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

Summary

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.

Abstract

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.

Introduction

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 photoclea....

Protocol

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- t....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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