Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Summary

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

Abstract

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Introduction

Growth factors are involved in a wide spectrum of cell functions, including proliferation, differentiation, migration, and apoptosis, and play pivotal roles in many biological events, including embryonic development, aging, and regulation of mental status^1,2,3,4,5. Many growth factors signal through complex intracellular signaling cascades. These signaling events are often operated by reversible protein phosphorylation in a precisely regulated fashion^6,7<....

Protocol

Animal research was conducted in accordance with guidelines set by the Illinois Institutional Animal Care and Use Committee (IACUC) and the University of Illinois Department of Animal Resources (DAR).

1. Optogenetic Induction of Protein Localization in BHK21 Mammalian Cell Culture

NOTE: Steps 1.1-1.3 provide a method to assemble a cell culture chamber for imaging with high-magnification objectives (e.g., 63X or 100X), which typically have short working distances. These objectives require a thin glass coverslip (e.g., #1.5, 170 µm thickness) as the imaging substrate. Alternatively, a glass-bottom c....

Results

Ratiometric expression of photoactivatable protein pairs: Figure 1A shows the design of a bicistronic optogenetic construct, CRY2-mCherry-Raf1-P2A-CIBN-CIBN-GFP-CaaX (referred to as CRY2-2A-2CIBN), based on the porcine teschovirum-1 2A (P2A) peptide, which shows the highest ribosome-skipping efficiency among mammalian cell lines⁴². In previous work, it has been determined that the optimal ratio for CIBN-GFP-CaaX:CRY2-mCherry-Raf1 is 2:.......

Discussion

When building the light box, the power of individual LEDs should be measured. Based on previous experience, the power output can vary between individual LEDs due to manufacturing variance. Select a set of LEDs that have a power output within 10% of each other. The number of LEDs, the current-limiting resistor, and the power input can be modified for different types of cell culture containers (e.g., a 6-well or 24-well plate). A 24 h of light illumination at a power of 0.2 mW/cm² does not induce detect.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glass coverslip	VWR	48393 230	Substrate for live cell imaging
Coverslip holder	Newcomer Supply	6817B	Holder for coverslips
Detergent	ThermoFisher	16 000 104	For cleaning coverslips
Boric acid	Sigma-Aldrich	B6768-500G	For making PLL buffer
Disodium tetraborate	Sigma-Aldrich	71996-250G	For making  PLL buffer
Plastic beaker	Nalgene	1201-1000	For cleaning coverslips
Sodium hydroxide	Sigma-Aldrich	221465-2.5KG	For adjust pH
Poly-L-lysine hydrobromide	Sigma-Aldrich	P1274-500MG	For coating coverslip
Diethylpyrocarbonate (DEPC)-Treated Water	ThermoFisher Scientific	750024	For DNA preparation
Cover Glass Forceps	Ted Pella	5645	Cover glass handling
Tissue cutlure dish	Thermofisher	12565321	Cell culture dish
Sterile centrifuge tubes	ThermoFisher	12-565-271	Buffer storage
Transfection Reagent	ThermoFisher	R0534	Transfection
CO2-independent medium	ThermoFisher	18045088	For live cell imaging
Polydimethylsiloxane (PDMS)	Ellsworth Adhesives	184 SIL ELAST KIT 0.5KG	Form make cell chamber
Plasmid Maxiprep kit	Qiagen	12965	Plasmid preparation
DMEM medium	ThermoFisher	11965-084	Cell culturing medium component
F12K medium	ThermoFisher	21127022	Cell culturing medium component
Horse serum	ThermoFisher	16050122	Cell culturing medium component
Fetal Bovine Serum	Signa-Aldrich	12303C-500 mL	Cell culturing medium component
Penicillin-Streptomycin-Glutamine	ThermoFisher	10378016	Cell culturing medium component
Trypsin (0.25%), phenol red	ThermoFisher Scientific	15050065	For mammalian cell dissociation
Agarose	Fisher Scientific	BP1356-100	For DNA preparation
Ficoll PM400	GE Heathcare Life Sciences	17-5442-02	For embryo buffer
L-Cysteine hydrochloride monohydrate	Sigma-Aldrich	1.02839.0025	Oocyte preparation
ApaI	ThermoFisher	FD1414	For linearization of plasmids
Dnase I	ThermoFisher	AM2222	For removing DNA template in the in vitro transcription assay
Index-match materials (immersion oil)	Thorlabs	MOIL-20LN	For matching the index between sample substrate and objective
Blue LED	Adafruit	301	Light source for optogenetic stimulation
Resistor kit	Amazon	EPC-103	current-limiting resistor
Aluminum boxes	BUD Industries	AC-401	light box
BreadBoard	Jekewin	837654333686	For making LED array
Hook up Wire	Electronix Express	27WK22SLD25	For making LED array
Relay Module	Jbtek	SRD-05VDC-SL-C	For intermittent light control
DC Power Supply	TMS	DCPowerSupply-LW-(PS-305D)	Power supply for LED
Silicon Power Head	Thorlabs	S121C	For light intensity measurement
Power meter	Thorlabs	PM100D	For light intensity measurement
Microscope	Leica Biosystems	DMI8	For live cell imaging
BioSafety Cabinet	ThermoFisher	1300 Series A2	For mammalian cell handling
CO2 incubator	ThermoFisher	Isotemp	For mammalian cell culturing
Stereo microscope	Leica	M60	For embryo micro-manipulation
Microinjector	Narishige	IM300	For embryo microinjection
Micropipette puller	Sutter Instruments	P87	Needle puller
in vitro transcription kit	ThermoFisher	AM1340	For in vitro transcription. The kit includes nuclease-free water, SP6 RNA  Polymerase, ribonucleotide mixture, cap analog, lithium choride precipitation solution, and spin column
RNA purfication kit	Qiagen	74104	Silica-membrane spin column for purification of synthesized RNA
Convection oven	MTI corporation	EQ-DHG-9015	PDMS curing
Centrifugal mixer and teflon container	THINKY	AR310	For mixing PDMS
Silicon wafer	UniversityWafer	452	Base for making PDMS  devices
Blade	Techni Edge	01-801	For cutting PDMS
Capillary glass	Sutter Instruments	BF100-58-10	For fabrication of injecting needles.