Fluorescent hydrogels for studying Ca2+-dependent reaction-diffusion processes

Article abstract of DOI:10.1039/c3cc49639b

Here, we report a convenient experimental platform to study the diffusion of Ca²⁺ in the presence of a Ca²⁺-binding protein (Calbindin D28k). This work opens up new possibilities to elucidate the physical chemistry of complex Ca²⁺-dependent reaction-diffusion networks that are abundant in living cells. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3cc49639b

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Fluorescent hydrogels for studying
Ca2+-dependent reaction–diffusion processes†
Cite this: Chem. Commun., 2014,
50, 3089
Sergey N. Semenov, Sjoerd G. J. Postma, Ilia N. Vialshin and Wilhelm T. S. Huck*
Received 19th December 2013,
Accepted 31st January 2014
DOI: 10.1039/c3cc49639b
www.rsc.org/chemcomm
Here, we report a convenient experimental platform to study the
We chose 10 wt% polyacrylamide (PAAm) and 9 wt% poly-
diffusion of Ca2+ in the presence of a Ca2+-binding protein (Calbin- ethylene glycol (PEG) cross-linked hydrogels as polymer matrices
din D28k). This work opens up new possibilities to elucidate the for our materials, since they are readily prepared and require
physical chemistry of complex Ca2+-dependent reaction–diffusion little modification of a Ca2+-sensing dye. The Ca2+ sensor Indo-1
networks that are abundant in living cells.
was chosen for the fluorescence read-out. Indo-1 is an ideal
sensing element, as a derivative of this Ca2+ indicator has already
Ca2+ is a pivotal second messenger involved in a wide variety of been successfully coupled to polymeric beads,14 and it displays
complex cellular networks.1,2 Reaction–diffusion (RD) processes an emission shift upon Ca2+ binding,15 allowing facile visualiza-
where interactions between molecules take place at similar time- tion of the diffusion front.
scales as their diffusion3,4 play an essential role in Ca2+ signalling.1,5
In order to covalently attach Indo-1 to a hydrogel matrix, we
In the highly crowded cytosol, Ca2+ diffusion profiles are influenced attached linker moieties to the carboxyl group of the indole ring
by both fixed and mobile Ca2+-binding proteins and non-specific (Scheme 1),14,16 as this site is not involved in the chelating
interactions with other proteins and charged surfaces.6 Although
the advent of organic Ca2+ sensors has enabled studies on Ca2+
dynamics in vivo,7 the determination of individual contributions of
Ca2+-binding proteins and other factors to RD networks requires an
artificial platform that avoids cellular complexity. Previous efforts
using 45Ca2+ could only measure the total amount of Ca2+ in a
sample, and thin slices of a frozen gel needed to be analysed, thus
precluding continuous measurements of Ca2+ diffusion.5,8 In a
different approach, Ca2+ sensors which diffused through a gel were
applied.9–11 However, this method results in a system in which not
only Ca2+, but also the indicator and the Ca2+-indicator complex
diffuse, thereby complicating the equations used to describe
the RD processes. Here, we present hydrogels with a covalently
linked fluorescent Ca2+-indicator. Our approach prevents diffusion
of the indicator whilst taking advantage of the facile probing of
Ca2+ diffusion in time by fluorescence microscopy. By applying
a wet stamping technique,12,13 we show that the influence of
a Ca2+-binding protein on Ca2+ diffusion can be monitored
and modelled, taking the first steps towards building complex
Ca2+-dependent RD networks in vitro.
Scheme 1 Synthesis of Indo-1-based Ca2+-sensing gels. (a) (3-Aminopropyl)-
methacrylamide hydrochloride, DIPEA, DMF (solv.), 2.5 h, r.t., 13%, (b) allylamine,
DMF (solv.), 2.5 h, r.t., 50%, (c) KOH, THF:MeOH (4:1, v/v), o.n., r.t.,
65%, (d) IndoL1, acrylamide, bisacrylamide, CaCl2, 2,20-azobis(2-methyl-
propionamidine) dihydrochloride (AAPH), UV (lex = 365 nm) and (e) IndoL2,
bis-acryl-PEG-10 000, thio-4ArmPEG-2000, CaCl2, lithium arylphospho-
nate (LAP), UV (lex = 365 nm).
Institute for Molecules and Materials, Radboud University Nijmegen,
Toernooiveld 1, 6525 ED, Nijmegen, The Netherlands.
E-mail: w.huck@science.ru.nl; Fax: +31 (0)24 3652929; Tel: +31 (0)24 3652138
† Electronic supplementary information (ESI) available: Including details of the
synthesis of Indo-1 and BAPTA derivatives, gel preparation and properties, RD
experiments, and modelling. See DOI: 10.1039/c3cc49639b
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properties of the 1,2-bis(2-aminophenoxy)ethane-N,N,N0,N0-
The response of the PAAmIndoL1 gel towards Ca2+ was visible
tetraacetic acid (BAPTA) fragment. We obtained the 5-[6-carboxy- by the naked eye (Fig. 1b) and reversible (see ESI†). To perform
indol-2-yl]-50-methyl-BAPTA ethyl ester following the protocols quantitative characterization of the gel response, pieces of
given by Bradley et al.14 Subsequent experiments showed that PAAmIndoL1 and PEGIndoL2 gels were equilibrated with buffers
the indole carboxyl group can be activated via the formation of an containing 0 to 39 mM of free Ca2+. The gels were then imaged by
N-hydroxysuccinimide (NHS) ester (1). Gratifyingly, compound 1 fluorescence microscopy (lex = 365 nm, lem = 405 or 520 nm).
was sufficiently stable to be purified by column chromatography. Fig. 1c depicts the calibration curves obtained in this way.
Activation of carboxylic acids by NHS ester formation is widely A detection range from 0.1 to 3 mM Ca2+ for PAAmIndoL1 could
used for attachment of target molecules to surfaces and proteins be assumed from the plot. The Kd of 660 ꢀ 50 nM calculated for
via free amines. Compound 1 is therefore a useful addition to the PAAmIndoL1 from this curve was in good agreement with our
toolbox of NHS-functionalized fluorophores.
Reactions of 1 with two different amines yielded Indo-1 not lead to changes in the Ca2+-sensing properties of IndoL1.
derivatives with either a methylacrylamide (IndoL1) or an alkene The calibration curves showed that the PEGIndoL2 gel has a
data for IndoL1 in solution, showing that copolymerization did
(IndoL2) functionality. Both derivatives show a blue shift of the similar detection range for Ca2+ as PAAmIndoL1, but the latter is
emission spectrum upon Ca2+ binding. Dissociation constants more accurate. Moreover, whereas the PAAmIndoL1 gel swelled by
(Kd) for IndoL1 and IndoL2 were determined to be 514 and 340 nM, 18% its initial volume during washings, for the PEGIndoL2 gel this
respectively, and the selectivity of IndoL1 towards Ca2+ was demon- value was 400%. Therefore, the PEGIndoL2 hydrogel is suitable for
strated (see ESI†). Next, both sensor molecules were incorporated applications requiring soft 3D gels, but is less convenient for
into hydrogels via co-polymerization. We prepared the hydrogels by accurate determination of Ca2+ concentrations. For this reason,
photoinitiation in the presence of indicator-saturating concentra- we chose to use the PAAmIndoL1 gel in further experiments.
tions of Ca2+ to reduce decomposition of the fluorophore.17 After
We demonstrated the application of the PAAmIndoL1 gel
initial washings, we placed the gel in a solution with a BAPTA- for quantitative studies on RD of Ca2+ in a wet stamping
functionalized PAAm gel (see ESI†). In this way, we removed residual experiment depicted in Fig. 2a.12,13 The great advantage of this
amounts of Ca2+ while avoiding the use of diffusing Ca2+ chelators method is that the initial conditions (i.e. starting time, points
that would complicate RD experiments.
of contact, and concentrations) are well-defined. A 6 wt%
To prove that the Ca2+ sensor was covalently attached to the agarose stamp with an array of parallel lines (500 mm wide,
PAAm gel, a fluorescence recovery after photobleaching (FRAP) 200 mm high and spaced by 1500 mm) was soaked in a CaCl2
experiment was performed (Fig. 1a). We compared two samples: solution for at least 15 hours, and placed feature-side-down
(i) the PAAm hydrogel doped with 10 mM free IndoL1 solution on top of a piece of PAAmIndoL1 gel (0.4 mm high). Next, a
after polymerization, and (ii) the PAAm hydrogel with 10 mM series of fluorescence images was acquired. At every 30 seconds
co-polymerized IndoL1. The fluorescence in the bleached region time point, ratios of intensities at 405 and 520 nm (I405/I520
)
was restored within 10 minutes in the first sample, while the were determined by dividing two images that were taken at
second sample did not show any recovery even after two days.
these different emission wavelengths within 1 second of each
other (Fig. 2b).
Fig. 1 Characterization of hydrogels. (a) False-colour images of the FRAP
experiment for the PAAmIndoL1 gel. (i) A gel with diffusing IndoL1,
10 minutes after bleaching. (ii) A gel with co-polymerized IndoL1, two
days after bleaching. Scale bars are 500 mm. (b) A real-colour photo of the
Fig. 2 (a) Schematic representation of the experimental setup. A Ca2+
-
PAAmIndoL1 gel with and without Ca2+
.
(c) Calibration curves for
soaked agarose stamp with pillars is placed on top of a PAAmIndoL1 gel. (b)
A typical I405/I520 image obtained by dividing two images of a Ca2+
diffusion experiment taken using different emission wavelength filters.
The gel was imaged from below.
PAAmIndoL1 and PEGIndoL2 gels. R is the ratio of emission intensities at
405 nm vs. 520 nm. Rmin and Rmax are the ratios for fully unsaturated and
saturated indicators, respectively.
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Fig. 3 Influence of the initial Ca2+ concentration on Ca2+ diffusion.
(a) The change of diffusion profiles in time for experiments with different
Ca2+ concentrations in the stamp. (b) Calculated [Ca2+] profiles for the
95 second time-point assuming a flat diffusion front. Considering the
sensitivity of our gel, 3 mM Ca2+ was the upper detection limit.
In the first series of experiments, we varied the initial
concentration of free Ca2+ in the stamp. As one should expect, we
observed an increase in the rate of propagation of the diffusion
front with higher initial concentrations of Ca2+ (Fig. 3a). I405/I520
values were converted to a profile of Ca2+ concentrations by using a
calibration curve (Fig. 3b). This transformation is valid if we assume
a flat diffusion front (pseudo-1D diffusion).
Fig. 4 Experimental data and modelling of RD of Ca2+ (25 mM in the
stamp) in the presence of Calbindin D28K (CalB, 30 mM). (a) Time–space
plots of Ca2+ RD with a pillar of the stamp at the center of the image in
(i) the absence and (ii) the presence of CalB. (b) The computer model of the
RD experiment 19 minutes after its start. The concentration of free Ca2+ is
depicted. The solid black border denotes that no flux is possible at those
points, and the gray border denotes that the Ca2+ concentration is
constant. (c) Comparison of Ca2+ diffusion profiles of the RD experiment
(green lines) to the simulations (black lines) at different time points.
Next, we demonstrated the potential of our platform for
in vitro studies on Ca2+-dependent RD processes by carrying out
an experiment involving Calbindin-D28k (CalB). CalB is a protein
abundant in neurons, and is able to bind up to four Ca2+ ions.18
We soaked the PAAmIndoL1 gel in a buffer containing CalB
(30 mM final concentration), and stamped it with an agarose
gel equilibrated in a buffer with 25 mM CaCl2. Time–space
plots allowed visualization of the diffusion process (Fig. 4a).
Compared to analogous experiments in the absence of the
Ca2+-binding protein, we clearly observed slower propagation
of the Ca2+ diffusion front when CalB was present. Because of
the slow diffusion, the front was not flat and a model of 2D
diffusion was required.12,13 The inherent control of the wet
stamping technique allowed us to simulate the RD experiment
using COMSOL (Fig. 4b and ESI†). A symmetry-independent
element of the experimental setup was constructed to model
reaction–diffusion through a half-pillar of the agarose stamp on
top of the PAAmIndoL1 gel (Fig. 4b). As the stamp used in the
RD experiment was much thicker than the pillar and the
PAAmIndoL1 gel, we maintained the Ca2+ concentration at
the top of the stamp at 25 mM (grey line in Fig. 4b) assuming
a continuous source of Ca2+. The reactions included in the
simulation were the four reversible binding events of Ca2+ to
CalB (30 mM) and one for Ca2+ binding to IndoL1. Ca2+, CalB and
the four Ca2+–CalB complexes were the only species allowed to
diffuse. As can be seen in Fig. 4c, there is an excellent fit between
the model and experimental data, especially at the early time
points. In addition to the concentration of free Ca2+ depicted in
Fig. 4b, all other components (Ca2+-bound or -free IndoL1 and
CalB) could be profiled in the model as well (see ESI†).
of the Ca2+-diffusion front in the presence of CalB is an indica-
tion that we can study ultrasensitivity and molecular titration
effects.13 In future experiments, mobile and stationary Ca2+
buffers, the influence of dimensionality, and photo-initiated
release of caged Ca2+ to mimic intracellular Ca2+ signals can
be studied using our platform. Moreover, we can exploit the
reversible binding of Ca2+ to the sensor hydrogel to investigate
the pattern formation in complex Ca2+-dependent RD networks
in order to increase our understanding of RD processes and
cellular complexity.
We thank Dr Wim Scheenen for fruitful discussions. We
acknowledge financial support from the European Research
Council (ERC; Advanced Grant 246812 Intercom), the Netherlands
Organisation for Scientific Research (NWO, VICI Grant 700.10.44
(W.T.S.H.)), the Marie Curie Intra-European Fellowship (Grant
300519 to S.N.S.) and funding from the Ministry of Education,
Culture and Science (Gravity program 024.001.035).
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