期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：3
页码：E267-E276
DOI：10.1073/pnas.1409667112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceRac1 activation involves two steps: translocation to plasma membrane and nucleotide exchange. Most previous studies focused on the nucleotide exchange cycle. Here we sought to understand membrane translocation dynamics by developing a single-particle tracking-based method. The labeled Rac1 molecules were further adapted for simultaneous FRET sensing of Rac1 nucleotide state, enabling a simultaneous comparison between Rac1 translocation dynamics and its nucleotide exchange dynamics. Elevated membrane recruitment can contribute significantly to polarized Rac1-signaling. This finding draws attention to the importance of spatial regulation of the Rac1 translocation process in the regulation of RhoGTPase signaling. Rac1 recruitment to membrane precedes its interaction with protein factors (e.g., GEFs) and is governed by phospholipid distributions. This finding resolves a long-standing question of the mechanism of Rac1 activation. Polarized Rac1 signaling is a hallmark of many cellular functions, including cell adhesion, motility, and cell division. The two steps of Rac1 activation are its translocation to the plasma membrane and the exchange of nucleotide from GDP to GTP. It is, however, unclear whether these two processes are regulated independent of each other and what their respective roles are in polarization of Rac1 signaling. We designed a single-particle tracking (SPT) method to quantitatively analyze the kinetics of Rac1 membrane translocation in living cells. We found that the rate of Rac1 translocation was significantly elevated in protrusions during cell spreading on collagen. Furthermore, combining FRET sensor imaging with SPT measurements in the same cell, the recruitment of Rac1 was found to be polarized to an extent similar to that of the nucleotide exchange process. Statistical analysis of single-molecule trajectories and optogenetic manipulation of membrane lipids revealed that Rac1 membrane translocation precedes nucleotide exchange, and is governed primarily by interactions with phospholipids, particularly PI(3,4,5)P3, instead of protein factors. Overall, the study highlights the significance of membrane translocation in spatial Rac1 signaling, which is in addition to the traditional view focusing primarily on GEF distribution and exchange reaction.
