Name: an integrated system to remotely trigger intracellular signal transduction by upconversion nanoparticle-mediated kinase photoactivation
Uploaded: 2017-08-30T14:00:00
Description: Watch this Scientific Journal Video about An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation at JoVE.com

We thank the Nano Science and Technology Program of Academia Sinica and the Ministry of Science and Technology of Taiwan for the funding (101-2113-M-001-001-MY2; 103-2113-M-001-028-MY2).

NOTE: The protocol describes a detailed instrumentation modification for upconversion-assisted photoactivation, a synthetic procedure to generate caged PKA-UCNP, transmission electron microscopy (TEM) of the silica-coated UCNP and caged PKA-UCNP samples, UV and NIR photolysis setup, cell preparation, PKA-UCNP microinjection, a photoactivation study, and the stress fiber staining of REF52 cells.
1. Fluorimeter Setup for Upconversion Spectrum Measurement
<ol>
	<li>Install a 4X objective lens n...

<ol>
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Previously, Hofmann and coworkers found that dramatic morphological changes were observed in REF52 cells after the microinjection of the free PKA19. In another study, the Lawrence group demonstrated that caged PKA can be activated in vivo, leading to morphological changes and the disintegration of stress fibers when subjected to UV photolysis20. Earlier reports on exploiting upconverted UV light for photoactivation showed the activation of the several UCNP-assisted...

Chemically modified proteins that can be photoactivated (e.g., PKA caged proteins) have been developed as an emerging field in chemical biology to non-invasively manipulate intercellular biochemical processes1,2,3. Using light as a stimulus provides excellent spatiotemporal resolution when activating these caged proteins. However, UV light can cause undesired morphological changes, apoptosis, and DNA damage to cells4,5. Hence, recent developments in the design of photocaging groups focus on enabling photocleavage upon longer-wavelength or two-photon excitation to reduce phototoxicity, as well as to increase deep-tissue penetration6,7. Caging groups that respond to longer wavelength allowing us to choose suitable uncaging wavelengths (i.e., channels) to selectively activate bioeffectors when two or more caging groups are present7. Given these useful features, developing new red-light photocaging groups is very important upstream work in photochemical methodologies for biological studies ranging from probing the mechanisms of reactions to controlling cellular activities8. Nonetheless, a two-photon caging group is normally too hydrophobic due to the fused aromatic ring structure, and a visible-light caging group is normally organometallic, with aromatic ligands. This hydrophobic/aromatic property is not suitable when the bioeffector is a protein or enzyme, as it denatures the activation site of the enzyme/protein and causes loss of function, even if the conjugation and photolysis still work on the chemical level2,9.
UCNPs are effective transducers that convert the NIR excitation light to UV.This unique and fascinating property of UCNPs has offered realistic resolutions to address the challenges associated with photoactivation and triggered controlled release of small molecules, including folic acid10, cisplatin derivatives11, DNA/siRNA12, copolymer vesicles13, and hollow particles14. However, to the best of our knowledge, the UCNP-assisted photoactivation of enzymes or proteins has not been tested so far. Because there is no successful case of using red light or NIR to photoactive an enzyme, we were prompted to perform the NIR-triggered activation of a protein/enzyme construct composed of chemically modified caged enzyme complexes with a silica-coated, lanthanide-doped UCNP15. In this study, the UCNP was conjugated with a rapidly reacting signal transduction kinase in the form of caged PKA. PKA controls glycogen synthesis and cytoskeletal regulation that responds to external stimuli via cyclic adenosine phosphate (cAMP) regulation in the cytosol16. We studied the feasibility of enzyme activation in temporal and spatial manners in a cellular experiment after NIR irradiation. This UCNP-assisted photoactivation platform is a new methodology to photoactivate an enzyme using NIR and avoids the undesired signal transduction response from cells caused by conventional UV irradiation2,4.
It is very difficult to translocate large bioeffectors (e.g., proteins) across the cell membrane to control cellular activity. Although particle-immobilized protein may be easier to translocate via endocytosis into the cytosol, endocytosis may be damaged or degraded via endosomal entrapment and the consequent lysosomal degradation2,4. Even if the caged protein is still functional after membrane translocation, the translocated amounts may not be enough to trigger the cellular response2,17. In sharp contrast, microinjection is a direct and quantitative approach to deliver large bioeffectors to the cytoplasm of the cell. Moreover, UCNP-immobilized bioeffector requires upconverted light to be activated. Therefore, the optical instrumentation requires further modification to measure, visualize, and utilize the upconversion light. In this work, the delivery of a caged PKA-UCNP complex to a cell using microinjection and the following essential spectroscopy and microscopy modifications for NIR photoactivation will be described in detail.

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			<td height="20" style="height:20px;width:431px;">Reagent</td>
			<td style="width:243px;"></td>
			<td style="width:119px;"></td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris(hydorxymethyl)aminomethane</td>
			<td>Sigma</td>
			<td>154563</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium chloride hexahydrate</td>
			<td>Sigma</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MOPS</td>
			<td>Sigma</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride&#160;</td>
			<td>Sigma</td>
			<td>31434</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium chloride</td>
			<td>Sigma</td>
			<td>12636</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Yttrium acetate&#160;hydrate</td>
			<td>Sigma</td>
			<td>326046</td>
			<td>Y(C2H3CO2)3 &#183; xH2O</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thulium(III) acetate hydrate</td>
			<td>Alfa Aesar</td>
			<td>14582</td>
			<td>Tm(CH3CO2)3 &#183; xH2O</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ytterbium(III) acetate tetrahydrate</td>
			<td>Sigma</td>
			<td>326011</td>
			<td>Yb(C2H3O2)3 &#183; 4H2O</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-Octadecene</td>
			<td>Sigma</td>
			<td>O806</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oleic acid</td>
			<td>Sigma</td>
			<td>364525</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol&#160;</td>
			<td>macron</td>
			<td>304168</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydroxide</td>
			<td>Sigma</td>
			<td>30620</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium fluoride</td>
			<td>J.T.Baker</td>
			<td>69804</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IGEPAL&#160;CO-520</td>
			<td>Sigma</td>
			<td>238643</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cyclohexane</td>
			<td>J.T.Baker</td>
			<td>920601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amomonium hydroxide (28%-30%)</td>
			<td>J.T.Baker</td>
			<td>972101</td>
			<td>Ammonia</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetraethyl orthosilicate (TEOS)</td>
			<td>Sigma</td>
			<td>8658</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DL-Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-hydroxymaleimide (NHM)</td>
			<td>Sigma</td>
			<td>226351</td>
			<td>PKA activity blocking reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Prionex protein stabilizer solution from hog collagen</td>
			<td>Sigma</td>
			<td>81662</td>
			<td>Protein stabilizer solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-nitrobenzyl bromide (NBB)</td>
			<td>Sigma</td>
			<td>107794</td>
			<td>PKA caging reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">8-(4-Chlorophenylthio)adenosine 3&#8242;,5&#8242;-cyclic monophosphate sodium salt</td>
			<td>Sigma</td>
			<td>C3912</td>
			<td>8-CPT-cAMP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle</td>
			<td>Sigma</td>
			<td>P0294</td>
			<td>PK/LDH</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adenosine 5&#39;-triphosphate disodium</td>
			<td>Sigma</td>
			<td>A2387</td>
			<td>ATP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-NADH reduced from dipotassium</td>
			<td>Sigma</td>
			<td>N4505</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphoenolpyruvate</td>
			<td>Sigma</td>
			<td>P7127</td>
			<td>PEP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coomassie Protein Assay Reagent, 950 ml</td>
			<td>Thermo Scientific</td>
			<td>23200</td>
			<td>Bradford assay reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cAMP-dependent protein kinase</td>
			<td>Promega</td>
			<td>V5161</td>
			<td>PKA activity control</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pET15b-PKACAT plasmid</td>
			<td>Addgene</td>
			<td>#14921</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pKaede-MC1 plasmid</td>
			<td>CoralHue</td>
			<td>AM-V0012</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline (PBS), pH 7.4</td>
			<td>Thermo Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, high glucose, pyruvate</td>
			<td>Gibco</td>
			<td>12800-017</td>
			<td>Cell culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leibovitz L-15 Medium</td>
			<td>Biological Industries</td>
			<td>01-115-1A</td>
			<td>Cell culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Biological Industries</td>
			<td>04-001-1A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>ACROS</td>
			<td>416785000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>Nucleus staining dye</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa 594-phalloidin</td>
			<td>Invitrogen</td>
			<td>A12381</td>
			<td>F-actin staining dye</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5(6)-Carboxyfluorescein</td>
			<td>Novabiochem</td>
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an integrated system to remotely trigger intracellular signal transduction by upconversion nanoparticle-mediated kinase photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

The design of the caged enzyme-UCNP construct is illustrated in Figure 1. The PKA enzyme was first reacted with 2-nitrobenzyl bromide to generate an inactive caged PKA, and it was then electrostatically immobilized on the surface of UCNP. UCNPs emit the upconverted light and consequently photolytically cleave the o-nitrobenzyl groups on Cys 199 and Cys 343, generating the activated PKA. TEM images and Bradford assay confirmed that the PKA and caged PKA were i...

Watch this Scientific Journal Video about An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation at JoVE.com

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

The goal of this experiment is to demonstrate the feasibility to photoactivate enzyme using near infrared irradiation. Caged-protein kinase A is immobilized on this surface of upconversion nanoparticles micro-injected into cells and kinase induced response in living cells is observed after near infrared irradiation. Traditional photoactivation require phototoxic UV light and inevitably, will co-induce undesirable chemical reaction and similar response, however, in our friendly options, like near infrared irradiation cannot trigger reaction due to its low energy.The upconversion nanoparticle can effectively upconvert bio-friendly low energy near IR photon into high energy UV photon to trigger a chemical reaction near the particle surface without direct UV irradiation. Kinase plays a key role in oncogenesis, this technique can help us examine more signal transduction information with much lower interfering noise from UV phototoxicity and its side reaction. Generally, individuals new to this field will encounter difficulties in modifying instruments and optical setup for upconversion application.To begin, install a 4x objective lens next to the cuvette holder of the fluorospectrometer, so that the laser can be focused in the middle of the cuvette. Place a tunable 980 nanometer laser diode 90 degrees against the objective lens. Install a full reflectance mirror and the light path intersection of the objective lens and laser.Don't forget to place an anodized metal plate to block the excitation window of the spectrometer to protect the interior parts. Then place a cuvette of silica coated upconversion nanoparticle solution in the holder. A bright upconversion beam can be visualized when the laser turns on.Switch the spectroscopy to luminescence mode, with the lowest PMT voltage during the alignments. Adjust the mirror and laser stages so that the beam is through the middle of the cuvette for the PMT to pick up the strongest signal. After installing a dichroic mirror and an excitation filter, install a 980 nanometer laser diode in the lower tier light path.Add a collimator in between the laser and the mirror to help diffuse and enlarge the NIR irradiating area in the focal plane. Then use an upconversion nanoparticle solution to visualize the near infrared beam. Adjust the position of laser stage to center the near infrared light beam to finish the alignment.Perform caged-PKA synthesis and demobilize the caged-PKA/UCNP, to generate caged-PKA/UCNP as described in the text protocol. To measure the activity of both constructs before UV photolysis, measure the decrease in NADH absorbents in the assay mixture over time after the addition of two microliters of PKA solution to the assay mixture. Repeat the measurement for PKA/UCNP.Next, perform UV photolysis at 200 milliwatts per square centimeter of caged-PKA and caged-PKA/UCNP, in an ice water bath, to make sure PKA can be activated. PKA activity is determined by a pyruvate kinase lactate dehydrogenase enzyme coupled assay. With a decrease in NADH absorbents, is measured over time after the addition of PKA solution.Incubate the cells in 10%fetal bovine serum containing L15 medium during the microinjection period for stable pH control outside the incubator. Configure the needle puller for a cone shaped tip with a diameter of around 1.5 microns for later tip opening. For opening the tip, install the needle on the microinjector move the needle tip in contact with the edge of a slide and follow with a mild knob on the microscope stage.This vibration force is sufficient to slightly break the tip and enlarge the opening. Examine the needle opening to ensure that it has an external diameter from 1.3 to 1.5 microns which can be confirmed by microscope images using a 40x objective lens. Next, set the injection pressure from 50 to 75 hectopascals with a pressurized interval of 200 to 300 milliseconds and set the constant compensation pressure at 15 to 30 hectopascals to prevent medium backflow into the needle.Load the caged-PKA/UCNP complex with TAMRA dye at the back position of the needle. The loaded needle is then set up on one axis hydraulic micromanipulator, with an angle from 30 degrees to 45 degrees. Check the needle with microscope to make sure no bubble generated after sample loading.After immersing the needle into the medium, switch to fluorescence mode, to confirm a burst release of dye when injection pressure is applied and a slow release of dye when compensation pressure was constantly applied. Then switch to phase contrasted bright field mode, to start cell microinjection. The membrane shockwave can be observed in successful injections.Wash the cells twice and check the injected cells for co-injected TAMRA fluorescence to locate successfully microinjected cells. Cell with UCNP admission but without a co-injected dye signal, indicate the cell membrane was ruptured during injection and should be excluded from further experiments. Irradiate the cells with a 980 nanometer laser diode in the lower tier light path focused by a 10x objective lens for 15 minutes.Allow for a five minute dark interval after every five minutes of irradiation to avoid heating. If a shorter activation time is needed, such as seconds or even subseconds, simply switch to a 40x objective lens for higher focus photon density. Following photolysis and cell recovery, fix the cells in one milliliter or 4%paraformaldehyde in PBS for 20 minutes, before further alexa 594 followed within staining.Finally, visualize the NIR induced stress fiber disintegration. After NIR irradiation, cells microinjected with caged-PKA/UCNP as indicated by white arrows, show upconversion emission. Stress fiber disintegration caused by a successful PKA NIR photoactivation, exposes more f actin and consequently results in strong green staining near UCNP.A negative control where the cells are microinjected but remain in the dark, show no disintegration of stress fibers. The level of f actin exposure after NIR photoactivation can be stained with alexa 594 followed within and quantified by image J.Intensity curves of f actin staining along the red arrow were plotted, providing quantitative information of stress fiber disintegration which can be correlated to NIR induced PKA activity. Once familiar with microinjection, this technique can be done within one day, including preparation and the quality control of caged-PKA/UCNP.With our instructions for our instrument modifications and the experiments, the desired reaction can be triggered by near IR, in highly spatial temporal resolution like, traditional UV for titration methods, but without the phototoxicity, it's not contagious. After watching this video, hope you have a good understanding of how to deliver enzyme nanoparticle complex into a cell by microinjection and photoactivate the enzyme, using bell compatible near IR light. This technique paved the way for researchers in the field of chemical biology, who need to use light to trigger desired enzymatic reaction in living cells for spatial temporal resolution when the cell cannot tolerate UV phototoxicity.Following this procedure, other bio affectors can be associated to upconversion nanoparticle, to answer additional questions that need spatial temporal photoactivation. If the bio affector works extracellularly, microinjection is not even needed. The near IR photoactivation platform can also provide more penetration depth in tissue and can therefore be applied in small animals to trigger desired reactions at the given time and the location.

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