the G2 phase-specific function of RBMX in nuclei, we performed spatiotemporal knockdown analyses by CALI using KillerRed fused with RBMX. We transfected a KillerRed-RBMX expression vector into HeLa cells expressing EGFP fused with histone-H1.2 and monitored chromosome dynamics by live cell imaging. At the consequence, we confirmed that RBMX is a novel cohesion regulator that maintains the proper cohesion of sister chromatids at G2 phase.
