Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid

Summary

This protocol involves transfecting cAMP sensors and bPAC-nLuc, an optogenetic protein, to accurately track its cellular distribution and response to light stimulation. The innovative approach of creating a cAMP response map using a point scanning system holds the potential for advancing research with optogenetic proteins in different fields.

Abstract

Our goal was to accurately track the cellular distribution of an optogenetic protein and evaluate its functionality within a specific cytoplasmic location. To achieve this, we co-transfected cells with nuclear-targeted cAMP sensors and our laboratory-developed optogenetic protein, bacterial photoactivatable adenylyl cyclase-nanoluciferase (bPAC-nLuc). bPAC-nLuc, when stimulated with 445 nm light or luciferase substrates, generates adenosine 3',5'-cyclic monophosphate (cAMP). We employed a solid-state laser illuminator connected to a point scanning system that allowed us to create a grid/matrix pattern of small illuminated spots (~1 µm²) throughout the cytoplasm of HC-1 cells. By doing so, we were able to effectively track the distribution of nuclear-targeted bPAC-nLuc and generate a comprehensive cAMP response map. This map accurately represented the cellular distribution of bPAC-nLuc, and its response to light stimulation varied according to the amount of protein in the illuminated spot. This innovative approach contributes to the expanding toolkit of techniques available for investigating cellular optogenetic proteins. The ability to map its distribution and response with high precision has far-reaching potential and could advance various fields of research.

Introduction

Optogenetics, born as a tool that revolutionized neurosciences, is now a growing research field and a rising technology routinely used by many laboratories worldwide and across various research areas in biology. We developed bPAC-nLuc, a versatile optogenetic protein, by fusing a light-sensitive adenylyl cyclase (AC) from Beggiatoa sp. (bacterial photoactivatable adenylyl cyclase; bPAC) to nanoluciferase (nLuc)^1,2,3. When stimulated with blue light, bPAC produces the second messenger 3',5'-cyclic adenosine monophosphate (cAMP). nLuc is a recently developed small luciferase that, in the presence of one of its substrates, can generate bioluminescence and activate cAMP production⁴. Thus, this optogenetic protein can be activated transiently by using brief light pulses or steadily with Furimazine or other luciferase substrates, allowing us to mimic different cAMP signaling patterns and assess cellular responses (activation of transcription factors, gene expression, cell proliferation, migration, etc.). Recent advances in 2^nd messenger signaling have emphasized the significance of events occurring in very restricted cytosolic regions (e.g., endosomal cAMP production for cAMP response element-binding protein (CREB) phosphorylation or Ca²⁺ microdomains for nuclear factor of activated T-cells (NFAT) translocation to the nucleus)^5,6. Therefore, developing consistent and systematic strategies to evaluate, mimic, and block signaling from these compartments in live cells is important. To show the ability of bPAC-nLuc to be specifically activated in different cell compartments, we co-transfected a hepatoma-derived cell line (HC-1) with the nuclear-targeted bPAC-nLuc and H208, a Förster resonance energy transfer (FRET) cAMP sensor (NLS-bPAC-nLuc; NLS-H208). HC-1 cells that derive from the HTC line are devoid of assayable AC activity, which results in very low basal cAMP levels, making it ideal to measure putative cAMP production while taking advantage of the full dynamic range of FRET sensors^7,8. Using a solid-state laser (445 nm, LDI-7, 89 North) connected to a point scanning system (UGA-42 Geo, Rapp OptoElectronic), we describe a protocol to systematically stimulate very small circular areas or spots (~1 µm²) within individual cells. The point scanning system was connected to one of the backports of a two-deck microscope, which allowed us to stimulate cells and perform FRET measurements simultaneously via an independent lightpath. We present a method in this protocol where the SysCon Geo software, supplied with the stimulation system by Rapp OptoElectronic, is employed to perform a comprehensive scan of the cytoplasm of cells. The approach involves generating a cAMP response map by setting up a sequence of illuminations that stimulate cells in a grid pattern (Figure 1).

Protocol

1. HC-1 cell culture and preparation for imaging Maintain HC-1 cells in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/L), streptomycin (100 mg/L), and L-glutamine in 10 cm dishes and incubated at 37 °C, 5% CO2, in 95% humidified air. Passage the cells every 2-4 days once cells are ~90% confluent using 1:5 or 1:10 dilutions. Seed cells for transfection on glass coverslips 2 days before the experiment. Working under sterile condit…

Representative Results

The results presented in Figure 1 show that only stimulations directed to the cell nucleus were able to generate measurable cAMP elevations. This confirms that NLS-bPAC-nLuc is expressed exclusively in the nuclear compartment of HC-1 cells. It is possible to precisely stimulate an optogenetic protein using this grid/matrix pattern to map its intracellular distribution. Additionally, the higher cAMP elevations towards the nuclear center reflect the higher mass…

Discussion

The objective of this study was to precisely monitor the intracellular distribution of an optogenetic protein and assess its performance within a particular cytoplasmic compartment. We also showed the precise stimulation capabilities of a point scanning system on cells expressing an optogenetic protein. To achieve this, we employed a nuclear-targeted bPAC-nLuc with high expression levels but a very confined distribution limited to the nucleus. The results showed that stimulation spots separated by only ~1 µm can eit…

Materials

13 W Amber compact fluorescence bulb – Low Blue Lights	Photonic Developments
3-Isobutyl-1-methylxanthine (IBMX)	Sigma	I7018
6-line multi-LED Lumencor Spectra X	Lumencor		6-line multi-LED light engine
Corning – DMEM	Thermo Fischer	MT10013CMEA
Corning – Regular fetal bovine serum	Thermo Fischer	MT35011CV
Cover glasses: circles	Thermo Fischer	12545102P
GBX-2 dark red safelight filter 5.5"	Kodak	1416827	Red safelight lamp
Hanks' balanced salt solution (HBSS) 10x	Thermo Fischer	14185052	Diluted to 1x, adjusted pH
LDI-7	89 North
L-Glutamine	Thermo Fischer	BW17605E
Lipofectamine 3000	Thermo Fischer	L3000001	Transfection kit
Olympus IX83 motorized two-deck microscope	Olympus		Motorized two-deck microscope
Opti-MEM, no phenol red	Thermo Fischer	11058021
ORCA-fusion digital CMOS camera	Hamamatsu	C14440-20UP
Penicillin-streptomycin (10,000 U/mL)	Thermo Fischer	15140122
Phosphate buffered solution (1x)	Lonza	17516F
Prior emission filter wheel and filter sets	Prior Scientific, Inc.		Emission filter wheel
Prior Proscan XY stage	Prior Scientific, Inc.		XY stage
Slidebook 6	Intelligent Imaging Innovations		Digital microscopy software
SysCon software	SysCon Software		Software provided by the stimulation system
UGA-42 Geo	Rapp OptoElectronic
UPlanSApo 100x	Olympus		100x/1.4 NA oil objective (∞/0.17/FN26.5)
ZT458rdc dichroic	Chroma Technology Corp		BS, Wavelength (CWL): 498 nm