Spatio-temporal manipulation of small GTPase activity at subcellular level and on timescale of seconds in living cells

• Published in: Journal of visualized experiments 2012-03 (61)
• Main Authors: DeRose, Robert, Pohlmeyer, Christopher, Umeda, Nobuhiro, Ueno, Tasuku, Nagano, Tetsuo, Kuo, Scot, Inoue, Takanari
• Format: Article
• Language: English
• Subjects: Animals; Bioengineering; Dimerization; Enzyme Activation; GTP Phosphohydrolases - chemistry; GTP Phosphohydrolases - metabolism; Mice; Microscopy, Confocal - instrumentation; Microscopy, Confocal - methods; NIH 3T3 Cells; Sirolimus - pharmacology; Subcellular Fractions - enzymology; Tacrolimus Binding Proteins - chemistry; Tacrolimus Binding Proteins - metabolism
• ISSN: 1940-087X; 1940-087X
• DOI: 10.3791/3794

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events(1, 2). Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time(3). In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac(4), Cdc42(4), RhoA(4) and Ras(5), in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells(6). Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level(6).