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1 2

Upgrading a Microplate Reader for Photobiology

3

and All-Optical Experiments

4 5

Florian Richter1,*, Ulrike S. Scheib2, Jennifer Mehlhorn2, Roman Schubert1, Jonas Wietek2,

6

Oliver Gernetzki2, Peter Hegemann2, Tilo Mathes2,3, Andreas Möglich1,*

7 8

1

Humboldt-Universität zu Berlin, Institut für Biologie, Biophysikalische Chemie, Berlin, Germany

9

2

Humboldt-Universität zu Berlin, Institut für Biologie, Experimentelle Biophysik, Berlin, Germany

10

3

Department of Physics, Faculty of Sciences, VU University Amsterdam, The Netherlands

11 12

*

13

F. R. Tel. +49-30-2093-8845. E-mail [email protected]

14

A. M. Tel. +49-30-2093-8850. Fax +49-30-2093-8948. E-mail [email protected]

to whom correspondence should be addressed:

1

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Table of contents entry

16

Using open-source electronics and optical components, we add programmable illumination

17

capabilities to a multimode microplate reader, thus enabling photobiological and all-optical

18

experiments at high throughput, reproducibility and fidelity.

19 20 21

Abstract

22

Automation can vastly reduce the cost of experimental labor and thus facilitate high

23

experimental throughput, but little off-the-shelf hardware for the automation of illumination

24

experiments is commercially available. Here, we use inexpensive open-source electronics to add

25

programmable illumination capabilities to a multimode microplate reader. We deploy this setup to

26

characterize light-triggered phenomena in three different sensory photoreceptors. First, we study

27

the photoactivation of Arabidopsis thaliana phytochrome B by light of different wavelengths.

28

Second, we investigate the dark-state recovery kinetics of the Synechocystis sp. blue-light sensor

29

Slr1694 at multiple temperatures and imidazole concentrations; while the kinetics of the W91F

30

mutant of Slr1694 are strongly accelerated by imidazole, the wild-type protein is hardly affected.

31

Third, we determine the light response of the Beggiatoa sp. photoactivatable adenylate cyclase

32

bPAC in chinese hamster ovary cells. bPAC is activated by blue light in dose-dependent manner

33

with a half-maximal intensity of 0.58 mW cm-2; intracellular cAMP spikes generated upon bPAC

34

activation decay with a half time of about 5 minutes after light switch-off. Taken together, we

35

present a setup which is easily assembled and which thus offers a facile approach to conducting

36

illumination experiments at high throughput, reproducibility and fidelity.

37 38 2

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Introduction

40

Laboratory automation and high-throughput experiments have a major impact on life-sciences

41

research. As a core piece of laboratory automation equipment, multimode microplate readers can

42

investigate multiple samples in parallel (usually in 96-, 384- or 1536-well format) with respect to

43

absorption, fluorescence, and luminescence, while at the same time controlling the temperature

44

and/or atmospheric composition of the sample environment. Such microplate readers can usually

45

be programmed to automate measurements, so as to facilitate high sample throughput.

46

In the photobiology field, numerous studies crucially require repetitive experimentation at

47

varying parameters, e.g., the analysis of responses to different light qualities and quantities1, or

48

the screening of variant libraries of light-responsive systems2. Usually, these studies mandate that

49

sample illumination and measurement be conducted in quick succession. Using a microplate

50

reader for such experiments therefore necessitates that individual wells can be illuminated inside

51

the instrument, and that illumination events can be precisely programmed as part of a

52

measurement protocol. Most commercially available microplate readers do not possess dedicated

53

illumination capabilities, and the conventional probe light used for absorption and fluorescence

54

measurements is generally of too low intensity to activate photoreceptors to significant extent

55

within reasonably short time intervals. To overcome this limitation, we used freely available open-

56

source electronics and optical components to design and implement an illumination device for a

57

widely used microplate reader. As illustrated in Fig. 1A, the Tecan Infinite M200 pro microplate

58

reader can be equipped with an external injector module which allows for injection of liquids into

59

individual wells at defined stages in a measurement protocol. This injector module consists of a

60

hollow metal cylinder (referred to as ‘injection cylinder’) that houses two injection tubes, denoted

61

A and B, which are connected to two pumps and two liquid reservoirs. The injection cylinder is

62

inserted into the microplate reader through a hole in the top. When the microplate reader 3

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63

receives an injection command for either pump, the microplate is positioned such that the desired

64

well is directly below the injection hole, and liquid from the respective reservoir (A or B) is injected

65

into said well. We reasoned that by shining light through the injection hole, this setup could be

66

repurposed for automatically ‘injecting’ light instead of liquid, and set out to implement an

67

illumination setup where both the duration and wavelength of illumination can be specified

68

through the standard iControl operating software of the Tecan microplate reader. The regular

69

injection capabilities from line A are retained in the repurposed microplate reader, thus allowing

70

experiments that rely on injection of both liquid and light. We demonstrate the utility of this setup

71

for studies on three photoreceptor systems.

72

Sensory photoreceptors are the biological agents that mediate vision and diverse light-driven

73

adaptive responses in numerous organisms3,4. Upon light absorption by its chromophore, the

74

photoreceptor undergoes structural and dynamic changes that culminate in a light-dependent

75

change of its biological activity. Based on the chromophore and the photochemistry employed for

76

light detection, photoreceptors are grouped into distinct classes. One class, BLUF domains

77

(sensors of blue light using flavin adenine dinucleotide) are widely distributed in prokaryotes and

78

protists where they control the activity of diverse physiological processes in response to blue-light

79

exposure5,6. Photon absorption in the blue spectral region leads to the formation of the signaling

80

state of the photoreceptor that then thermally reverts to the dark state over the course of

81

seconds to minutes depending on BLUF domain. The molecular details of BLUF photochemistry are

82

still under intense debate but both photoactivation and thermal inactivation are apparently

83

accomplished by a rearrangement of the hydrogen-bonding network near the flavin-nucleotide

84

cofactor7. Another class, phytochromes (Phy) are biological red-/far-red-light receptors that occur

85

in plants, (cyano)bacteria, fungi, and diatoms8. Phys exist in intrinsic equilibrium between red-

86

light-absorbing and far-red-light-absorbing forms, Pr and Pfr, which differ in the isomerization

87

state of their bilin chromophore. Illumination with red and far-red light drives the Pr→Pfr and 4

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Pfr→Pr transiƟons, respecƟvely; that is, Phys are photochromic and can be toggled between two

89

states by exposure to different light colors. In higher plants, five phytochromes (PhyA through

90

PhyE) control various aspects of physiology, e.g., photomorphogenesis and shade avoidance8,9.

91

In recent years, photoreceptors have garnered increased attention due to their use in

92

optogenetics10. For these applications, photoreceptors are introduced as DNA templates to target

93

cells, tissues and organs which are thereby rendered light-sensitive. Application of light can hence

94

be used to control cellular events with supreme spatiotemporal resolution, minimal invasiveness

95

and full reversibility. As a case in point, the blue-light-activated adenylate cyclase bPAC from

96

Beggiatoa sp. possesses a BLUF domain and catalyzes formation of the second messenger 3’,5’-

97

cyclic adenosine monophosphate (cAMP) upon blue-light exposure11,12. Since cAMP is involved in

98

the regulation of manifold biological processes, bPAC represents a particularly versatile and widely

99

used optogenetic tool13–15. In a similar vein, the light-induced association of Arabidopsis thaliana

100

PhyB with its interacting factors PIF16 has been exploited in the construction of light-responsive

101

cellular circuits, e.g., to control by light gene expression17 or cell motility18.

102 103 104

Results and discussion

105

Design and calibration of automated illumination setup

106

To enable efficient and versatile studies on sensory photoreceptors and other light-responsive

107

systems, we sought to equip the off-the-shelf Tecan Infinite M200 pro microplate reader with

108

programmable illumination capabilities by using readily available optical and electrical

109

components (Fig. 1A-C). Two separate aspects had to be realized: i) light delivery through the

110

injection hole; and ii) control of an LED through the plate reader operating software. For i), a fiber-

111

optics waveguide (1.5 mm diameter) was threaded through an empty injection cylinder and was 5

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connected to a light source containing eight separate LEDs that are coupled into a single fiber

113

output. For ii), the communication between the microplate reader and its injection module was

114

captured and processed using an Arduino microcontroller (Fig. 1B). We configured the setup such

115

that injection commands issued to pump B of the injector are re-interpreted to control the LED

116

fiber optics; in this way, illumination events can be conveniently implemented into measurement

117

protocols using the standard iControl software (version 1.9) of the microplate reader. Notably, we

118

also fitted an injection tube (connected to pump A) into the cylinder alongside the waveguide,

119

thus enabling experiments that require both illumination and dispensing of liquid from line A.

120

Details on the setup are provided in the Experimental section and as supplementary material.

121

We first measured the light power of the eight LEDs included in the light source (peak emission

122

at 385, 420, 470, 530, 590, 617, 680, 850 nm, respectively) at maximum output and found that it

123

ranged from about 2 to 10 mW cm-2 (Fig. 1D); lower intensities can be set in 0.1 % increments via

124

the control software of the LED light source.

125

Next, we assessed whether light application via our new illumination setup can be implemented

126

into standard iControl measurement protocols and whether the applied light intensities suffice to

127

trigger photochemical reactions in sensory photoreceptors. To this end, we resorted to A. thaliana

128

PhyB which absorbs light across the entire UV/visible spectral region and which can be reversibly

129

switched between its Pr and Pfr states by illumination with different wavelengths. In its dark-

130

adapted form, PhyB predominantly populates the Pr state which is characterized by an absorption

131

maximum at 652 nm (Fig. 2A). Illumination with 385, 420, 470, 530, 590, 617 nm could be used to

132

also populate the Pfr state, which maximally absorbs around 714 nm, to varying extent, where 590

133

and 617 nm were most efficient in driving the Pr→Pfr conversion. By contrast, illumination with

134

680 nm efficiently promoted the reverse Pfr→Pr conversion, and the resultant absorpƟon

135

spectrum largely corresponded to that of the dark-adapted state. Illumination with 850 nm elicited

136

no spectral changes. 6

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To assess whether illumination via the fiber-optics setup can be confined to a single well

138

without affecting adjacent wells on the microplate, we put nine equivalent aliquots of PhyB in

139

individual wells in a 3-by-3 arrangement. Absorption spectra were recorded for all nine wells prior

140

to and after illumination of the center well with 590-nm light at maximum intensity for 16 s (Fig.

141

2B). In the center well, conversion to the Pfr state occurred to the same extent as before; by

142

contrast, in the surrounding wells the absorption spectra before and after illumination closely

143

overlapped. These data show that individual wells can be addressed with minimal cross-talk, even

144

when relatively high light doses are applied.

145 146

Temperature and imidazole effects on dark-state recovery kinetics in Slr1694

147

Having established the principal function of the customized microplate reader illumination

148

setup, we analyzed the dark-state recovery kinetics after blue-light exposure of the Synechocystis

149

sp. PCC 6803 Slr1694 BLUF protein at multiple, systematically varied experimental conditions (Fig.

150

3). While the response of these kinetics towards perturbations could well provide incisive insight

151

into the still contentious mechanism of the BLUF photocycle, corresponding systematic studies

152

have so far been hampered by the sizeable number of measurements required. However, using

153

our newly implemented, automated illumination setup, only modest amounts of experimental

154

labor are required to set up and conduct multiple measurements in reproducible and efficient

155

fashion.

156

It was previously reported that imidazole dramatically accelerates the dark recovery kinetics in

157

the BLUF protein AppA from R. sphaeroides19, similar to observations for the related class of light-

158

oxygen-voltage class of photoreceptors20. Base catalysis thus might generally play an important

159

role in the dark-state recovery mechanism, although the precise molecular basis remains unclear.

160

To get a better understanding of this effect and to glean additional insight into the mechanism of 7

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BLUF photochemistry, we recorded dark-state recovery kinetics as a function of imidazole

162

concentration (0, 5, 25, 50, 100, 170 and 250 mM) and temperature (27, 29, 31, 33, 35 and 37 °C).

163

Surprisingly, the single-exponential dark-state recovery kinetics of Slr1694 (rate constant for dark

164

recovery k = 0.08 s-1 at 27 °C) were largely insensitive towards imidazole addition up to

165

concentrations of 250 mM (Fig. 3B, 3C). Evidently, the strongly accelerating effect of imidazole is

166

not universally shared among all BLUF domains.

167

Inspection of the structures of AppA21,22 and Slr169423 pinpoints the histidine residue 73 in

168

Slr1694 which is absent in AppA (Fig. 3A). Interestingly, His73 is situated in immediate vicinity of

169

the strictly conserved histidine 72 which probably plays a crucial role in BLUF photochemistry

170

because of its involvement in a proton-relay pathway between the exterior solvent and the flavin

171

chromophore24. We reasoned that His73 might serve as an imidazole proxy which would account

172

both for the intrinsically faster recovery kinetics of Slr1694 compared to AppA and for its low

173

sensitivity towards imidazole addition. To test this notion, we generated the H73S mutant of

174

Slr1694 which exhibited slightly slower recovery kinetics than wild-type (k = 0.04 s-1 at 27 °C).

175

However, the sensitivity towards imidazole was only marginally enhanced (Fig. 3D). By contrast,

176

we discovered that the previously generated Slr1694 mutant W91F25,26 displays a very strong

177

effect of imidazole on the lifetime of the signaling state (Fig. 3E). The intrinsic dark recovery in

178

Slr1694 W91F is around 10-fold slower than in wild-type (k = 8·10-3 s-1 at 27 °C) but is accelerated

179

by about 30-fold in the presence of 250 mM imidazole (Fig. 3F).

180

To better characterize the imidazole effects among the different Slr1694 variants, we recorded

181

recovery kinetics at several temperatures and calculated activation energies (Fig. 3G). In the

182

absence of imidazole, wild-type Slr1694 and the H73S mutant showed closely similar activation

183

energies of EA = 30 kJ mol-1 and EA = 32 kJ mol-1, respectively. By contrast, the recovery kinetics in

184

the W91F mutant displayed stronger temperature dependence with EA amounting to 69 kJ mol-1.

185

At elevated imidazole concentrations ≥ 25 mM, the activation energy for dark recovery in W91F 8

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reached an asymptotic value of 49 kJ mol-1. These data can be explained by assuming two parallel

187

pathways for dark recovery, one that is independent of imidazole, and one that is accelerated by

188

imidazole. In the absence of imidazole, only the first pathway contributes to the observable

189

kinetics, whereas at higher concentrations the kinetics are increasingly dominated by the

190

imidazole-dependent pathway. Related observations were also made for the recovery kinetics of

191

Slr1694 wild-type and H73S. However, in these cases, imidazole addition led to an increase of EA to

192

about 50 kJ mol-1 and 60 kJ mol-1, respectively, at 250 mM imidazole. Due to the lower imidazole

193

sensitivity of these variants, the accelerating effect cannot be saturated, and both recovery

194

pathways contribute to the observable kinetics at all tested imidazole concentrations.

195

In the absence of structural information on the Slr1694 mutants and on the signaling state, a

196

molecular interpretation of these findings is difficult. However, Fourier transform infrared

197

spectroscopy showed that mutation of tryptophan 91 to phenylalanine increases the flexibility of

198

the β5 strand of the BLUF domain which has been centrally implicated in signal transduction24,26.

199

Resultant changes in protein dynamics could govern solvent accessibility to the flavin

200

chromophore and could thereby affect the stability and lifetime of the signaling state. More

201

experiments are needed to clarify the mechanism of dark reversion in BLUF photoreceptors.

202

Doubtless, the automated microplate reader illumination setup we present here will greatly

203

facilitate pertinent measurements.

204 205

Light-activated adenylate cyclase activity in eukaryotic cell culture

206

As detailed above, we configured the microplate reader such that both light and liquid can be

207

dispensed in a single experiment. We exploited this previously unavailable setup to efficiently map

208

the blue-light-dependent activity of bPAC in eukaryotic cells. To readily visualize adenylate cyclase

209

activity, we resorted to a previously constructed chinese hamster ovary (CHO) reporter cell line27,28 9

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(Fig. 4A). Briefly, this cell line stably expresses a cyclic-nucleotide-gated (CNG) ion channel and the

211

aequorin luciferase from Aequoria victoria. An increase of the cAMP concentration inside these

212

cells triggers opening of the CNG channel which allows influx of Ca2+ ions from the extracellular

213

medium. Once aequorin binds Ca2+, it rapidly oxidizes its cofactor coelenterazine to

214

coelenteramide, with concomitant release of CO2 and emission of a photon in the blue-green

215

spectral region. The resultant, transient luminescence signal can be measured with great

216

sensitivity via the dedicated luminescence channel of the microplate reader.

217

To assess the light-regulated adenylate cyclase activity of bPAC, the CHO reporter cell line was

218

transiently transfected with bPAC-encoding DNA. Following seeding into 96-well microplates and

219

incubation in the dark with the cofactor coelenterazine, the transfected cells were exposed for 8 s

220

to different intensities of 470-nm light (0, 0.17, 0.30, 0.77, 1.51, 1.96, 2.50 mW cm-2) to stimulate

221

bPAC activity11,12. Immediately after light exposure, CaCl2 was added to a final concentration of 3

222

mM, and resultant luminescence signals were recorded over time (Fig. 4B). The integrated

223

luminescence signal, reflective of intracellular adenylate cyclase activity, increases with applied

224

light intensity but approaches saturation for intensities ≥ 2 mW cm-2. A hyperbolic fit was used to

225

determine the half-maximal light dose for bPAC activation of E50 = (0.58 ± 0.12) mW cm-2 (Fig. 4C).

226

This number corresponds to a previously reported value of (0.40 ± 0.04) mW cm-2 for the half-

227

maximal light dose although the underlying experiments were performed in vitro on purified

228

bPAC, and high-performance liquid chromatography was used for detection12.

229

Empowered by the efficient microplate reader illumination setup, we sought to investigate for

230

how long cAMP spikes persist in the CHO cells after blue-light exposure ceases (470 nm, 2.5 mW

231

cm-2). To this end, we successively increased the time lag between light application and CaCl2

232

injection from 3 seconds to 40 minutes. With increasing delay time, the observable luminescence

233

rapidly decayed, and at delay times larger than 20 minutes, luminescence had returned to baseline

10

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234

values (Fig. 4D, E). Based on these data, we estimate that intracellular cAMP spikes that result

235

from blue-light-triggered bPAC activity decay with a half life of approximately 5 minutes.

236

Taken together, our experiments provide valuable benchmarks for optogenetic applications of

237

bPAC. More broadly, the newly developed illumination system allows to assess the light sensitivity

238

of bPAC and intracellular cAMP persistence in a time-resolved and automated way, thereby

239

complementing single-cell electrophysiological activity assays12. The ability to dispense both light

240

and liquid within the same measurement protocol proved of decisive advantage, as it enables

241

multiplexing and ensures reproducibility.

242 243 244

Experimental

245

Design of the automated illumination setup

246

The rejigging of the Tecan Infinite M200 pro microplate reader (Tecan Group Ltd., Männedorf)

247

for illumination comprised two principal steps: i) delivering light into the microplate reader

248

through the injection hole; and ii) turning an LED on and off in response to an injection signal sent

249

by the microplate reader iControl 1.9 software.

250

For i), an empty injection cylinder was generously provided by Tecan, into which a 1.5-mm

251

waveguide (Thorlabs, Dachau) and an injection tube were fitted. The injection tube was connected

252

to pump A of the injector. The waveguide was hooked up to an LED light source (Mightex Systems,

253

Toronto) containing eight different LEDs (385 [full-width at half maximum: 10 nm], 420 [13], 470

254

[20], 530 [32], 590 [17], 617 [18], 680 [21], 850 [32] nm), thus allowing simultaneous delivery of

255

light of several wavelengths through the injection hole into the microplate reader.

256

For ii), we exploited the fact that in the Tecan Infinite series, the injection module is connected

257

to the microplate reader through an external cable. Through this cable, the control software and 11

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the injector module pass messages back and forth using a so-called ‘Controller-Area-Network’, or

259

CAN-bus protocol (http://en.wikipedia.org/wiki/CAN_bus). The graphical user interface of the

260

iControl software allows users to specify the volume (in μL), the speed (in μL s-1) and the source

261

reservoir/pump (A or B) of an injection event. We used an Arduino-Mega microcontroller (EXP

262

GmbH, Saarbrücken) in combination with a Seeed-Studio CAN Bus shield (EXP GmbH, Saarbrücken)

263

to intercept and record message traffic on the CAN-Bus in response to an injection command.

264

Analysis of these recorded messages indicated that the control software passes the three user-

265

specified parameters of pump identity, injection volume and injection speed to the injector which

266

then performs a corresponding injection and reports back to the control software. The quotient of

267

injection volume over injection speed yields the injection duration, i.e. the time that the injection

268

target well is situated below the injection hole, and consequently the time that this well would be

269

illuminated if light were delivered through the injection hole. We next connected the Arduino

270

microcontroller to the TTL (transistor-transistor logic) trigger port of the Mightex light source, thus

271

enabling the LEDs to be controlled through signals generated by the Arduino controller. Finally, we

272

programmed the Arduino controller to, in response to a pump-B injection command passed by the

273

iControl software, turn on the LED for the injection duration. As an added feature, the Arduino

274

controller was programmed to turn on different LEDs depending on the last digit of the user-

275

specified injection volume, meaning that both the duration and color of illumination can be

276

encoded in a single injection command. The Arduino source code for decoding microplate-reader

277

communications

278

https://gist.github.com/flosopher/6fd3ff661df9db708632. A list of parts used for the illumination

279

upgrade as well as photographs that illustrate the setup are provided as supplementary

280

information.

and

switching

LEDs

in

response

has

been

deposited

at

281

In summary, by fitting a waveguide through the injection hole, and splicing a microcontroller

282

into the data connection between microplate reader and injector, we were able to illuminate 12

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283

individual microplate wells in response to injection commands, thus enabling the variable

284

programming of illumination events during measurement protocols through the regular

285

microplate reader iControl software. Since the injector is equipped with two pumps that are

286

separately addressable by the control software, measurement protocols that feature decoupled

287

illumination and injection events are also feasible. All experiments reported presently were

288

conducted with 96-well microplates; while the plate reader also handles 384-well plates, we have

289

not tested the illumination setup for such plates.

290 291

Experiments on A. thaliana phytochrome B

292

The PAS-GAF-PHY photosensory core module of A. thaliana PhyB (residues 1-651) was co-

293

expressed in Escherichia coli BL21 with heme oxygenase and PcyA from Synechocystis sp. which

294

together provide the phycocyanobilin chromophore29. After protein expression for 20 h at 18 °C,

295

cells were lysed by sonication, and PhyB was purified by immobilized metal ion affinity

296

chromatography (Protino Ni-NTA, Macherey-Nagel, Düren) using an Äkta pure system (GE

297

Healthcare, Freiburg). Fractions containing PhyB were pooled and further purified via anion

298

exchange (HiTrap Q, GE Healthcare). The PhyB sample was concentrated and dialyzed into storage

299

buffer (25 mM Tris pH 8, 200 mM NaCl, 10 % (v/v) glycerol); sample purity and chromophore

300

incorporation were confirmed by gel electrophoresis and zinc-acetate staining30. Concentration of

301

PhyB was determined by absorption spectroscopy using an extinction coefficient of 93,000 M-1 cm-

302

1

303

closely similar to the previously reported value of 1.05 for a corresponding PhyB construct31.

304

Microplate reader measurements were conducted at 30 °C in 96-well μClear microplates (Greiner

305

BioOne, Frickenhausen) on 100 μL solution containing 5 µM PhyB. Absorption spectra were

at 652 nm. The ratio of the absorption at 652 nm and at 280 nm amounted to 1.12 which is

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recorded prior to and following illumination for 16 s at maximum intensity of the respective LED in

307

the light source.

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Experiments on Synechocystis sp. PCC 6803 Slr1694

310

Expression constructs for Slr1694 wild-type and the W91F variant were prepared as previously

311

described32,33; the corresponding construct for the H73S variant was obtained by site-directed

312

mutagenesis (QuikChange, Stratagene, Agilent, Waldbronn). Protein expression in a fermenter and

313

subsequent purification were carried out as described32,33. Spectroscopic measurements were

314

performed in 96-well μClear microplates with each well containing 100 μL of 100 μM Slr1694

315

protein and varying imidazole concentrations (0, 5, 25, 50, 100, 170 and 250 mM). Samples were

316

exposed to 470-nm light (7.7 mW cm-2) for 8 s, and dark-state recovery kinetics were followed by

317

measuring absorption at 490 nm. Measurements were performed at temperatures of 27, 29, 31,

318

33, 35 and 37 °C; temperature fluctuations over time were on the order of ± 0.2 °C or less. For

319

data analysis, all obtained curves were fitted to a single-exponential decay using the ‘leastsq’

320

optimization algorithm implemented in the Python module SciPy (http://scipy.org/). The reported

321

rate constants for dark recovery, k, are mean ± s.d. of triplicate measurements.

322 323

Experiments on Beggiatoa sp. bPAC

324

Light-triggered alterations of intracellular cAMP levels mediated by bPAC were detected in a

325

CHO cell line that stably expresses a cAMP-sensitive heterotetrameric CNG channel and the

326

luminescence reporter aequorin (provided by Dr. F. Wunder, Bayer Pharma)28. Cells were cultured

327

at 37 °C and 5 % (v/v) CO2 in Dulbecco’s modified Eagle’s medium/NUT mix F-12 with L-glutamine,

328

supplemented with 10 % (v/v) inactivated fetal calf serum, 50 U mL-1 penicillin, 50 µg mL-1

329

streptomycin, 2.5 µg mL-1 amphotericin B, 0.6 mg mL-1 hygromycin B, and 0.25 mg mL-1 zeocin. In 14

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330

preparation of activity measurements, 2.5-3.0·104 cells were seeded into single wells of 96-well

331

μClear microplates containing 100 μL medium. One day after seeding, wells were supplied with 60

332

µL medium-transfection mix, containing 400 ng DNA encoding bPAC-mCherry12 and 1.4 µL FuGENE

333

HD transfection reagent (Promega). After 48 h, cells were washed once with Ca2+-free Tyrode

334

solution (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl2, 4.8 mM NaHCO3 pH 7.4) and

335

incubated at 37 °C with 0.6 µg µL-1 coelenterazine (Promega). After 1-1.5 hours, cells were washed

336

and supplemented with 100 µL Ca2+-free Tyrode buffer per well.

337

To analyze light-activated adenylate cyclase activity, wells were illuminated for 8 s with

338

different intensities of 470-nm light provided by the fiber-optics light source. After a defined delay,

339

CaCl2 was injected to a final concentration of 3 mM; for experiments in Fig. 4B this delay was held

340

constant at 3 s, for experiments in Fig. 4D this delay was varied between 3 s and 40 min. Resultant

341

luminescence was monitored over time using a (520 ± 20) nm bandpass emission filter.

342

Luminescence values were integrated over 33 s and corrected for background; reported values

343

represent mean ± s.e.m. of four independent measurements. The light-dose dependency of the

344

luminescence signals was fitted to a hyperbolic function using the non-linear least squares

345

algorithm in Origin Pro 8.0.

346 347 348

Conclusion

349

In summary, we have presented an easily implementable strategy for adding illumination

350

capabilities to a widely used, commercially available multimode microplate reader. All optical and

351

electronic components are readily obtainable; notably, the LED light source used presently can

352

easily be substituted by any other light source that can be controlled by TTL signals. The newly

353

established, programmable illumination setup offers several decisive benefits: samples can be 15

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354

illuminated with multiple wavelengths within one measurement (cf. Fig. 2); data acquisition is

355

greatly facilitated thus enabling high throughput, reproducibility and fidelity (cf. Fig. 3);

356

experiments that rely on precisely synchronized injections of both liquid and light are feasible (cf.

357

Fig. 4).

358

We expect our setup to be broadly applicable to studies on sensory photoreceptors as

359

demonstrated here. Of particular interest, the setup is suitable for optogenetic studies as it

360

supports all-optical experiments in which a system is both perturbed and probed by light. For

361

example, light-gated ion channels34 can be combined with fluorescent voltage sensors to achieve

362

continuous and precise online control over membrane voltage35. In a similar vein, bPAC11,12 (or,

363

other light-activated nucleotide cyclases36,37) can be combined with a newly available, red-light-

364

activated cAMP/cGMP-specific phosphodiesterase38 and suitable cNMP indicator dyes to establish

365

corresponding, tight control over intracellular cNMP levels.

366

More generally, the approach we present here not only applies to photosensory signal

367

transduction but also to any other biological, physical or chemical reaction that can be triggered

368

by light and whose progress can be monitored spectroscopically.

369 370 371

Acknowledgements

372

We thank members of the Hegemann and Möglich groups for discussion. Helpful advice and

373

support by H. W. Stotz and Dr. D. Truckenbrodt (Tecan Deutschland GmbH) are greatly

374

appreciated, as is stellar support by G. Schmitt of the mechanical workshop at Humboldt-

375

Universität. Dr. M. Stierl provided valuable advice on plate reader luminescence measurements;

376

the CHO reporter cell line was generously provided by Dr. F. Wunder (Bayer Pharma); and

377

plasmids used for expression of PhyB were received from Dr. W. Weber (Universität Freiburg). 16

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378

Funding through a Sofja-Kovalevskaya Award by the Alexander-von-Humboldt Foundation (A.M.),

379

by the Einstein Foundation grant IPF-2012-148 (F.R. & A.M.), by Deutsche Forschungsgemeinschaft

380

grants RI2468/1-1 (F.R.) and HE3824/24-1 (P.H. & T.M.), and by the Leibniz Graduate School of

381

Molecular Biophysics (R.S. & A.M.) is gratefully acknowledged.

382 383 384

Bibliographic references and notes

385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417

1. N. C. Rockwell, D. Duanmu, S. S. Martin, C. Bachy, D. C. Price, D. Bhattacharya, A. Z. Worden, and J. C. Lagarias, Proc Natl Acad Sci U S A, 2014, 111, 3871–3876. 2. A. Möglich and K. Moffat, Photochem Photobiol Sci, 2010, 9, 1286–1300. 3. P. Hegemann, Annu Rev Plant Biol, 2008, 59, 167–189. 4. A. Möglich, X. Yang, R. A. Ayers, and K. Moffat, Annu Rev Plant Biol, 2010, 61, 21–47. 5. M. Iseki, S. Matsunaga, A. Murakami, K. Ohno, K. Shiga, K. Yoshida, M. Sugai, T. Takahashi, T. Hori, and M. Watanabe, Nature, 2002, 415, 1047–1051. 6. M. Gomelsky and G. Klug, Trends Biochem Sci, 2002, 27, 497–500. 7. M. Gauden, I. H. van Stokkum, J. M. Key, D. C. Lührs, R. van Grondelle, P. Hegemann, and J. T. Kennis, Proc Natl Acad Sci U S A, 2006, 103, 10895–900. 8. N. C. Rockwell, Y. S. Su, and J. C. Lagarias, Annu Rev Plant Biol, 2006, 57, 837–858. 9. J. Hughes, Annu Rev Plant Biol, 2013, 64, 377–402. 10. K. Deisseroth, G. Feng, A. K. Majewska, G. Miesenböck, A. Ting, and M. J. Schnitzer, J Neurosci, 2006, 26, 10380–10386. 11. M.-H. Ryu, O. V. Moskvin, J. Siltberg-Liberles, and M. Gomelsky, J Biol Chem, 2010, 285, 41501–41508. 12. M. Stierl, P. Stumpf, D. Udwari, R. Gueta, R. Hagedorn, A. Losi, W. Gärtner, L. Petereit, M. Efetova, M. Schwarzel, T. G. Oertner, G. Nagel, and P. Hegemann, J Biol Chem, 2011, 286, 1181–1188. 13. R. J. De Marco, A. H. Groneberg, C.-M. Yeh, L. A. Castillo Ramírez, and S. Ryu, Front Neural Circuits, 2013, 7, 82. 14. M. Efetova, L. Petereit, K. Rosiewicz, G. Overend, F. Haußig, B. T. Hovemann, P. Cabrero, J. A. T. Dow, and M. Schwärzel, J. Cell. Sci., 2013, 126, 778–788. 15. A. Hartmann, R. D. Arroyo-Olarte, K. Imkeller, P. Hegemann, R. Lucius, and N. Gupta, J. Biol. Chem., 2013, 288, 13705–13717. 16. M. Ni, J. M. Tepperman, and P. H. Quail, Cell, 1998, 95, 657–667. 17. S. Shimizu-Sato, E. Huq, J. M. Tepperman, and P. H. Quail, Nat Biotechnol, 2002, 20, 1041– 1044. 18. A. Levskaya, O. D. Weiner, W. A. Lim, and C. A. Voigt, Nature, 2009, 461, 997–1001. 19. W. Laan, M. Gauden, S. Yeremenko, R. van Grondelle, J. T. M. Kennis, and K. J. Hellingwerf, Biochemistry, 2006, 45, 51–60. 20. M. T. Alexandre, J. C. Arents, R. van Grondelle, K. J. Hellingwerf, and J. T. Kennis, Biochemistry, 2007, 46, 3129–3137. 17

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21. S. Anderson, V. Dragnea, S. Masuda, J. Ybe, K. Moffat, and C. Bauer, Biochemistry, 2005, 44, 7998–8005. 22. A. Jung, T. Domratcheva, M. Tarutina, Q. Wu, W. H. Ko, R. L. Shoeman, M. Gomelsky, K. H. Gardner, and I. Schlichting, Proc Natl Acad Sci U S A, 2005, 102, 12350–5. 23. H. Yuan, S. Anderson, S. Masuda, V. Dragnea, K. Moffat, and C. Bauer, Biochemistry, 2006, 45, 12687–94. 24. T. Majerus, T. Kottke, W. Laan, K. Hellingwerf, and J. Heberle, Chemphyschem, 2007, 8, 1787– 1789. 25. C. Bonetti, M. Stierl, T. Mathes, I. H. M. van Stokkum, K. M. Mullen, T. A. Cohen-Stuart, R. van Grondelle, P. Hegemann, and J. T. M. Kennis, Biochemistry, 2009, 48, 11458–11469. 26. S. Masuda, K. Hasegawa, and T. Ono, Plant Cell Physiol., 2005, 46, 1894–1901. 27. F. Wunder, J.-P. Stasch, J. Hütter, C. Alonso-Alija, J. Hüser, and E. Lohrmann, Anal. Biochem., 2005, 339, 104–112. 28. F. Wunder, A. Rebmann, A. Geerts, and B. Kalthof, Mol. Pharmacol., 2008, 73, 1235–1243. 29. L. O. Essen, J. Mailliet, and J. Hughes, Proc Natl Acad Sci U S A, 2008, 105, 14709–14714. 30. T. R. Berkelman and J. C. Lagarias, Anal. Biochem., 1986, 156, 194–201. 31. K. Mukougawa, H. Kanamoto, T. Kobayashi, A. Yokota, and T. Kohchi, FEBS Lett, 2006, 580, 1333–1338. 32. T. Mathes, C. Vogl, J. Stolz, and P. Hegemann, J Mol Biol, 2009, 385, 1511–1518. 33. J. Mehlhorn, H. Steinocher, S. Beck, J. T. M. Kennis, P. Hegemann, and T. Mathes, PLoS ONE, 2013, 8, e79006. 34. G. Nagel, D. Ollig, M. Fuhrmann, S. Kateriya, A. M. Musti, E. Bamberg, and P. Hegemann, Science, 2002, 296, 2395–2398. 35. D. R. Hochbaum, Y. Zhao, S. L. Farhi, N. Klapoetke, C. A. Werley, V. Kapoor, P. Zou, J. M. Kralj, D. Maclaurin, N. Smedemark-Margulies, J. L. Saulnier, G. L. Boulting, C. Straub, Y. K. Cho, M. Melkonian, G. K.-S. Wong, D. J. Harrison, V. N. Murthy, B. L. Sabatini, E. S. Boyden, R. E. Campbell, and A. E. Cohen, Nat. Methods, 2014, 11, 825–833. 36. G. M. Avelar, R. I. Schumacher, P. A. Zaini, G. Leonard, T. A. Richards, and S. L. Gomes, Curr Biol, 2014, 24, 1234–1240. 37. M.-H. Ryu, I.-H. Kang, M. D. Nelson, T. M. Jensen, A. I. Lyuksyutova, J. Siltberg-Liberles, D. M. Raizen, and M. Gomelsky, Proc. Natl. Acad. Sci. U.S.A., 2014, 111, 10167–10172. 38. C. F. Gasser, S. Taiber, C.-M. Yeh, C. H. Wittig, P. Hegemann, S. Ryu, F. Wunder, and A. Möglich, Proc Natl Acad Sci U S A, 2014, 111, 8803–8808.

452 453

Figure legends

454

Fig. 1 - Microplate reader illumination setup

455

(A) The Tecan Infinite M200 pro multimode microplate reader can inject liquids into defined

456

wells using an injection cylinder that is inserted through an injection hole in the top of the

457

instrument.

18

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458

(B) Schematic of the automated illumination setup: the CAN-bus communication between the

459

microplate reader and the injector module is captured via an Arduino microcontroller and a CAN-

460

bus extension shield. The microcontroller is programmed to translate injection commands into TTL

461

signals that control a light source containing up to 8 LEDs of different wavelengths. The optical

462

output from the LEDs is coupled into a fiber-optics waveguide fitted into the injection cylinder. In

463

this setup, light application to individual wells can conveniently be programmed via the iControl

464

software of the microplate reader.

465

(C) Illumination through the injection cylinder in the newly designed setup.

466

(D) Using a 1.5-mm waveguide, maximal light intensities of up to 10 mW cm-2 are obtained,

467

depending upon wavelength.

468 469 470 471

Fig. 2 - Photoactivation of PhyB with different light colors. (A) Absorption spectra of PhyB in its dark-adapted state (solid lines) and after illumination with 385, 530, 617 and 680 nm (dashed lines).

472

(B) Equivalent PhyB samples were put in the adjacent wells D7-D9, E7-E9 and F7-F9 of a

473

microtiter plate. Absorption spectra were recorded prior to (solid lines) and after (dashed lines)

474

illumination of the center well E8 with 590-nm light.

475 476

Fig. 3 - Temperature and imidazole effects on dark recovery in Slr1694 variants

477

(A) Structural model of Slr1694 in the W91in configuration (orange) according to Yuan et al.23

478

(B) Dark-state recovery kinetics monitored by absorption measurements at 490 nm as a

479

function of imidazole concentration (from left to right: 0, 5, 25, 50, 100, 170 and 250 mM). The

480

inset shows a close-up view of a portion of the data.

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484 485 486

(C-E) Temperature and imidazole dependence of rate constants for dark-state recovery in Slr1694 variants: wild-type (C), H73S (D), and W91F (E). (F) Factor acceleration of dark-state recovery as a function of imidazole concentration in Slr1694 wild-type (black), H73S (green) and W91F (orange). (G) Activation energies for dark-state recovery as a function of imidazole concentration in Slr1694 wild-type (black), H73S (green) and W91F (orange).

487 488

Fig. 4 - Dose response of bPAC and cAMP decay kinetics in eukaryotic cells

489

(A) bPAC was transfected into CHO reporter cells bearing a cyclic-nucleotide-gated ion channel

490

(CNG) and the aequorin luciferase. Upon application of 470-nm light via the newly developed

491

illumination device, bPAC is stimulated to produce cAMP which triggers opening of the CNG

492

channels. Subsequent injection of CaCl2 allows influx of Ca2+ ions through the CNG channel which

493

promotes an increase in aequorin luminescence.

494

(B) bPAC-transfected cells were exposed for 8 s to 470-nm light of varying intensities (from

495

bottom to top: 0, 0.17, 0.30, 0.77, 1.51, 1.96, 2.50 mW cm-2), CaCl2 was injected, and resultant

496

luminescence was recorded over time. Background-corrected luminescence data are reported as

497

relative luminescence units (RLU) and represent mean ± s.e.m. of n = 4 measurements.

498

(C) Integrated luminescence data from (B) as a function of applied light dose. bPAC activity

499

increases with light dose in a hyperbolic manner; the half-maximal light dose, E50, amounts to

500

(0.58 ± 0.12) mW cm-2.

501

(D) Experiments were conducted as described in (B) using a light intensity of 2.50 mW cm-2. To

502

assess for how long intracellular cAMP spikes persist after bPAC activation, the delay between

503

light exposure and CaCl2 injection was varied between 3 seconds and 40 minutes (from bottom to

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top: 0.05, 0.5, 1, 5.5, 8, 10, 22, 40 min). Background-corrected luminescence data are reported as

505

relative luminescence units (RLU) and represent mean ± s.e.m. of n = 4 measurements.

506

(E) Integrated luminescence data from (D) as a function of delay time between light application

507

and CaCl2 injection. The initial cAMP spike after bPAC activation decays with a half life of roughly

508

five

minutes.

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Upgrading a microplate reader for photobiology and all-optical experiments.

Automation can vastly reduce the cost of experimental labor and thus facilitate high experimental throughput, but little off-the-shelf hardware for th...
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