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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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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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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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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
13
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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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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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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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DOI: 10.1039/C4PP00361F
418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451
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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(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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A
Photochemical & Photobiological Sciences
B injection.tube
CAN-BUS View Article Online LED.1
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DOI: 10.1039/C4PP00361F LED.3
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TTL
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530 590 617 wavelength9[nm]
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3859nm
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B
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