title: Single-molecule tracking of small GTPase Rac1 uncovers spatial regulation of membrane translocation and mechanism for polarized signaling.
abstract: 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.
authors.affiliation: Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT 06030.
authors.forename: Sulagna
authors.initials: S
authors.lastname: Das
authors.affiliation: Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT 06030.
authors.forename: Taofei
authors.initials: T
authors.lastname: Yin
authors.affiliation: Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT 06030.
authors.forename: Qingfen
authors.initials: Q
authors.lastname: Yang
authors.affiliation: Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT 06030.
authors.forename: Jingqiao
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT 06030.
authors.forename: Yi I
authors.initials: YI
authors.lastname: Wu
authors.affiliation: Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT 06030 jyu@uchc.edu.
authors.forename: Ji
authors.initials: J
authors.lastname: Yu
mesh_terms: D006801:Humans; D007700:Kinetics; D061986:MCF-7 Cells; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D015398:Signal Transduction; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
chemical_list: D020830:rac1 GTP-Binding Protein
keywords: Rac1; RhoGTPase; cell polarization; sptPALM; super-resolution microscopy