Bioluminescent Low-Affinity Ca2+ Indicator for ER with Multicolor

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Bioluminescent low-affinity Ca indicator for ER with multicolor calcium imaging in single living cells Md Nadim Hossain, Kazushi Suzuki, Megumi Iwano, Tomoki Matsuda, and Takeharu Nagai ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b01014 • Publication Date (Web): 01 Mar 2018 Downloaded from http://pubs.acs.org on March 2, 2018

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ACS Chemical Biology

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Bioluminescent low-affinity Ca2+ indicator for ER with multicolor calcium

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imaging in single living cells

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Md Nadim Hossain1, Kazushi Suzuki1, Megumi Iwano2, Tomoki Matsuda1,2, and

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Takeharu Nagai1,2,*

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Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-

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1 Yamadaoka, Suita 565-0871, Japan

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Department of Biomolecular Science and Engineering, The Institute of Scientific and

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Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki 567-0047, Japan

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*Corresponding author: [email protected]

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Abstract

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The sarco/endoplasmic reticulum (SR/ER) is the foremost intercellular Ca2+ store (at

24

sub-millimolar concentrations), playing a crucial role in controlling intracellular Ca2+

25

levels. For the investigation of SR/ER Ca2+ dynamics in cells, fluorescent protein-

26

based genetically encoded calcium indicators (GECIs) with low Ca2+ affinity have

27

been used. Recently, bioluminescent protein-based GECIs with high brightness have

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been reported to counter the constraints of fluorescence imaging, such as

29

phototoxicity. However, their Ca2+ affinity is high and limited for imaging in the

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cytosol, nucleus, or mitochondria. In this study, we developed a novel cyan color,

31

low-affinity (Kd = 110 µM) intensiometric bioluminescent GECI, which enables

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monitoring of the Ca2+ dynamics in the ER of HeLa cells and the SR of C2C12-

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derived myotubes. To facilitate the broad concentration range of Ca2+ in cellular

34

organelles, we additionally developed an intermediate affinity (Kd = 18 µM), orange

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color, bioluminescent GECI, which enables monitoring of Ca2+ dynamics in the

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mitochondria of HeLa cells. With these indicators, in conjunction with an existing

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high-affinity,

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bioluminescent Ca2+ imaging in three distinct organelles (nuclei, mitochondria, and

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ER) simultaneously. The multicolor, live, bioluminescent Ca2+ imaging demonstrated

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here can be used to stably reveal the ER Ca2+ homeostasis and cooperative Ca2+

41

regulation among organelles. This will lead to the further understanding of Ca2+-

42

related physiological functions and pathophysiological mechanisms.

green,

bioluminescent

GECI,

we

43

44


succeeded

in

multicolor

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Ca2+ is widely recognized as the most ubiquitous intercellular signaling molecule

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involved in a variety of biological functions, such as fertilization, muscle contraction,

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secretion, cell proliferation, and apoptosis1,2. In the process of regulation of

48

intracellular Ca2+ homeostasis, the sarco/endoplasmic reticulum (SR/ER) has a

49

predominant role in releasing and restoring Ca2+ levels. The SR/ER lumen accounts

50

for ~10% of the total cell volume and stores more than 90% of the total amount of

51

intracellular

52

concentration of Ca2+ than the cytosol5,6.

Ca2+3,4.

These

organelles

store

5,000-

to

10,000-fold

higher

53

To investigate the SR/ER Ca2+ dynamics, different types of low-affinity indicators,

54

such as synthetic chemical Ca2+ indicators, fluorescent protein-based genetically

55

encoded Ca2+ indicators (FP-GECIs), and bioluminescent protein-based GECIs (BP-

56

GECIs) have been reported for live cell Ca2+ imaging7,8. Although low-affinity

57

chemical indicators conjugated with acetoxymethyl (AM)-ester were applied to ER

58

Ca2+ imaging, they showed difficulty for accurate targeting and long term retention in

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the ER5,9,10. In contrast, GECIs could be targeted to the ER precisely by fusing them

60

to proper signal sequences. Several low-affinity FP-GECIs with high signal-to-noise

61

ratios and large dynamic ranges, such as CEPIA11, have been reported to

62

successfully demonstrate ER Ca2+ dynamics. However, owing to the necessity of

63

excitation using intense light, fluorescence imaging has the potential for causing

64

chromophore photobleaching, cell phototoxicity, specimen autofluorescence, and

65

can be difficult to use together with other optogenetic tools12,13. To overcome these

66

problems, BPs are drawing attention as genetically encoded tools for cellular Ca2+

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imaging14.

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bioluminescence in the presence of Ca2+, can be employed as a calcium imaging

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tool. The bioluminescence intensity of aequorin is very low and aequorin-GFP fusion

The

Ca2+-dependent

photoprotein


aequorin15,

which

emits


70

proteins can improve bioluminescence resonance energy transfer (BRET) intensity

71

by 50-fold16. However, the intensity of aequorin-GFP is still not sufficiently high

72

because of the low light quantum yield of aequorin and the slow turnover rate of

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aequorin, making it unfavorable for long-term imaging, especially for high Ca2+

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environments such as the SR/ER16,17.

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In contrast, a growing number of BP-based GECIs have been facilitated for

76

bioimaging with a high bioluminescence intensity and spatiotemporal resolution,

77

which could be comparable to FP-GECIs18. A recent advance was the development

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of BPs such as NanoLuc (Nluc), which is derived from the deep-sea shrimp

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Oplophorus gracilirostris19 that have been used in revolutionary molecular and

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cellular imaging20. We reported BP-GECIs (Nano-lantern(Ca2+)18 and enhanced

81

Nano-lantern(Ca2+)12) with a series of high Ca2+ affinity (low dissociation constant

82

(Kd)) variants and demonstrated the imaging of Ca2+ dynamics in the cytosol,

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nucleus, and mitochondria. The development of low-affinity BP-GECIs for

84

bioluminescence Ca2+ imaging in the SR/ER is a current necessity.

85

In this study, we developed a novel, low-affinity, cyan color BP-GECI,

86

CeNL(Ca2+), as a member of enhanced Nano-lantern (Ca2+) (eNL(Ca2+)) and

87

demonstrated SR/ER Ca2+ dynamics in mammalian cells. In addition, an orange

88

color variant of intermediate-affinity BP-GECI was developed for bioluminescence

89

imaging of mitochondria storing sub-micromolar concentrations of Ca2+. To show

90

their applicability, we demonstrated triple color bioluminescence Ca2+ imaging with

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three distinct Ca2+ tools in the nucleus, mitochondria, and ER.

92

93


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RESULTS AND DISCUSSION

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Molecular design and construction of CeNL(Ca2+) variants

96

Recently, our group reported green color BP-GECI, a tool in the green enhanced

97

Nano-lantern Ca2+ (GeNL(Ca2+)) series, which employs Nluc as a BP12. This tool

98

contains the calcium binding protein calmodulin (CaM) and its binding peptide M13

99

inserted between Gly66 and Leu67 of the Nluc sequence and the fluorescent protein

100

mNeonGreen (with the C-terminal sequence deleted) fused at its N-terminus. Ca2+

101

binding-dependent compaction of the CaM-M13 moiety induces reconstitution of the

102

split Nluc moiety, which causes an increase in the bioluminescence intensity. Based

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on the construction framework of the GeNL(Ca2+) series, we developed a cyan color

104

variant CeNL(Ca2+) so that a low-affinity variant, which is available for multicolor-

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multi-affinity imaging with GeNL(Ca2+), could be created. We made a fusion protein

106

of cyan color fluorescent protein mTurquoise2 (mTQ2)23, Nluc without the start

107

codon, and the calcium sensing domain CaM-M13 derived from YC3.621. CaM-M13

108

was inserted into the Nluc moiety at the same splitting site (Gly66 and Leu67) as the

109

GeNL(Ca2+) series (Figure 1A).

110

In order to weaken the affinity of CeNL(Ca2+), mutations were introduced into the

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CaM moiety (Figure 1B). For the development of low-affinity CEPIAer, mutations

112

from glutamate to aspartate were introduced at the 12th residues in the EF-hand

113

motifs (each of which is composed of 12 residues). Furthermore, F92W and D133E

114

mutations (their residue numbering scheme is for a single CaM) were introduced13.

115

All of these mutations, except for the F92W mutation, are known to form the chelate

116

bond with Ca2+. Following the generation of R-CEPIA1er (Kd = 565 µM),

117

E31D/E67D/F92W/E104D/D133E mutations were introduced into the CaM moiety of



Page 6 of 32

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CeNL(Ca2+). That variant showed decreased bioluminescence intensity with

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increasing concentrations of Ca2+, meaning that its dynamic range was a small and

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inversely related to calcium levels. To improve the dynamic range, we made

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derivatives, for which single mutation sites were reverted to the original CaM amino

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acid. The D133E mutation is known to contribute to the drastic affinity reduction and

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so

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E67D/F92W/E104D/D133E

125

E31D/F92W/E104D/D133E had greater than 100% dynamic ranges (118% and

126

108%, respectively). Affinity of CeNL(Ca2+)_Vb, which has the same mutations as G-

127

CEPIA1er, showed a much lower affinity (Kd = 110 µM, Hill coefficient 2.18) (Figure

128

1C, Table 1). This affinity is within the possible range to monitor the Ca2+ levels in

129

the ER. The affinity of CeNL(Ca2+)_Va (Kd = 19 µM, Hill coefficient 1.15) is

130

appropriate to monitor the Ca2+ dynamics in mitochondria (Supporting Information

131

Figure S1). Additionally, another reversion mutation of CeNL(Ca2+)_Vc to generate

132

the variant CeNL(Ca2+)_Vc (E67D/E104D/D133E) resulted in an intermediate affinity

133

(106% dynamic range, Kd = 1.2 µM, Hill coefficient 0.98) (Figure 1C, Table 1). This

134

affinity allows for monitoring mitochondrial Ca2+ dynamics. We designated

135

CeNL(Ca2+)_Va,

136

CeNL(Ca2+)_110µ, and CeNL(Ca2+)_1.2µ, respectively (Supporting Information

137

Figure S2A− −C). In the presence of saturating Ca2+ concentrations, the relative

138

bioluminescence

139

CeNL(Ca2+)_110µ to Nluc were 2.4-, 2.9-, and 1.8-fold, respectively (Figure 1D).

140

Ca2+ imaging in ER/SR with the low-affinity CeNL(Ca2+)_110µ construct

was

not

reverted11.

Among

these and

CeNL(Ca2+)_Vb,

intensities

of

and

variants,

CeNL(Ca2+)_Va containing

CeNL(Ca2+)_Vb

CeNL(Ca2+)_Vc

CeNL(Ca2+)_1.2µ,


as

containing

CeNL(Ca2+)_19µ,

CeNL(Ca2+)_19µ,

and

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The

low-affinity

CeNL(Ca2+)_110µ

construct

fused

with

the

N-

142

terminal calreticulin signal sequence and a C-terminal KDEL retention signal

143

sequence was expressed in HeLa cells and successful localization to the ER was

144

confirmed by colocalization with the ER marker ER-tracker Red (Figure 2A). To test

145

the ability of the construct to detect Ca2+ concentration changes in the ER, cells were

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stimulated with histamine to induce Ca2+ release from the ER through the IP3

147

receptor. After adding histamine, a transient decrease and a subsequent recovery of

148

the bioluminescence intensity were observed (Figure 2B and C). As an alternative

149

approach to affect Ca2+ concentration in the ER, ATP, or thapsigargin (an inhibitor of

150

the SERCA pump) was added. 10 µM ATP caused a decrease and oscillations

151

(Figure 2D and E). Thapsigargin caused a continuous decrease of bioluminescence

152

intensity (Figure 2F and G, Supporting Information Movies S1 and S2). We

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additionally successfully observed Ca2+ release from the ER in HEK293 cells

154

(Supporting Information Figure S3).

155

We demonstrated SR Ca2+ imaging in myotubes derived from the mouse

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myogenic cell line C2C12 (8 days after culture) with localized CeNL(Ca2+)_110µ

157

(Figure 3A and B). Because Ca2+ release from the SR through IP3R and RynR upon

158

acetylcholine (ACh) stimulation is known to be caused by activation of nicotinic ACh

159

receptors24, we added ACh to induce reduction of Ca2+ concentration in SR. We

160

observed a rapid decrease (within a few seconds) of the bioluminescence signal,

161

recovering to approximately 100% within 50 s (Figure 3C and Supporting

162

Information Movie S3).

163

The concentration range of Ca2+ in the SR/ER is approximately 100

164

micromolar in the resting state and in the low micromolar range in the depletion

165

state11,25. There are previous reports of FP-GECIs, such as D1ER, CatchER, and



166

LAR-GECO1, which are categorized as low-affinity indicators25-26. Although they

167

allowed for SR/ER Ca2+ imaging in single cells, their dissociation constants (60, 200,

168

and 24 µM, respectively) are not sufficiently low for monitoring SR/ER Ca2+ dynamics

169

well25-26. In contrast, CEPIAer family members (Kd = 500−700 µM) have lower

170

affinities and were successfully used to monitor the SR/ER Ca2+ dynamics with high

171

spatiotemporal resolution11. Although CeNL(Ca2+)_110µ was developed based on

172

the construction of CEPIA, its Kd for Ca2+ is almost five times lower than that of the

173

original CEPIA, which successfully detected oscillation. We expect Ca2+ oscillations

174

to be observed with variants having a higher Kd than CeNL(Ca2+)_110µ generated by

175

further improvement for lower affinity. Another fluorescent probe, D1ER, which has a

176

lower Kd (60 µM) than CeNL(Ca2+)_110µ, successfully detected an oscillatory

177

intensity change upon ATP treatment25. Additionally, we treated cells expressing

178

CeNL(Ca2+)_110µ with ATP (Figure 2D and E) and similar oscillation was detected.

179

However, there is possibility that additional obstacles to be overcome still exist, such

180

as slower kinetics owing to complicated sensing mechanisms including the

181

reconstitution of the luciferase moiety. Consideration of such matters will lead to

182

further improvement.

183

Development of an intermediate-affinity OeNL(Ca2+) construct

184

Investigations of how ER Ca2+ release or uptake in one organelle relates to

185

Ca2+ levels in other organelles, such as the mitochondria and nucleus, are crucial to

186

reveal detailed mechanisms of cellular functions. For this purpose, multicolor Ca2+

187

imaging in different subcellular compartments by expressing BP-GECIs which emit

188

different colors is indispensable. To monitor intermediate ranges of Ca2+ dynamics in

189

mitochondria in combination with the low-affinity CeNL(Ca2+) and the high-affinity


Page 8 of 32

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190

GeNL(Ca2+), we developed an orange color emitting intermediate-affinity variant. By

191

replacing the mTQ2 moiety in the CeNL(Ca2+)_1.2µ with mKOκ, we created an

192

orange eNL(Ca2+), which we termed OeNL(Ca2+). However, the energy transfer from

193

the BRET donor Nluc to the acceptor mKOκ was not efficient as an orange

194

bioluminescence indicator owing to the dominant bioluminescent spectrum peak of

195

the Nluc. Thus, we attempted to improve the BRET efficiency. As it has been

196

previously reported that the eNL series was modified by changing the number of N-

197

terminal amino acids in the Nluc protein to improve BRET efficiency12, we deleted

198

four amino acids (MVFT) from the N-terminus of Nluc moiety. The improved

199

OeNL(Ca2+) showed a Kd value of 18 µM with a dynamic range of 114% (Figure 4 A

200

and B) and we termed this indicator OeNL(Ca2+)_18µ (Supporting Information

201

Figure S2D).

202

We expressed OeNL(Ca2+)_18µ with a mitochondrial localization sequence

203

(CoxVIII×2) in HeLa cells. It properly localized to the mitochondria and showed

204

changes in bioluminescence intensity in response to changes in mitochondrial Ca2+

205

levels upon histamine stimulation (Figure 4 C− −E, Supporting Information Movie

206

S4).

207

Multicolor Ca2+ imaging in the ER, nucleus, and mitochondria

208

We first performed dual color Ca2+ imaging using CeNL(Ca2+)_110µ with an ER

209

targeting signal and GeNL(Ca2+)_520 with a nuclear localization tag (histone H2B).

210

Upon histamine stimulation, Ca2+ levels in the ER continuously decreased and

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reached equilibrium at a lower level. Conversely, the nuclear Ca2+ signal increased

212

transiently and a subsequent oscillation was observed (Figure 5, Supporting

213

Information Movie S5).



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After successfully observing dual color Ca2+ dynamics in the ER and nucleus, we

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conducted triple color imaging of Ca2+ dynamics in the ER, mitochondria, and

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nucleus with co-expression of the low-affinity CeNL(Ca2+)_110µ, the intermediate-

217

affinity OeNL(Ca2+)_18µ, and the high-affinity GeNL(Ca2+)_520. The signal from

218

each organelle was separated by optical filtering and linear unmixing following the

219

method of five-color imaging with eNL12. Validity of the linear unmixing for

220

simultaneous Ca2+ imaging of multiple subcellular compartments can be confirmed

221

by the image of each channel with proper subcellular localization free from serious

222

bleed-thorough in Figure 6B compared with the images before unmixing (Figure

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6A). After adding histamine, bioluminescence signal decreased in the ER and

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increased in the mitochondria and nucleus resulting from Ca2+ release from ER was

225

observed (Figure 6C and D, Supporting Information Movie S6). However, some

226

signal deterioration associated with the linear unmixing process caused distortion in

227

the time course in Figure 6D compared with the single color imaging (Figure 2B

228

and C). This issue should be solved by improvement of spectra separation among

229

indicators, with improvement in BRET efficiency, or development of a more

230

sophisticated analytical algorithm for the linear unmixing calculation.

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Bioluminescent indicators have advantages for imaging under the stimulation

232

of multiple optogenetic actuators, which need excitation with different wavelengths of

233

light, as has been shown with the imaging of bioluminescent voltage indicators under

234

actuation

235

(ChR2(H134R) and eNpHR3.0)33. The combination of multicolored bioluminescent

236

Ca2+ imaging with multi-optogenetic stimulation enables further detailed analysis of

237

the influence of optogenetic stimulation. This would contribute to understanding

238

physiological phenomena involving cooperative Ca2+ regulation among organelles.

with

the

depolarizing

and

hyperpolarizing


optical

control

tools

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239

For example, the ER and mitochondria, which are known to form an ER-

240

mitochondria junction, and the role of mitochondrial Ca2+ uptake being investigated

241

in different tissues and organs are an interesting area of research where multicolor

242

bioluminescence Ca2+ imaging can be utilized. Additionally, it is advantageous to

243

advance

244

photochemical reactions, such as circadian rhythms and photosynthesis in plant

245

cells18.

246

METHODS

247

Molecular Biology

ER

Ca2+

imaging

to

investigate

physiological

events

involving

248

DNA oligonucleotides were purchased from Hokkaido System Science. All of

249

the DNA oligonucleotides sequences are provided in Table S1. KOD-Plus (Toyobo

250

Life Science) was used for non-mutagenesis PCR amplification. PCR products were

251

purified using a phenol-chloroform standard protocol and restriction enzyme

252

digestion products were purified from agarose electrophoresis gel using the QIAEX II

253

gel extraction kit (QIAGEN). Restriction digestion was performed by endonucleases

254

(Takara Bio or New England Biolabs) following the manufacturer’s recommended

255

protocol. DNA fragments extracted from gels were ligated with T4 ligase in Rapid

256

Ligation Buffer (Promega). Small-scale plasmid DNA was obtained from 1.5 mL of

257

LB-liquid bacterial culture by alkaline lysis and ethanol precipitation. Large-scale

258

plasmid DNA was obtained from bacterial pellets from 200 mL of LB-liquid culture by

259

alkaline lysis, PEG-8000 precipitation, two rounds of phenol/chloroform extraction,

260

and isopropanol precipitation. DNA sequencing of the cDNA constructs was

261

performed using the BigDye Terminator v1.1 Cycle Sequencing kit (Life

262

Technologies).



263

Page 12 of 32

Gene construction of bacterial expression vectors

264

Following the construction strategy of GeNL(Ca2+)_52012, cDNA of mTQ2 with

265

BamHI and KpnI sites was amplified by PCR and digested with restriction enzymes.

266

The cDNA of the bioluminescent protein Nluc without the N-terminal start codon was

267

likewise amplified by PCR and digested with KpnI and EcoRI. For bacterial

268

expression, the digested PCR fragments were gel-purified, ligated, and cloned in-

269

frame between BamHI of EcoRI sites of pRSETB (Invitrogen). Then, NcoI and SacI

270

restriction sites were introduced between the 66th and 67th residues of the Nluc

271

moiety in CeNL_pRSETB by inverse PCR techniques. Two restriction sites (NcoI and

272

SacI) were added through PCR amplification at the 5ʹ and 3ʹ ends of the CaM-M13

273

domain at YC3.621. Then, restriction digested CaM-M13 was inserted into the Nluc

274

split moiety27 in CeNL_pRSETB by ligation. Plasmid DNA was purified from E. coli

275

colonies transformed with ligation product and DNA sequences were determined.

276

To generate the affinity variants of CeNL(Ca2+), we introduced site-directed

277

mutagenesis in the

CaM-M13 moiety.

The

designated oligoprimers were

278

phosphorylated and then inverse-PCR was performed. The fragments of PCR

279

products were then ligated. E. coli transformed with the ligation product were

280

cultured overnight and plasmids were subsequently purified.

281

To develop color variants of eNL(Ca2+), we replaced the mTQ2 domain in the

282

CeNL(Ca2+) construct with mKOκ. mKOκ was amplified by PCR with primers

283

containing BamHI and KpnI restriction sites. The PCR product and CeNL(Ca2+) were

284

digested

285

OeNL(Ca2+)/pRSETB.

with

BamHI

and

KpnI

independently


and

ligated

to

construct

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286

To improve the BRET efficiency, four amino acids (MVFT) were eliminated

287

from the N-terminus of the Nluc moiety (Nluc∆N4) by PCR. Then, these fragments

288

were digested with EcoRI and KpnI was cloned in-frame between the BamHI and

289

EcoRI sites of pRSETB (Invitrogen).

290

Gene construction of mammalian expression vectors

291

For the construction of variants for ER localization, ReNL in the pcDNA3-

292

ReNL-ER12

293

(MLLSVPLLLGLLGLAAAD) and a KDEL signal for ER retention located at the N-

294

terminus and C-terminus of ReNL, was replaced with CeNL(Ca2+) variants. For this

295

purpose, three DNA fragments were prepared and ligated: 1) A DNA fragment from

296

mTQ2 to the N-terminus of Nluc was excised from the bacterial expression vector

297

CeNL(Ca2+)_110µ/pRSETB by its BamHI and NcoI sites. 2) A DNA fragment from

298

CaM to the C-terminus of Nluc in the CeNL(Ca2+)_110µ construct was amplified by

299

PCR and extended NcoI and KpnI sites were digested with restriction enzymes. 3)

300

pcDNA3-ReNL-ER was digested with BamHI and KpnI to remove ReNL. These three

301

fragments were then ligated and cloned.

vector,

which

contains

a

signal

peptide

from

calreticulin

302

For mitochondrial localization, duplicate mitochondrial targeting signals of

303

subunit VIII of human cytochrome c oxidase (CoxVIII) were fused at the N-terminus

304

of the intermediate-affinity variants CeNL(Ca2+)_1.2µ, CeNL(Ca2+)_19µ, and

305

OeNL(Ca2+)_18µ.

306

ReNL_pcDNA312 was replaced with each variant amplified by PCR and CoxVIIIx2-

307

CeNL(Ca2+)_1.2µ-pcDNA3, CoxVIIIx2-CeNL(Ca2+)_19µ-pcDNA3, and CoxVIIIx2-

308

OeNL(Ca2+)_18µ-pcDNA3 were created (Figure S2).

ReNL

between

BamHI

and


XhoI

sites

in

CoxVIIIx2-


309

For nuclear localization, histone 2B (H2B) was fused at the C-terminus of the

310

high-affinity variant GeNL(Ca2+)_520. H2B between BamHI and XhoI sites in

311

pcDNA3-GeNL_H2B12 was replaced with GeNL(Ca2+)_520 amplified by PCR and

312

pcDNA3-GeNL(Ca2+)_520-H2B was created.

313

Bacterial expression and purification of the proteins

314

CeNL(Ca2+) and OeNL(Ca2+) constructs with N-terminal polyhistidine tags

315

were expressed in E. coli strain JM109 (DE3) at 23°C for 69 h in 200 mL LB bacterial

316

growth medium supplemented with 0.1 mg/mL carbenicillin. Cells were collected and

317

lysed with 0.5 µg/µL lysozyme, followed by five freeze-thaw cycles. The lysate was

318

clarified by centrifugation (8,000 rpm at 4°C for 20 min). The recombinant proteins

319

were purified from the supernatant using Ni-NTA agarose affinity columns (Qiagen),

320

followed by buffer-exchange to 20 mM HEPES, pH 7.4 with the desalting column

321

PD-10 (GE Healthcare)12,28. The protein purification process after lysis was

322

conducted on ice to avoid protein degradation. Protein concentrations were

323

determined by the Bradford assay (Figure 1D) and the alkaline-denature method29.

324

We used the molar extinction coefficient of mTQ2 on purified proteins, which is

325

reported to be 30,000 M−1 cm-1 30.

326

Characterization of CeNL(Ca2+) and OeNL(Ca2+) constructs

327

Emission spectra were measured by a micro-plate reader SH-9000 (Corona

328

Electric). A final concentration of 2 nM of protein and 5 µM of the bioluminescent

329

substrate coelenterazine-h (Wako chemicals) was used for these measurements.

330

Experiments were performed at least three times and the averaged data was used

331

for further analysis. For the Ca2+ titrations, we performed reciprocal dilution of Ca2+-


Page 14 of 32

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332

saturated and Ca2+-free buffers containing 10 mM MOPS, 100 mM KCl, and 10 mM

333

EGTA with or without 10 mM Ca2+ added as CaCO3 at pH 7.2, 25°C. The free Ca2+

334

concentrations were calculated using 0.15 µM for the apparent Kd value of EGTA for

335

Ca2+. For the low-affinity variants, Ca2+ solutions prepared by dilution of small

336

aliquots of concentrated CaCl211. A Ca2+ titration curve was used to calculate the

337

apparent Kd value by non-linear regression analysis. Sigmodal binding curves were

338

fitted to the data to extract the single Hill equation using Origin7 software

339

(OriginLab).

340

Mammalian cell culture and transfection

341

HeLa cells (purchased from RIKEN BioResource Center) with Dulbecco’s

342

Modified Eagle’s Medium (DMEM) (Sigma-Aldrich) supplemented with 10% fetal

343

bovine serum (FBS) were cultured on collagen-coated, 35-mm, glass-bottom dishes.

344

HeLa cells were cultured until reaching ~80% confluency (~24 to 30 h). Then, HeLa

345

cells were transfected with 5.0 µg plasmid DNA using a calcium phosphate

346

transfection protocol. The DMEM (with 10% FBS) medium was changed after 12 h

347

and the cells were grown for an additional 18 h in a CO2 incubator (Sanyo) at 37°C in

348

5% CO2. The cells were then washed with phenol red–free DMEM/F12 and used for

349

imaging. HEK293 cells were imaged following the same protocol.

350

The C2C12 mouse skeletal muscle cell line was obtained from American Type

351

Culture Collection (ATCC, The Global BioResource Center). C2C12 cells were

352

cultured in DMEM supplemented with 10% fetal FBS on collagen-coated 35 mm

353

glass-bottom dishes at 37°C in an atmosphere of 95% air and 5% CO2 in a CO2

354

incubator (SANYO). One day after plating, the medium was replaced with fresh

355

DMEM with 10% FBS and when the culture reached ~80% confluency, cells were



356

transfected with 4 µg plasmid DNA using Lipofectamine 2000 Transfection Reagent

357

(Life Technologies) according to the manufacture’s recommended protocol. Medium

358

was replaced after 4 to 5 h to differentiation medium DMEM (high glucose +

359

glutamine, no sodium pyruvate) (GIBCO) with 2% Donor Equine Serum (HyClone)

360

containing 100 µg/mL penicillin-streptomycin31,32. Differentiation medium was

361

changed every 24 to 36 h. Cell differentiation has been studied in this system at 3, 5,

362

7, 10, and 13 days after being exposed to differentiation medium32 and generally

363

occurs 3 days after differentiation is initiated. We obtained myogenic C2C12 cells

364

after 10 days of culture31.

365

Bioluminescence Ca2+ imaging

366

For bioluminescence imaging of HeLa and C2C12 cells, the culture medium was

367

exchanged with phenol red–free DMEM/F12 in advance of the imaging. To perform

368

bioluminescence Ca2+ imaging, we added furimazine (Promega) at a final

369

concentration of 20 µM to the culture medium. An inverted LUMINOVIEW

370

microscope LV-200 (Olympus) equipped with a 60× oil-immersion objective

371

(Olympus, UPlanSApo, numerical aperture 1.35) and a 0.5× relay lens was used.

372

Emission signal was detected using an EM-CCD camera ImagEM (Hamamatsu

373

Photonics) with an exposure time of 1 s, binning of 1×1, and EM gain of 400. We

374

used a 100× oil-immersion objective (Olympus, UPLSAPO 100, numerical aperture

375

1.4) for ER Ca2+ imaging for thapsigargin stimulation. At 3 min after adding

376

furimazine, the stimulants for calcium flux were added. For CeNL(Ca2+),

377

GeNL(Ca2+)_520, and OeNL(Ca2+) constructs, emission filters FF01-483/32

378

(Semrock), FF01-520/35 (Semrock), and FF01-593/40 (Semrock) were used,

379

respectively. For the ER-tracker Green (ThermoFisher Scientific) and MitoTracker

380

Red, (Thermo Fisher Scientific) we used the emission filters FF01-624/40 and FF01ACS Paragon Plus Environment

Page 16 of 32

Page 17 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


381

520/35 (Semrock). For multicolor bioluminescence Ca2+ imaging of the HeLa cells

382

expressing

383

mito_OeNL(Ca2+)_18µ, three images were acquired with each of the three emission

384

filters described above and signals from each indicator was separated by linear

385

unmixing using these coefficients by PrizMage software (Molecular Devices)

386

following a previously reported method12. All imaging studies were performed at

387

25°C.

ER_CeNL(Ca2+)_110µ,

H2B-GeNL(Ca2+)_520,

388


and


389

ASSOCIATED CONTENT

390

Supporting Information

391

The Supporting Information is freely available on the ACS Publications website.

392

Supporting Figures S1−S3, Tables S1 (PDF), Supporting Movies S1−S6 (AVI).

393

Accession code

394

The nucleotide sequences of the CeNL(Ca2+) and OeNL(Ca2+) constructs were

395

deposited in DDBJ under accession numbers LC334051, LC334052, LC334053,

396

LC334055.

397

AUTHOR INFORMATION

398

Corresponding Author

399

Phone: +81-6-6879-8480.

400

E-mail: [email protected]

401

Notes: The authors declare no competing financial interest.

402

ORCID

403

Md Nadim Hossain: https://orcid.org/0000-0002-8086-8939

404

405

Funding

406

This work was supported by a Grant-in-Aid for Scientific Research on Innovative

407

Areas ‘Spying minority in biological phenomena’ (No. 3306) of MEXT (No. 23115003,

408

No. 23115001), a Grant-in-Aid for Scientific Research (A) of MEXT (No. 26251018),

409

the JST-SENTAN program, A. Advanced Research Networks, the Uehara Memorial


Page 18 of 32

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410

Foundation, and the Naito Foundation to T.N.; T.M. was supported by a grant on the

411

‘Interplay of developmental clock and extracellular environment in brain formation’

412

(No. JP16H06487). We additionally are thankful to the Bionanophotonics Consortium

413

(BNPC) for assistance with microscopy.

414

ACKNOWLEDGEMENTS

415

We thank to M. Iino (University of Tokyo) for his kind suggestions and fruitful

416

comments on our work. We are very grateful to the Japan Government and the

417

Ministry of Education, Culture, Sports, Science, and Technology (MEXT) for

418

supporting Md Nadim Hossain through the scholarships during this study.

419



420

REFERENCES

421

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Chem. 61, 251–366.

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(2) Reddish, F. N., Miller, C. L., Gorkhali, R., and Yang, J. J. (2017) Calcium

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dynamics mediated by the endoplasmic/sarcoplasmic reticulum and related

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diseases. Int. J. Mol. Sci. 18, E1024.

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(3) Martonosi, A.N. (1984) Mechanisms of Ca2+ release from sarcoplasmic reticulum

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of skeletal muscle. Physiol Rev 64, 1240–1320.

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Harvey, B. K. (2015) A low affinity GCaMP3 variant (GCaMPer) for imaging the

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multicolour bioimaging. Nat. Commun. 7, 1–10.

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aequorin. Nature 256, 236–238.

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Brulet, P., and Brúlet, P. (2000) Chimeric green fluorescent protein-aequorin as

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bioluminescent Ca2+ reporters at the single-cell level. Proc. Natl. Acad. Sci. U. S. A.

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speed single-cell and whole-body imaging. Nat. Commun. 3, 1262.

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utilizing a novel imidazopyrazinone substrate. ACS Chem. Biol. 7, 1848–1857.

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animals. Mol. Imaging 12, 1–13.

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Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted

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yellow fluorescent proteins. Proc. Natl. Acad. Sci. U. S. A. 101, 10554–9.

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(22) Horikawa, K., Yamada, Y., Matsuda, T., Kobayashi, K., Hashimoto, M., Matsu-

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ura, T., Miyawaki, A., Michikawa, T., Mikoshiba, K., and Nagai, T. (2010)

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Spontaneous network activity visualized by ultrasensitive Ca2+ indicators, yellow

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Cameleon-Nano. Nat. Methods 7, 729–732.

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(23) Goedhart, J., van Weeren, L., Hink, M. A., Vischer, N. O. E., Jalink, K., and

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Gadella, T. W. J. (2010) Bright cyan fluorescent protein variants identified by

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fluorescence lifetime screening. Nat. Methods 7, 137–139.


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Sperimentale, C. R. (1994) Ca2+ signalling pathways activated by acetylcholine in

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mouse C2C12 myotubes. Eur. J. Physiol. 428, 340–345.

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(25) Palmer, A. E., Jin, C., Reed, J. C., and Tsien, R. Y. (2004) Bcl-2-mediated

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alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically

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encoded fluorescent sensor. Proc. Natl. Acad. Sci. 101, 17404–17409.

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(26) Wu, J., Prole, D. L., Shen, Y., Lin, Z., Gnanasekaran, A., Liu, Y., Chen, L., Zhou,

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H., Chen, S. R. W., Usachev, Y. M., Taylor, C. W., and Campbell, R. E. (2014) Red

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fluorescent genetically encoded Ca2+ indicators for use in mitochondria and

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endoplasmic reticulum. Biochem. J. 464, 13–22.

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(27) England, C. G., Ehlerding, E. B., and Cai, W. (2016) NanoLuc: A Small

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Luciferase Is Brightening Up the Field of Bioluminescence. Bioconjug. Chem. 27,

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1175–1187.

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(28) Tiwari, D. K., Arai, Y., Yamanaka, M., Matsuda, T., Agetsuma, M., Nakano, M.,

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Fujita, K., and Nagai, T. (2015) A fast- and positively photoswitchable fluorescent

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protein for ultralow-laser-power RESOLFT nanoscopy. Nat. Methods 12, 515–518.

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(29) Kain, M. C. and S. R. Green fluorescent protein: properties, applications, and

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protocols-2nd ed. Methods of Biochemistry analysis_47.

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(30) Goedhart, J., Stetten, D. Von, Noirclerc-savoye, M., Lelimousin, M., Joosen, L.,

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Hink, M. A., Weeren, L. Van, Jr, T. W. J. G., and Royant, A. (2012) Structure-guided

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evolution of cyan fluorescent proteins towards a quantum yield of 93% Nat. Commun

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3, 751.

511

(31) Fisher-Aylor, K., and Williams, B. (1977) Cell Growth Protocol and



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Differentiation treatment for the C2C12 Cell Line From: Wold mouse ENCODE. Nat.

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Commun. Blau al. Sci. 270, 725–727.

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(32) Burattini, S., Ferri, R., Battistelli, M., Curci, R., Luchetti, F., and Falcieri, E.

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(2004) C2C12 murine myoblasts as a model of skeletal muscle development:

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Morpho-functional characterization. Eur. J. Histochem. 48, 223–233.

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(33) Inagaki, S., Tsutsui H., Suzuki K., Agetsuma M., Arai Y., Jinno Y., Bai G., Daniels

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M. J., Okamura Y., Matsuda T., and Nagai T. (2017) Genetically encoded

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bioluminescent voltage indicator for multi-purpose use in wide range of bioimaging.

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522

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Page 25 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


533 534

Figure 1. Development and properties of BP-GECI CeNL(Ca2+) constructs.

535

(A) A schematic description of Ca2+-sensing mechanism of CeNL(Ca2+). Components

536

of CeNL(Ca2+) from the N- to C-terminals are: mTQ2, the N-terminus of Nluc

537

(Nluc(N)), the calcium sensing domain CaM-M13, and the C-terminus of Nluc

538

(Nluc(C)). Ca2+ and substrate are indicated as magenta and blue circles,

539

respectively. (B) Amino acid sequences around four EF hands (EF1 to 4) in the CaM

540

of CeNL(Ca2+) are shown. Residues that have hydrogen bonds with Ca2+ are

541

connected by lines with Ca2+, which is indicated by the magenta circle. Residues,

542

where mutations (E31D, E67D, F92W, E104D, and D133E) are introduced to reduce

543

affinity for Ca2+ are shown in yellow. (C) Normalized in vitro Ca2+ titration curves of

544

CeNL(Ca2+)_19µ (Orange), CeNL(Ca2+)_110µ (magenta) and CeNL(Ca2+)_1.2µ



545

(black) are shown with fitted Hill plot curves. (D) Relative bioluminescence intensities

546

of Nluc and CeNL(Ca2+) series.

547


Page 26 of 32

Page 27 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


548

Figure 2. ER Ca2+ imaging with CeNL(Ca2+)_110µ in HeLa cells.

549

Fluorescent and bioluminescent images of CeNL(Ca2+)_110µ fused with an N-

550

terminal calreticulin signal sequence and the C-terminal ER retention signal KDEL in

551

HeLa cells. (A) From left to right, fluorescence of CeNL(Ca2+)_110µ, ER-Tracker Red

552

(co-stained), and merged image of them. Scale bar 5 µm. (B, D, F) Bioluminescence

553

images of HeLa cells expressing CeNL(Ca2+)_110µ in ER. (C, E, G) Time course of

554

the normalized bioluminescence intensity of multiple regions of interests (ROIs)

555

indicated in B, D and F. (C) Time course with 20 µM histamine stimulation (black:

556

ROI 1 and green: ROI 2) or mock stimulation by medium (gray). (E) Time course with

557

10 µM ATP (black: ROI 1 and blue: ROI 2) or mock (gray) stimulation. (G) Time

558

course with 3 µM thapsigargin treatment to inhibit ER Ca2+ influx (black: ROI 1 and

559

green ROI: 2). See Supplementary Movie S1, and S2. Scale bar 10 µm for B, D

560

and 5 µm for F.

561 562

Figure 3. SR Ca2+ dynamics in C2C12-derived myotubes monitored by

563

CeNL(Ca2+)_110µ.

564

Bright field (A) and bioluminescence (B) images of myotubes expressing SR-

565

targeted CeNL(Ca2+)_110µ. Scale bar 10 µm. (C) Time course of normalized

566

bioluminescence intensity change indicated several ROIs by stimulation with 50 µM



567

acetylcholine (green: ROI 1, and blue: ROI 2) and mock stimulation by medium

568

(gray). See Supplementary Movie S3.

569 570

Figure 4. Characterization of OeNL(Ca2+)_18µ. (A) Domain structure of

571

intermediary affinity OeNL(Ca2+)_18µ. (B) In vitro Ca2+ titration curve of

572

OeNL(Ca2+)_18µ fitted with a Hill plot curve (C) Fluorescence of OeNL(Ca2+)_18µ,

573

MitoTracker Green FM (co-stained), and merged image of them in HeLa cells are

574

shown. Scale bar 10 µm (E) Time course of mitochondrial Ca2+ imaging of

575

OeNL(Ca2+)_18µ following stimulation with histamine (20 µM) (blue: ROI 1 and

576

black: ROI 2) and mock stimulation by medium (gray) in HeLa cells. ROIs for the

577

analysis are indicated in D by white circles. See Supplementary Movie S4.


Page 28 of 32

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578 579

Figure 5. Simultaneous analysis of Ca2+ dynamics in nucleus and ER.

580

(A) Time-lapse bioluminescence images of CRsig_CeNL(Ca2+)_110µ expressed in

581

ER (cyan) and GeNL(Ca2+)_520 in nucleus (magenta) of HeLa cells with histamine

582

stimulation following linear unmixing. (B) Merged image of cyan and green channels

583

after stimulation, Scale bar 5 µm. (C) Time course of normalized bioluminescence

584

intensity from ER (ROIs 3 and 4) and nuclear (ROIs 1 and 2) following stimulation

585

with 20 µM histamine (green: ROI 1, magenta: ROI 2, black: ROI 3, and cyan: ROI 4)

586

and mock stimulation by medium (gray) in a HeLa cell. See Supplementary Movie

587

S5.



588 589

590

Figure 6. Simultaneous multicolor bioluminescence Ca2+ imaging in ER,

591

nucleus, and mitochondria.


Page 30 of 32

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592

Bioluminescence

images

of

each

emission

channel

for

ER-localized

593

CeNL(Ca2+)_110µ, nuclear-localized GeNL(Ca2+)_520, and mitochondrial-localized

594

OeNL(Ca2+)_18µ in HeLa cells, before (A) and after (B) linear unmixing. Scale bar 10

595

µm. (C) Merged image of bioluminescence from the three subcellular compartments.

596

Scale bar 10 µm. (D) Time course of normalized fluorescence intensity in ER (cyan),

597

nucleus (magenta), and mitochondria (green) following 20 µM histamine stimulation

598

or mock stimulation by medium (gray). Regions of interest for measurements are

599

shown in C: mitochondria (ROI 1), nucleus (ROI 2), and ER (ROI 3). See

600

Supplementary Movie S6.

601

602

603

604

Table 1 Properties of CeNL(Ca2+) variants and OeNL(Ca2+)_18µ

Indicator name

Mutation in CaM

Kd for Ca2+ (µM)

Hill coefficient

Dynamic range (%)

Types of affinity

CeNL(Ca2+)_1.2µ

E67D, E104D, D133E

1.2

0.98

106

Intermediate

CeNL(Ca2+)_19µ

E67D, F92W, E104D, D133E

19

1.15

118

Intermediate

CeNL(Ca2+)_110µ

E31D, F92W, E104D. D133E

110

2.18

108

Low

OeNL(Ca2+)_18µ

E67D, E104D, D133E

18

1.5

114

Intermediate

605

606

607



TOC graphic 269x263mm (300 x 300 DPI)


Page 32 of 32

Bioluminescent Low-Affinity Ca2+ Indicator for ER with Multicolor

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