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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 processes^1,2,3. 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 cells^4,5. 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 penetration^6,7. 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 level^2,9.

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 irradiation^2,4.

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 degradation^2,4. Even if the caged protein is still functional after membrane translocation, the translocated amounts may not be enough to trigger the cellular response^2,17. 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.

Protokół

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

1. Install a 4X objective lens next to the cuvette holder of the fluorescence spectrometer so that the laser is focused on the middle of the cuvette.
2. Place a tunable 0.5- to 8-W 980-nm laser diode 90° against the objective lens.
   CAUTION: The operator needs to have laser safety knowledge and wear NIR laser goggles.
3. Install a full reflectance mirror in the light path intersection of the objective lens and laser. Place a black anodized metal plate to block the excitation window of spectroscopy and to protect the interior parts.
4. Place the cuvette of silica-coated UCNP solution (0.2 - 0.6 mg/mL) in the cuvette holder so that a bright upconversion beam can be visualized and can be used to adjust the best light alignment for mirror installation.
5. Switch the spectroscopy to luminescence mode with lowest PMT voltage setting (adjust to a higher setting if the signal is too weak). Adjust the mirror to the best alignment, where the beam is on the middle of cuvette and the PMT picks up the strongest signal.
6. Measure the upconversion spectrum, after alignment, with the desired power setting. Use a thermal pile power meter to build a calibration curve of laser power against the electrical current reading on the laser power supply. Constantly check the laser performance to make sure that the performance is within the calibrated range.

2. Microscope Setup

NOTE: The following setup works for microscopes equipped with a two-tier optical path. If the user intends to install an excitation light source switch in the back port to make the NIR laser and the halide lamp share the same port, the collimator in the back port will significantly block the NIR because it is designed to reduce sample heating. The user must make a difficult choice: keep the collimator in and suffer from very low upconversion efficiency, or remove the collimator and sacrifice the intensity of excitation light for the epifluorescence.

NOTE: A cutaway diagram showing the light path scheme for a modified spectrofluorometer, the microscopy for upconversion luminescence and NIR photolysis mode and for epifluorescence and UV photolysis mode, and the experimental setup used for this experiment are illustrated in Figure 2.

1. Equip the fluorescence microscope with a metal halide light guide, directed into the back port.
   NOTE: In this upper-tier light path where there is a cube turret, one cube position must be empty to allow for NIR coming from the lower-tier light path.
2. Install a tunable 0.5 to 8.0 W 980 nm laser diode in the right-side port, with the side-port collimator open.
   NOTE: This enlarges the laser light spot to about 500 µm in diameter when a 10X objective lens is used. Removing this collimator results in a micrometer-range light spot and makes NIR cell irradiation impractical. This collimator is NIR-transparent.
3. Install a dichroic mirror (850-nm short-pass) and an excitation filter (950 nm long-pass) in the lower-tier light path.
4. Use a UCNP solution to visualize the NIR light spot. Adjust the position of laser to center the NIR light beam in the field of view to finish the alignment.

3. Preparation and Characterization of Caged PKA-UCNP Construct

1. Incubate recombinant PKA (9 µM) with 0.5 mM 2-nitrobenzylbromide (NBB) in caging buffer (20 mM Tris-HCl, 100 mM NaCl, and 10 mM MgCl₂; pH 8.5) for 1 - 2 h at 25 °C. Quench the excess NBB with 10 mM dthiothreitol (DTT) and then dialyze against 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer (10 mM, pH 7.5) to remove excess reagents and salts.
   NOTE: The pH of the caging solution is lowered from 8.5 to 7.5 during dialysis with HEPES pH 7.5 for the subsequent immobilization reaction.
2. Mix the lanthanide salts-Y(CH₃CO₂)₃ hydrate (99.9%, 0.4527 g, 1.38 mmol), Yb(CH₃CO₂)₃ hydrate (99.9%, 0.2533 g, 0.60 mmol), and Tm(CH₃CO₂)₃ hydrate (99.9%, 0.0168 g, 0.02 mmol)-in octadecene (30 mL) and oleic acid (12 mL), heat the mixture to 115 °C for 30 min, and cool them to 50 °C. Next, dissolve the reaction mixture in a methanolic solution (20 mL) with 0.2 g (5.0 mmol) of NaOH pellet and 0.2964 g (8 mmol) of ammonium fluoride by sonication for 15 min. Increase the reaction temperature to 300 °C to get the UCNP core (β-NaYF₄:1.0% Tm³⁺/30% Yb³⁺) nanoparticles.
3. Dissolve the core nanoparticles (25 mg) with CO-520 (0.5 mL) in cyclohexane (10 mL). Add ammonia (wt 30% v/v, 0.08 mL) and tetraethylorthosilicate (TEOS, 0.04 mL) and apply constant stirring for 12 h at room temperature to get the silica-coated β-NaYF₄:1.0%Tm³⁺/30%Yb³⁺ core nanoparticles.
4. Incubate the PKA (6 nmol) or caged PKA (6 nmol) with UCNP (1 mg)¹⁵ in HEPES buffer (10 mM, pH 7.5) at 4 °C for 3 h to immobilize the PKA on silica-coated UCNP via electrostatic interactions.
5. Filter the mixture using PVDF filter (0.45 µm), centrifuge at 17,000 x g for 10 min at 4 °C, and redisperse the pellet in HEPES (50 µL, 10 mM, pH 7.5) containing 0.1% protein stabilizer to obtain the purified PKA-UCNP complex. Centrifuge at 17,000 x g for 10 min at 4 °C and collect the supernatant for a Bradford assay in a 1.5-mL tube.
6. Analyze the unbounded PKA in supernatant saved from step 3.5 with a Bradford assay to back-calculate the PKA substitution level on the UCNP particles, which normally have a substitution level of ~ 4.5 nmol of PKA per mg of particle.
   NOTE: The Bradford assay reveals a strong blue color on the pellets, which suggests a high level and stable protein immobilization without desorption, while the supernatant fraction of caged PKA-UCNP solution shows a color similar to HEPES buffer (Figure 4c-4e).

4. Characterization

NOTE: The kinase assay is used to quantify the specific activity of pyruvate kinase. In brief, the phosphorylation of the peptide, which is coupled to pyruvate kinase and lactate dehydrogenase, results in the oxidation of NADH. Formation of the latter is monitored by measuring the decrease in absorbance of NADH at 340 nm.

1. Determine the activity of PKA and PKA-UCNP in a 60 µL total volume of assay mixture containing 4-morpholinepropanesulfonic acid (MOPS, 100 mM, pH 7.5), KCl (100 mM), phosphoenolpyruvate (PEP, 1 mM), adenosine triphosphate (ATP, 1 mM), β-nicotinamide adenine dinucleotide, reduced form (NADH, 0.8 mM), magnesium chloride (MgCl₂, 1 mM), kemptide (0.4 mM), and a pyruvate kinase/lactate dehydrogenase mixture (30/40 units of PK/LDH).
2. Measure the decrease in NADH absorbance over time after the addition of PKA solution (2 µL) to the assay mixture. Calculate the slope of the line to directly reflect the activity of the kinase being measured.
3. Measure the activity of PKA-UCNP¹⁵ and the overall activity, which should be well matched with the multiplication product of native PKA-specific activity and the calculated PKA surface-coated amount.

5. Photolysis Setup

1. Measure all power of the light sources using a thermal pile sensor and calculate power densities (PD = Power (W)/Area (cm²))¹⁸ based on the light spot area.
2. Perform in vitro UV photolysis experiments on a commercial system with a 365 nm LED (200 mW/cm²)¹⁵.
3. For UV photoactivation on the microscope stage, illuminate the PKA-UCNP solution with the excitation light filtered by a commercial cube¹⁵.
   NOTE: The power density is ~15 mW/cm², without objective lens focusing.
4. For in vitro NIR photoactivation on the microscope stage, illuminate the PKA-UCNP solution using a 980 nm laser with customized upconversion filter/mirror settings installed on the lower-tier light path, a dichroic mirror (850 nm short-pass), and an excitation filter (950 nm long-pass).
   NOTE: The output power density is 5 W/cm² before 4X objective lens focusing. The power density is estimated to be ~ 68 W/cm² after 4X objective lens focusing.

6. Cell Sample Preparation and Microinjection of PKA-UCNP Complexes

1. Pre-filter the samples through a 0.45 µm filter after PKA immobilization (step 3.5).
2. Incubate the cells in 10% fetal bovine serum (FBS) containing L-15 medium during the microinjection period for stable pH control outside the incubator.
   NOTE:To confirm microinjection completion, co-inject the native PKA-UCNP, caged PKA-UCNP, or PEGylated UCNP (7.5 mg/mL)¹⁵ with 5(6)-carboxytetramethylrhodamine (TAMRA) (10 µM) in to cells.
3. Adjust the concentration of the PKA-UCNP particles such that, after microinjection, the cytosolic concentration is 1 - 3 μM PKA - a physiological concentration range for PKA in a cell, with the assumption that the injection volume is 1/10 of cellular volume.
4. Perform the microinjection into the cells (step 6.2) using a microinjector device coupled with a one-axis hydraulic micromanipulator (Figure 3).
5. Use a digital pressure gauge to monitor the pressure applied by the microinjector is in the operating range (~50-200 hPa) and seal the dispenser valve in a customized pressuring chamber to provide compensation pressure for microinjection. Pull back the needle using a puller.
6. Maintain the injection pressure between 50 and 75 hPa, with an injection time of 200-300 ms and the compensation pressure at 15 hPa to prevent culture medium backflow into needle by capillary action.
7. Examine the tip openings of the needles to ensure that they have an external diameter between 1.3 and 1.5 µm, which can be confirmed by taking images using a 40x objective lens.
8. Wash the cells with 2 mL of L-15 medium containing 10% FBS after the microinjection. Observe the cellular fluorescence imaging from 5(6)-carboxytetramethyl-rhodamine (TAMRA) to confirm microinjection completion.

7. Photoactivation of Caged PKA-UCNP Using UV or NIR Light in Living Cells

1. For UV photoactivation after the PKA-UCNP microinjection, grow the REF52 cells on a 3.5 µm dish, place them on the microscope stage, and irradiate them with UV light filtered by the cube for 3 min without objective lens focusing.
   NOTE: REF52 cells were maintained in DMEM containing 10% FBS at 37 °C in a 5% CO₂ incubator, and cells were subcultured when the confluency reached to 90% every 2-3 days.
2. For NIR photoactivation after PKA-UCNP microinjection, illuminate the cells on a 980-nm laser from lower-tier filter/mirror settings, as described in step 5.4.
   NOTE: The power density output is 5 W/cm² before 10X objective lens focusing and is estimated to be 300 W/cm² after 10x objective lens focusing. Upconversion is a process that requires a high photon density, especially when a larger anti-Stoke shift is needed. Hence, focusing is needed during photoactivation.
3. When a larger light spot (650 µm x 520 µm) is needed, irradiate these cells with NIR focused by a 10x objective lens with a collimator for 15 min. Allow for a 5 min dark interval after every 5-min irradiation to avoid heating.
   NOTE: When a high spatial-resolution (subcellular light spot) is needed, use the 40X objective lens with a pinhole installed in the exit end of the light guide to generate a subcellular-dimension light spot (5 µm x 4 µm). In this case, 1 s of irradiation is enough for NIR photoactivation due to the high flux of photons.
4. After UV or NIR photolysis, allow the cells to recover in a 5% CO₂ incubator at 37 °C for 1 h in complete medium to generate cellular responses. Subsequently, fix them in 1 mL of 4% paraformaldehyde/phosphate-buffered saline (PBS) for 20 min before further staining.
   CAUTION: Paraformaldehyde is highly toxic; avoid contact with skin, eyes, or mucous membranes. Avoid breathing the powder during measuring and preparation.

8. Visualization of Stress Fiber Disintegration Caused by NIR Photoactivation

1. After fixation, use Alexa 594-phalloidin (add 5 µL of 6.6 µM stock solution to 200 µL of PBS for staining; incubate for 25 min at room temperature) to stain and visualize the stress fibers. Visualize the images of Alexa 594-phalloidin (Ex 581 nm/Em 609 nm) using a filter set.
2. Incubate the cells for 5 min in 4',6-diamidino-2-phenylindole (DAPI) at room temperature. After staining, wash the samples with PBS and separately take fluorescent images of the stress fibers and nuclei.

Wyniki

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...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
Reagent
Tris(hydroxymethyl)aminomethane	Sigma	154563
Magnesium chloride hexahydrate	Sigma	M9272
MOPS	Sigma	M1254
HEPES	Sigma	H4034
Sodium chloride	Sigma	31434
Potassium chloride	Sigma	12636
Yttrium acetate hydrate	Sigma	326046	Y(C2H3CO2)3 · xH2O
Thulium(III) acetate hydrate	Alfa Aesar	14582	Tm(CH3CO2)3 · xH2O
Ytterbium(III) acetate tetrahydrate	Sigma	326011	Yb(C2H3O2)3 · 4H2O
1-Octadecene	Sigma	O806
Oleic acid	Sigma	364525
Methanol	macron	304168
Sodium hydroxide	Sigma	30620
Ammonium fluoride	J.T.Baker	69804
IGEPAL CO-520	Sigma	238643
Cyclohexane	J.T.Baker	920601
Ammonium hydroxide (28% - 30%)	J.T.Baker	972101	Ammonia
Tetraethyl orthosilicate (TEOS)	Sigma	8658
DL-Dithiothreitol (DTT)	Sigma	D0632
N-hydroxymaleimide (NHM)	Sigma	226351	PKA activity blocking reagent
Prionex protein stabilizer solution from hog collagen	Sigma	81662	Protein stabilizer solution
2-nitrobenzyl bromide (NBB)	Sigma	107794	PKA caging reagent
8-(4-Chlorophenylthio)adenosine 3′,5′-cyclic monophosphate sodium salt	Sigma	C3912	8-CPT-cAMP
Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle	Sigma	P0294	PK/LDH
Adenosine 5'-triphosphate disodium	Sigma	A2387	ATP
β-NADH reduced from dipotassium	Sigma	N4505
Phosphoenolpyruvate	Sigma	P7127	PEP
Coomassie Protein Assay Reagent, 950 mL	Thermo Scientific	23200	Bradford assay reagent
cAMP-dependent protein kinase	Promega	V5161	PKA activity control
pET15b-PKACAT plasmid	Addgene	#14921
pKaede-MC1 plasmid	CoralHue	AM-V0012
Phosphate buffered saline (PBS), pH 7.4	Thermo Scientific	10010023
DMEM, high glucose, pyruvate	Gibco	12800-017	Cell culture medium
Leibovitz L-15 Medium	Biological Industries	01-115-1A	Cell culture medium
Fetal Bovine Serum	Biological Industries	04-001-1A
Paraformaldehyde	ACROS	416785000
DAPI	Invitrogen	D1306	Nucleus staining dye
Alexa 594-phalloidin	Invitrogen	A12381	F-actin staining dye
5(6)-Carboxytetramethylrhodamine	Novabiochem	8.51030.9999
Pierce Coomassie (Bradford) Protein Assay Kit	Thermo Scientific	23200
CelluSep T4 Tubings/Nominal filter rating MWCO 12,000 - 14,000 Da	Membrane Filtration Products, Inc.	1430-33	Dialysis membrane
Millex-HV Syringe Filter Unit, 0.45 µm, PVDF, 13 mm, gamma sterilized	EMD Milipore	SLHVX13NL
Equipment
Dynamic Light Scattering/Zetapotential Zetasizer nano-ZS	Malvern	M104
Transmission Electron Microscope	JEOL	JEM-1400
Fluorescence Spectrophotometer	Agilent Technologies	10075200	Cary Eclipse
UV-Vis Spectrophotometer	Agilent Technologies	10068900	Cary 50
Fluorescence Microscope	Olympus	IX-71
950 nm longpass filter	Thorlabs	FEL0950
850 nm dichroic mirror shortpass	Chroma	NC265609
RT3 color CCD system	SPOT	RT2520
Fluorescence Illumination	PRIOR	Lumen 200
980 nm Infra-red diode laser	CNI	MDL-N-980-8W
UV LED Spot Light Source	UVATA	UVATA-UPS412	With a UPH-056-365 nm LED at 200 mW/cm2
Thermal pile sensor	OPHIR	12A-V1-ROHS
Picospritzer III	Parker Hannifin	052-0500-900	Intracellular Microinjection Dispense Systems
PC-10 Needle puller	Narishige	PC-10
MANOMETER Digital pressure gauge	Lutron	PM-9100
One-axis Oil Hydraulic Micromanipulator	Narishige	MMO-220A
Heraeus Fresco 17 Centrifuge, Refrigerated	Thermo Scientific	75002421