This study was supported by NIH research funding (DK090868 and GM092930 to T.I.). There is a pending patent on a caged rapamycin analog.

1. Transfection of Plasmid DNA
<ol>
	<li>Add plasmid DNAs, 0.5 &mu;g membrane-tethered FRB (hereafter referred to as LDR) and 0.5 &mu;g FKBP-Tiam1 (T-cell lymphoma invasion and metastasis-inducing protein 1: Rac activator) tagged with fluorescent protein to 37.5 &mu;l dH2O. Add 1 &mu;l FuGENE HD.</li>
	<li>Vortex and incubate at room temperature for 20 minutes. Meanwhile, proceed to steps 1.3-1.8.</li>
	<li>Wash each well of an 8-well chamber with 50 &mu;l poly-D-Lysine.</li>
	<li>Wash the center of each wel...

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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 ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Rapamycin</td>
 <td>Tecoland</td>
 <td>RAPA99</td>
 </tr>
 <tr>
 <td>FuGENE HD</td>
 <td>Roche</td>
 <td>04709691001</td>
 </tr>
 <tr>
 <td>Avidin</td>
 <td>Sigma</td>
 <td>A9275-10MG</td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>Sigma</td>
 <td>D2650-5X5ML</td>
 </tr>
 <tr>
 <td>Size-exclusion column</td>
 <td>GE Healthcare</td>
 <td>Spin Trap G-25</td>
 </tr>
 <tr>
 <td>Glass bottom 8-well chamber</td>
 <td>Thermo Scientific</td>
 <td>12-565-47</td>
 </tr>
 <tr>
 <td>Inverted Fluorescence microscope</td>
 <td>Zeiss</td>
 <td>Axiovert200M</td>
 </tr>
 <tr>
 <td>UV LED light source</td>
 <td>Rapp Opto Electronics</td>
 <td>UVILED</td>
 </tr>
 <tr>
 <td>Confocal spinning disk</td>
 <td>Yokogawa Electric Corp.</td>
 <td>CSU10</td>
 </tr>
 <tr>
 <td>CCD camera</td>
 <td>Hamamatsu Photonics</td>
 <td>ORCA-ER</td>
 </tr>
 <tr>
 <td>100x Objective lens</td>
 <td>Zeiss</td>
 <td>Plan Apochromat</td>
 </tr>
 </tbody>
</table>
 Synthetic scheme of cRb (Copyright ACS2011) 6
 <a href="/files/ftp_upload/3794/3794scheme1_lg.jpg"><img src="/files/ftp_upload/3794/3794scheme1.jpg" alt="Scheme 1" /></a> 
Synthetic conditions: (a) BnBr, K2CO3, DMF (b) f.HNO3, AcOH (c) TFA (d) Propargyl-Br, K2CO3, DMF, (e) glycerol, cat. pTsA, Toluene (f) NaBH3CN, TiCl4, MeCN (g) Tf2O, Pyridin (h) methanol HCl, (i) LiAlH4, THF (j) TsCl, Pyridine, CH2Cl2 (k) NaN3, DMF (l) 8, 2,6-di-t-butylpyridine, CH2Cl2 (m) 13, CuSO4, Ascorbate, 2-propanol, H2O, CH2Cl2.

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

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 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.

Watch this Scientific Journal Video about Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells at JoVE.com

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

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.

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

spatio-temporal-manipulation-small-gtpase-activity-at-subcellular

Research

JoVE Journal

Bioengineering

10.7K visualizações.  Johns Hopkins University. Um método para espácio-temporal controlo da actividade GTPase pequena pela luz é descrito. Este método baseia-se em rapamicina induzida heterodimerização FKBP-FRB e foto-enjaulamento sistemas. Otimização de luz irradiação permite a ativação espaço-temporalmente controlada de pequenas GTPases no nível subcelular.