@article {69223,
	title = {Fluorescence complementation-based FRET imaging reveals centromere assembly dynamics.},
	journal = {Molecular Biology of the Cell},
	year = {2024},
	month = {2024 Feb 21},
	pages = {mbcE23090379},
	abstract = {<p>Visualization of specific molecules and their assembly in real time and space is essential to delineate how cellular dynamics and signaling circuit are orchestrated during cell division cycle. Our recent studies reveal structural insights into human centromere-kinetochore core CCAN complex. Here we introduce a method for optically imaging trimeric and tetrameric protein interactions at nanometer spatial resolution in live cells using fluorescence complementation-based F{\"o}rster resonance energy transfer (FC-FRET). Complementary fluorescent protein molecules were first used to visualize dimerization followed by FRET measurements. Using FC- FRET, we visualized centromere CENP-SXTW tetramer assembly dynamics in live cells, and dimeric interactions between CENP-TW dimer and kinetochore protein Spc24/25 dimer in dividing cells. We further delineated the interactions of monomeric CENP-T with Spc24/25 dimer in dividing cells. Surprisingly, our analyses revealed critical role of CDK1 kinase activity in the initial recruitment of Spc24/25 by CENP-T. However, interactions between CENP-T and Spc24/25 during chromosome segregation is independent of CDK1. Thus, FC-FRET provides a unique approach to delineate spatiotemporal dynamics of trimerized and tetramerized proteins at nanometer scale and establishes a platform to report the precise regulation of multimeric protein interactions in space and time in live cells.</p>
},
	issn = {1939-4586},
	doi = {10.1091/mbc.E23-09-0379},
	author = {Dou, Zhen and Liu, Ran and Gui, Ping and Fu, Chuanhai and Lippincott-Schwartz, Jennifer and Yao, Xuebiao and Liu, Xing}
}
