Abstract:

Objective To detect the mobility of a series of green fluorescence protein (GFP) tagged wild-type and mutant promyelocytic leukemia zinc finger-retinoic acid receptor α (PLZF-RARα) expressing proteins by fluorescence recovery after photobleaching (FRAP). Methods The electrophoretic mobility shift assay (EMSA) was adopted to detect the ability of GFP tagged wild-type and mutant PLZF-RARα fusion protein binding with retinoic acid response elements (RARE). The intranuclear mobility of PLZF-RARα and its mutant expressing proteins was analyzed by FRAP and confocal fluorescence microscopy. GFP-RARα and GFP-PLZF-RARα expressing cells were treated by transcriptional inhibitor actinomycin D (Act D) and/or ATRA. Variations of the intranuclear mobility of PLZF-RARα and wild-type RARα under these treatments was observed by FRAP. Results POZ and zinc finger domains within PLZF moiety not only contributed to the reduction of intranuclear mobility of PLZF-RARα, but also caused the decrease of ligand-induced intranuclear mobility. Act D could effectively immobilize the intranuclear mobility of PLZFRARα and RARα protein molecules. Treatment with differentiation inducer all-trans retinoic acid (ATRA) could reverse Act D induced immobilization of the intranuclear mobility of RARα, but could not reverse the immobilization of intranuclear mobility of PLZF-RARα treated by Act D. Conclusion Decreased intranuclear mobility of PLZF-RARα fusion protein detected by FRAP may rely on the existence of ligand and its transcription activity.

Key words: promyelocytic leukemia zinc finger-retinoic acid receptor α, retinoic acid receptor α, green fluorescence protein, fluorescence recovery after photobleaching, transcription