Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Summary

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

Abstract

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 time³. 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 Rac⁴, Cdc42⁴, RhoA⁴ and Ras⁵, 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 cells⁶. 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 level⁶.

Protocol

1. Transfection of Plasmid DNA

1. Add plasmid DNAs, 0.5 μg membrane-tethered FRB (hereafter referred to as LDR) and 0.5 μg FKBP-Tiam1 (T-cell lymphoma invasion and metastasis-inducing protein 1: Rac activator) tagged with fluorescent protein to 37.5 μl dH₂O. Add 1 μl FuGENE HD.
2. Vortex and incubate at room temperature for 20 minutes. Meanwhile, proceed to steps 1.3-1.8.
3. Wash each well of an 8-well chamber with 50 μl poly-D-Lysine.
4. Wash the center of each well with dH₂O.
5. Trypsinize 80-85% confluent NIH-3T3 cells and dilute to 10 ml culture medium (DMEM with 10% FBS).
6. Remove 375 μl of suspen....

Discussion

We described a technique that employs a novel caged compound together with the FKBP-FRB heterodimerization system in order to manipulate Rho GTPase activity at a precise subcellular location on a time scale of seconds.

There are three limitations of the present approach. Firstly, because the method is based on preventing or allowing diffusion of dimerizer across the plasma membrane, the target cellular location needs to be plasma membrane or its vicinity. Further optimization of the chemical .......