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

This video demonstrates a method in which UV light is used to manipulate the activity of a protein at the plasma membrane. To do this, cells are cot transfected with membrane anchored FRB protein and the FK BP fused protein of interest. A photo caged rapamycin derivative is added to the medium.

Then during live cell imaging, UV light is applied to a specific region of the cell. The linker between rapamycin and biotin is cleaved releasing chemical dimerizer and its byproduct the released dimerizer diffuses into cells and induces dimerization between the FKBP labeled protein of interest and plasma membrane anchored FRB in the irradiated region. Without the UV radiation, FKBP and FRB do not associate, hence producing no effect.

Analysis of data reveals highly localized recruitment of the FKBP fused protein of interest based on its physiological effect. The advantage of this technique over existing methods such as pharmacology and genetics approach, is the combination of speed of action, target specificity, and spatial resolution. The implications of this technique extend toward therapy of cancer metastatic is a consequence of migration by cancer cells.

Therefore, manipulation of activity of key migration factors may reveal its precise molecular mechanism, which is prerequisite for its treatment and cure. Demonstrating procedure will be Chris Paul Meyer, a graduate student and Bob de Rose, a technician from my laboratory. Begin this protocol by combining the following in a sterile micro centrifuge tube plasmid, DNA, membrane tethered FRB FLUORESCENTLY tagged FKBP, TIA one and deionized water add one microliter a few gene HD to the mixture and vortex.

Incubate the tube at room temperature for 20 minutes. During the incubation, add 50 microliters of poly de lycine to each well of a glass eight well chamber side and incubate for one minute to coat the surface, then aspirate the solution and wash the center of each well with 50 of deionized water. Next trypsin is 80 to 85%confluent NIH three T three cells.

After the cells detach, add 10 milliliters of DMEM containing 10%FBS transfer 375 microliters of the suspension to a 15 milliliter sterile tube and centrifuge at 1, 500 G for three minutes. Following the spin, carefully aspirate the medium. Add 750 microliters of DMEM with 10%FVS and Resus.

Suspend the pellet by gently pipetting up and down several times. Finally, add the cell suspension to the transfection solution and using the pipette tip stir gently, add 250 microliters of the cell transfection suspension to each well of the chamber. Slide and incubate it.

37 degrees Celsius for 15 to 18 hours. To synthesize cage rapamycin, biotin adduct. Begin by preparing a one millimolar stock solution of CRB in DMSO in a sterile fuge tube in a separate 1.5 milliliter micro centrifuge tube dissolve one milligram of avadon in 250 microliters of PBS.

Next to conjugate the caging moiety to rapamycin. Add 0.5 microliters of the CRB stock solution to dissolved AVID in mixed by inverting several times and then incubated room temperature for 15 minutes. To yield CRB AVID in conjugate solution to exclude unbound small molecules and purify CRBA equilibrate a G 25 size exclusion column with 300 microliters of PBS.

Repeat five times after the column has equilibrated. Load 125 microliters of CRBA solution onto the center bed of the column. Then spin down the solution for two minutes at 800 G At room temperature, collect the flow through in a 1.5 milliliter micro centrifuge tube following transfection of the cells.

Exchange the medium to DMEM without FBS to serum starve the cells incubate for 12 hours. After the incubation, gently aspirate the medium and add 250 microliters of the prepared one micromolar CRBA solution. Next to visualize protein dynamics in living cells, use a spinning disc confocal microscope with UV illumination to couple optics for UV LED light at 365 nanometers.

With epi fluorescence light. Define the coordinates and size of an illumination spot by using a glass plate coated with fluorescent dyes excited by UV light. This permits fine tuning of the area to be irradiated.

Place the chamber slide with the cells on the stage using a 100 x objective and epi fluorescence. Bring the cells into focus and find a cell. For imaging, begin imaging the cells after determining the background levels of the physiological effect.

Here, membrane ruffling is measured irradiate UV light in one chosen quadrant of the cell, dissolve uncaged, thus original rapamycin in DMSO and then in PBS to a final concentration of 400 nanomolar. Add 100 microliters of the solution to the imaging chamber to induce global effects. Capture fluorescence images every 15 seconds for 20 minutes to determine whether localized activation of RAC can induce local ruffle formation.

NIH three T three cells were transfected with YFP tagged RAC activator FK bp, TIA one and LDR, which is a membrane anchored. FRB localized irradiation at the edge of the cell indicated by the yellow circle, was followed by global illumination with a mercury lamp for one second starting at 1063 seconds. As can be seen here, almost no membrane ruffling was present at the beginning.

However, following localized illumination spatially confined ruffling was observed after global illumination and addition of rapamycin, which causes translocation of FK bp TIA one to the plasma membrane. The cell showed global ruffle formation indicating that the previously observed local ruffle formation was caused by spatially confined activation of rack. For a quantitative analysis, target cells were divided into four quadrants and the frequency of ruffle formation in each quadrant upon local as well as following global stimulation was determined as seen in this graph.

None of the observed cells showed ruffle formation with CRBA without uve a radiation or local uve, a radiation with or without Aden in bulk medium. However, when CRBA was added and UV irradiation was performed, ruffle formation was seen at about the same, roughly 90%in all quadrants with global radiation when only quadrant one was irradiated, the frequency of ruffle formation was 100%compared to 30%25%and 30%in quadrants two through four. These results indicate that localized uncaging of rapamycin can be used to induce highly localized physiological effects in a single cell.

In this case, membrane ruffling caused by rack activation. After watching this video, you should have a good understanding of how to achieve spatial temporal control of small g TPAs activity by light. This method could be applied to any physiological process that is dependent on specific proteins translocating to the plasma membrane.

While attempting this procedure, it is important to remember to freshly prepare solutions, reagents and cells for each procedure. Don't forget that working with UV light can be extremely hazardous and precautions such as UV glasses should always be worn.