Difference between revisions of "Team:Slovenia/Mechanosensing/CaDependent mediator"

m
 
(69 intermediate revisions by 6 users not shown)
Line 2: Line 2:
 
<html>
 
<html>
 
<head>
 
<head>
−
     <title>Home</title>
+
     <title>Ca-dependent mediator</title>
 
     <link rel="stylesheet"
 
     <link rel="stylesheet"
 
           href="//2016.igem.org/Team:Slovenia/libraries/semantic-min-css?action=raw&ctype=text/css">
 
           href="//2016.igem.org/Team:Slovenia/libraries/semantic-min-css?action=raw&ctype=text/css">
Line 13: Line 13:
 
     <script type="text/javascript"
 
     <script type="text/javascript"
 
             src="https://2016.igem.org/Team:Slovenia/libraries/bibtexparse-js?action=raw&ctype=text/javascript"></script>
 
             src="https://2016.igem.org/Team:Slovenia/libraries/bibtexparse-js?action=raw&ctype=text/javascript"></script>
−
<!-- MathJax (LaTeX for the web) -->
+
    <!-- MathJax (LaTeX for the web) -->
 
     <script type="text/x-mathjax-config">
 
     <script type="text/x-mathjax-config">
 
         MathJax.Hub.Config({
 
         MathJax.Hub.Config({
Line 35: Line 35:
 
         });
 
         });
 
     </script>
 
     </script>
−
<script type="text/javascript" async
+
    <script type="text/javascript" async
 
             src="//2016.igem.org/common/MathJax-2.5-latest/MathJax.js?config=TeX-AMS-MML_HTMLorMML">
 
             src="//2016.igem.org/common/MathJax-2.5-latest/MathJax.js?config=TeX-AMS-MML_HTMLorMML">
 
     </script>
 
     </script>
 
</head>
 
</head>
 
<body>
 
<body>
−
+
 
−
+
 
−
<div id="example">
+
<div id="example">
−
<div class="pusher">
+
    <div class="pusher">
−
<div class="full height">
+
        <div class="full height">
−
<div class="banana">
+
            <div class="banana">
−
<a href = "//2016.igem.org/Team:Slovenia">
+
                <a href = "//2016.igem.org/Team:Slovenia">
−
<img class="ui medium sticky image" src="//2016.igem.org/wiki/images/d/d1/T--Slovenia--logo.png">
+
                    <img class="ui medium sticky image" src="//2016.igem.org/wiki/images/d/d1/T--Slovenia--logo.png">
−
</a>
+
                </a>
−
<div class="ui vertical sticky text menu">
+
                <div class="ui vertical sticky text menu">
−
<a class="item" href="#intro" style="margin-left: 10%">
+
                    <a class="item" href="//2016.igem.org/Team:Slovenia/Mechanosensing/Gas_vesicles">
−
<i class="selected radio icon"></i>
+
                        <i class="chevron circle left icon"></i>
−
<b>Project</b>
+
                        <b>Gas vesicles</b>
−
</a>
+
                    </a>
−
<a class="item" href="#achievements" style="margin-left: 10%">
+
                    <a class="item" href="//2016.igem.org/Team:Slovenia/Mechanosensing/CaDependent_mediator" style="color:#DB2828;">
−
<i class="selected radio icon"></i>
+
                        <i class="selected radio icon"></i>
−
<b>Achievements</b>
+
                        <b>Ca-dependent mediator</b>
−
</a>
+
                    </a>
−
<a class="item" href="#requirements" style="margin-left: 10%">
+
                    <a class="item" href="#intro" style="margin-left: 10%">
−
<i class="selected radio icon"></i>
+
                        <i class="selected radio icon"></i>
−
<b>Medal requirements</b>
+
                        <b>Achievements</b>
−
</a>
+
                    </a>
−
<a class="item" href="idea">
+
                    <a class="item" href="#mot" style="margin-left: 10%">
−
<i class="chevron circle right icon"></i>
+
                        <i class="selected radio icon"></i>
−
<b>Idea</b>
+
                        <b>Introduction</b>
−
</a>
+
                    </a>
−
+
                    <a class="item" href="#cam" style="margin-left: 10%">
−
</div>
+
                        <i class="selected radio icon"></i>
−
+
                        <b>CaMeleon</b>
−
</div>
+
                    </a>
−
<div class="article" id="context">
+
                    <a class="item" href="#split" style="margin-left: 10%">
−
<!-- menu goes here -->
+
                        <i class="selected radio icon"></i>
−
<!-- content goes here -->
+
                        <b>Split calcium sensors</b>
−
<div>
+
                    </a>
−
<div class="main ui citing justified container">
+
                    <a class="item" href="//2016.igem.org/Team:Slovenia/Protease_signaling/Overview">
−
<h1 class = "ui left dividing header"><span class="section">&nbsp;</span>Calcium-dependent mediator</h1>
+
                        <i class="chevron circle right icon"></i>
−
<div class = "ui segment" style = "background-color: #ebc7c7; ">
+
                        <b>Protease signaling</b>
−
<p><b><ul><li>A new split calcium sensing/reporting system based on split firefly luciferase linked to M13 and calmodulin was designed that
+
                    </a>
−
is able to report the increase of the cytosolic calcium ions induced by mechanoreceptor stimulation by emitted light.</ul></b></p>
+
 
−
</div>
+
                </div>
−
<div class = "ui segment">
+
            </div>
−
<p>While calcium influx could be detected by exogenous fluorescent dyes such as the FuraRed and Fluo-4, we needed a genetically encoded calcium sensor that
+
            <div class="article" id="context">
−
would couple a change in the calcium concentration to a biologically relevant output, such as the luciferase reporter or reconstitution of a split protease the  
+
                <!-- menu goes here -->
−
initiation of the signaling pathway. The ideal calcium sensor should be inactive at intracellular concentration of calcium and have a high response to calcium
+
                <!-- content goes here -->
−
concentrations above physiological levels and should be detected by a quick and easy readout. For our intended application, the calcium sensor should also have  
+
                <div>
−
the potential to act as the reconstitution mechanism for split proteins so that a conformational rearrangement in the presence of calcium would bring the two  
+
                    <div class="main ui citing justified container">
−
split protein fragments together and reconstitute the protein’s activity.
+
                        <div>
−
</p>
+
                            <h1 class = "ui left dividing header"><span id = "intro" class="section colorize">&nbsp;</span>Ca-dependent reporter and mediator</h1>
−
</div>
+
                            <div class = "ui segment" style = "background-color: #ebc7c7; ">
−
<h1><span id = "results" class="section"> &nbsp; </span>Results</h1>
+
                                <p><b><ul><li>A new split calcium sensing/reporter system based on split firefly luciferase fused to M13 and calmodulin was designed and tested.
−
+
<li>The designed sensor was able to report the increase of the cytosolic calcium ions induced by mechanoreceptor stimulation via emitted light.</ul></b></p>
−
<div class="ui segment">
+
                            </div>
−
+
                        </div>
−
<p>A wide pallet of genetically encoded calcium sensors are used for mapping intracellular calcium concentration <x-ref>Whitaker2010</x-ref>,  
+
                        <div class = "ui segment">
−
including calmodulin, troponin C and aequorin <x-ref>Wilms2014</x-ref>. These reporters are based on different mechanisms of detection. From this abundant  
+
                            <h3><span id = "mot" class = "section colorize"> &nbsp; </span></h3>
−
collection we chose the calmodulin (CaM)-based calcium sensors (in particular CaMeleons), since their mechanism is based on a large conformational change, allowing
+
                            <p>While calcium influx can be detected by exogenous fluorescent dyes such as the FuraRed and Fluo-4, we needed a genetically encoded calcium sensor for the purposes of our project. Our sensor should couple a change in calcium concentration to a biologically relevant output, such as the luciferase reporter or reconstitution of a split protease for the
−
reconstitution of split proteins <x-ref>Whitaker2010</x-ref>.
+
                                initiation of the signaling pathway. The ideal calcium sensor should be inactive at resting intracellular concentrations of calcium, have a high response to calcium
−
</p>
+
                                concentrations above physiological levels and be detected by a quick and easy readout. For our intended application, the calcium sensor should also have
−
<div class="ui styled fluid accordion">
+
                                the potential to act as a reconstitution mechanism for split proteins so that a conformational rearrangement in the presence of calcium would bring the two
−
<div class="title">
+
                                split protein fragments together and reconstitute the protein’s activity.
−
<i class="dropdown icon"></i>
+
                            </p>
−
Further explanation ...
+
                        </div>
−
</div>
+
                        <h1><span class="section colorize"> &nbsp; </span>Results</h1>
−
<div class="content">
+
 
−
<p>CaMeleons are based on a genetic fusion of a recombinant calcium binding protein with a pair of fluorescent proteins, forming a FRET (Förster resonance energy  
+
                        <div class="ui segment">
−
transfer) based sensor. Yellow CaMeleon 2.12 is a CaMeleon composed of calmodulin and a CaM-binding domain of the skeletal muscle myosin light chain kinase  
+
                            <div>
−
(M13), forming the backbone of the sensor, and a FRET pair linked to the termini of the construct. The binding of calcium causes calmodulin to wrap around the  
+
                                <h3><span id = "cam" class="section colorize">&nbsp;</span>CaMeleon based reporters</h3>
−
M13 domain, bringing the two fluorescent proteins closer to each other, thus producing FRET <x-ref>Whitaker2010</x-ref>.
+
                                <p>A wide pallet of genetically encoded calcium sensors, based on different mechanisms of detection, are used for mapping intracellular calcium concentration<x-ref>Whitaker2010</x-ref>,
−
</p>
+
                                    including calmodulin, troponin C and aequorin<x-ref>Wilms2014</x-ref>. From this abundant
−
</div>
+
                                    collection we chose calmodulin (CaM)-based calcium sensors (in particular CaMeleons), since their mechanism is based on a large conformational change, allowing
−
</div><br/>
+
                                    reconstitution of split proteins <x-ref>Whitaker2010</x-ref>.
−
<div style = "float:left">  
+
                                </p>
−
<figure data-ref="1">
+
                                <div class="ui styled fluid accordion">
−
<img class="ui medium image" src="https://static.igem.org/mediawiki/2016/c/cf/T--Slovenia--3.6.1.png" >
+
                                    <div class="title">
−
<figcaption><b>A luminescence based calcium sensor fLuc2.12 has high activity already at the resting levels of calcium.</b><br/>
+
                                        <i class="dropdown icon"></i>
−
HEK293T cells were transfected with 50 ng fLuc2.12. 24 h after transfection luciferase activities were measured immediately after addition of calcium  
+
                                        Further explanation ...
−
ionophore A23187 (10 µM). Scheme: The chimeric protein M13-calmodulin fused to N- and C- fragments of split luciferase changes conformation upon calcium  
+
                                    </div>
−
binding.</figcaption>
+
                                    <div class="content">
−
</figure>
+
                                        <p>CaMeleons are based on a genetic fusion of a recombinant calcium binding protein with a pair of fluorescent proteins, forming a FRET (Förster resonance energy
 +
                                            transfer) based sensor. Yellow CaMeleon 2.12 is a CaMeleon composed of calmodulin and a CaM-binding domain of the skeletal muscle myosin light chain kinase
 +
                                            (M13), forming the backbone of the sensor, and the FRET pair EYFP-ECFP linked to the termini of the construct. The binding of calcium causes calmodulin to wrap around the
 +
                                            M13 domain, bringing the two fluorescent proteins closer to each other, resulting in the FRET effect<x-ref>Whitaker2010</x-ref>.
 +
                                        </p>
 +
                                    </div>
 +
                                </div><br/>
 +
                                <div style = "float:left; width:50%">
 +
                                    <figure data-ref="1">
 +
                                        <img src="https://static.igem.org/mediawiki/2016/c/cf/T--Slovenia--3.6.1.png" >
 +
                                        <figcaption><b>A luminescence based calcium sensor fLuc2.12 is highly active already at the resting levels of calcium.</b><br/>
 +
                                            <p style="text-align:justify">HEK293T cells were transfected with 50 ng of the plasmid encoding fLuc2.12. 24 hours after transfection luciferase activity was measured immediately after addition of 10 µM  calcium ionophore A23187. Scheme: The fLuc2.12 chimeric protein (M13-calmodulin fused to N- and C- fragments of split luciferase) changes conformation upon calcium
 +
                                                binding.
 +
                                            </p>
 +
                                        </figcaption>
 +
                                    </figure>
 +
                                </div>
 +
                                <p>We replaced the FRET pair of Yellow CaMeleon 2.12 by a split firefly luciferase (fLuc), as split luciferase provides a distinct signal even in small amounts and has a remarkable
 +
                                    signal-to-noise ratio. The new luciferase based calcium sensor was named fLuc2.12. The fLuc2.12 construct was tested on HEK293T cells, but we found that the sensor was active
 +
                                    already in resting cells (<ref>1</ref>). We hypothesized that activation was a consequence of the close proximity of calmodulin and M13 in the fusion even in the absence
 +
                                    of calcium binding. In order to resolve this problem we set out to test a similar sensor based on two separate molecules. Two-molecule-based CaM sensors have not been
 +
                                    widely studied, however lower leakage in comparison to a single molecule FRET sensor has been reported by Miyawaki <i>et al.</i> <x-ref>Miyawaki1997</x-ref>.
 +
                                </p>
 
</div>
 
</div>
−
<p>We replaced the FRET pair of CaMeleon2.12 by a split firefly luciferase (fLuc) as it provides a distinct signal even in small amounts and has a remarkable
+
<div>
−
signal-to-noise ratio. The new luciferase based calcium sensor was named fLuc2.12. The fLuc2.12 was tested on HEK293 cells, but we found that the sensor was active
+
                                <h3 style="clear:both"><span id = "split" class="section colorize">&nbsp;</span>Split calcium sensor</h3>
−
already in resting cells (<ref>1</ref>). We hypothesized the activation was a consequence of a close proximity of calmodulin and M13 in the fusion even in the absence
+
                                <p style="clear:both">An inspection of the crystal structure of the CaM-M13 complex in its closed state (PDB code: 2BBM) suggested that reporter
−
of calcium binding. In order to resolve this problem we set out to test a similar sensor based on two separate molecules. Two-molecule-based CaM sensors have not been
+
                                    proteins could be directly fused to the termini of these two interacting proteins (<ref>2</ref>).</p>
−
widely studied, but lower leakage in comparison to a single molecule sensor has been reported by Miyawaki <i>et al.</i> <x-ref>Miyawaki1997</x-ref>.
+
                                </p>
−
</p>
+
 
 +
                                <div style="margin-left:auto; margin-right:auto; width:70%">
 +
                                    <figure data-ref="2">
 +
                                        <img src="https://static.igem.org/mediawiki/2016/3/38/T--Slovenia--3.4.6.png" >
 +
                                        <figcaption><b>The Calmodulin and M13 peptide complex upon calcium binding.</b><br/>
 +
                                            <p style="text-align:justify">The complex CaM-M13 in its closed state upon calcium binding (<a href="http://www.rcsb.org/pdb/explore.do?structureId=2BBM">PDB 2BBM</a>) (left) and rotated by 90° (right).
 +
                                                The M13 peptide is in color scale from red to yellow (N-term to C-term), while calmodulin is in color scale from blue to cyan (N-term to C-term).
 +
                                                Calcium is shown in green, and the side chains of residues E31Q and E104Q are visible. The C-terminus of calmodulin (red)
 +
                                                and the N-terminus of the M13 peptide (blue) are brought together in the closed state.
 +
                                            </p>
 +
                                        </figcaption>
 +
                                    </figure>
 +
                                </div>
 +
 
 +
 
 +
                                <p>We therefore decided to fuse the N-terminal fragment of the split firefly luciferase to the N-terminus of M13 (nLuc:M13, <a href="http://parts.igem.org/Part:BBa_K1965014">BBa_K1965014</a>) and the C-terminal fragment of the split firefly luciferase to the C-terminus of calmodulin (CaM:cLuc, <a href="http://parts.igem.org/Part:BBa_K1965015">BBa_K1965015</a>). The split calcium sensor is represented in <ref>2.1</ref>A. When transfected into HEK293T cells the sensor was expressed in the cytosol
 +
                                    (<ref>2.1</ref>B).
 +
                                </p>
 +
                                <div style = "float:left; width:100%">
 +
                                    <figure data-ref="2.1">
 +
                                        <img src="https://static.igem.org/mediawiki/2016/9/9c/T--Slovenia--3.6.1-2x.png" >
 +
                                        <figcaption><b>Cytosolic expression of the split calcium sensor.</b><br/>
 +
                                            <p style="text-align:justify">(A) Scheme of the function of the split calcium sensor. Calmodulin is fused to the C-terminal fragment and M13 is fused to N-terminal fragment of split
 +
                                                firefly luciferase. Free calcium ions trigger binding of M13 to calmodulin and the formation of active luciferase. (B) Split calcium sensor is expressed in
 +
                                                the cytosol. HEK293T cells were transfected with plasmids encoding nLuc:M13 and CaM:cLuc in the ratio 5:1. 24 hours after transfection cells were fixed, stained with anti-HA
 +
                                                and anti-Myc antibodies and localization was confirmed with confocal microscopy.
 +
                                            </p>
 +
                                        </figcaption>
 +
                                    </figure>
 +
                                </div>
 +
                            <div>
 +
 
 +
                                <div style="float:right; width:50%">
 +
                                    <figure data-ref="4">
 +
                                        <img src="https://static.igem.org/mediawiki/2016/0/01/T--Slovenia--S.3.6.1.png" >
 +
                                        <figcaption><b> Detection of calcium influx by the split calcium sensor.</b> </figcaption>
 +
                                    </figure>
 +
                                </div>
 +
                                <p>The split calcium sensor was tested on live cells (<ref>4</ref>) but the ratio of the outputs from stimulated and non-stimulated cells remained low,
 +
                                    because the sensor was still highly activated at the resting cell calcium concentration. In order to decrease the activation of the sensor at resting
 +
                                    calcium levels, we introduced mutations E31Q and E104Q in the EF hand motifs, previously reported to decrease the affinity of calmodulin to calcium <x-ref>Evans2009</x-ref>.
 +
                                    We prepared the single mutant CaM<sup>E104Q</sup> and the double mutant CaM<sup>E31Q, E104Q</sup> split calcium sensors and tested them on HEK293T cells (<ref>5</ref>).
 +
                                </p>
 +
 
 +
                                <p>Split calcium reporter with a single mutation (E104Q) exhibited significantly reduced activation at resting calcium levels and high activation when cells were induced with ionophore. However, the sensor with two mutations (E31Q, E104Q) generated a low signal at induction. The sensor with the E104Q mutation resulted in the highest signal-to-noise ratio, therefore representing the best calcium sensor (<ref>5</ref>).
 +
                                    We additionally tested whether the ratio between the two calcium sensor constructs  affects the fold change between the signal of non-stimulated and stimulated cells.
 +
                                    The response of the calcium sensor depended on the ratio, favoring an excess of the nLuc:M13 construct, with the highest fold change close to 10-fold (<ref>6</ref>).
 +
                                </p>
 +
 
 +
                                <div style = "float:left; width:50%">
 +
                                    <figure data-ref="5">
 +
                                        <img src="https://static.igem.org/mediawiki/2016/4/4a/T--Slovenia--3.6.2.png">
 +
                                        <figcaption><b>The split calcium sensor with a single mutation E104Q within calmodulin demonstrated the highest signal-to-noise ratio.</b><br/>
 +
                                            <p style="text-align:justify">(A) Schemes of split calcium sensors corresponding to the graphs below. (B) Mutations within the calmodulin construct of the split calcium sensor affected
 +
                                                the response to calcium. HEK293T cells were transfected with 50ng of both plasmids encoding split calcium sensors. 24 hours after transfection, luciferase activitiy was measured
 +
                                                immediately after addition of 10 µM calcium ionophore A23187.
 +
                                            </p>
 +
                                        </figcaption>
 +
                                    </figure>
 +
                                </div>
 +
 
 +
                                <div style = "float:left; width:50%">
 +
                                    <figure data-ref="6">
 +
                                        <img src="https://static.igem.org/mediawiki/2016/5/50/T--Slovenia--3.6.3.png" >
 +
                                        <figcaption><b>Response of the split calcium sensor depending on the ratio of both protein components.</b><br/>
 +
                                            <p style="text-align:justify">Fold activation of the split calcium sensor depended
 +
                                                on ratio between both components. HEK293T cells were transfected with plasmids encoding the split calcium sensor CaM<sup>E104Q</sup>:cLuc and nLuc:M13. 24 h after transfection
 +
                                                luciferase activity was measured immediately after the addition of 10 µM calcium ionophore A23187. The dots represent fold increase, which were obtained by division of value with corresponding mock.
 +
                                            </p>
 +
                                        </figcaption>
 +
                                    </figure>
 +
                                </div>
 +
 
 +
                                <p style="clear:both">Our newly designed calcium sensor was later used to detect activation of mechanoreceptors based on ultrasound stimulation and mechanical stress, where it enabled a clear
 +
                                    differentiation between stimulated and unstimulated cells only a few minutes after stimulation and could be used for real time monitoring.
 +
                                </p>
 
 
−
<p style="clear:both">Based on the inspection of the 3D structure of the CaM-M13 complex (<a href="http://www.rcsb.org/pdb/explore.do?structureId=2BBM">PDB code: 2BBM</a>), we fused the
 
−
N-terminal fragment of the split firefly luciferase to the N-terminus of M13 (nLuc:M13) and the C-terminal fragment of the split firefly luciferase to the C-terminus
 
−
of calmodulin (CaM:cLuc). The split calcium sensor is represented in <ref>2</ref>A. When transfected into HEK293T cells the sensor was expressed in the cytosol
 
−
(<ref>2</ref>B).
 
−
</p>
 
−
<div style = "float:left;">
 
−
<figure data-ref="2">
 
−
<img onclick="resize(this);" class="ui medium image" src="https://static.igem.org/mediawiki/2016/9/9c/T--Slovenia--3.6.1-2x.png" >
 
−
<figcaption><b>Split calcium sensor is expressed in the cytosol.</b><br/>
 
−
(A) Scheme of the function of the split calcium sensor. Calmodulin is fused to the C-terminal fragment and M13 is fused to N-terminal fragment of split
 
−
firefly luciferase. Free calcium ions trigger binding of M13 to calmodulin and formation of active luciferase. (B) Split calcium sensor is expressed in
 
−
the cytosol. HEK293T cells were transfected with 50 ng of nLuc:M13 and 10 ng of CaM:cLuc. 24 h after transfection cells were fixed, stained with anti-HA
 
−
and anti-Myc antibodies and localization was confirmed on the confocal microscope.</figcaption>
 
−
</figure>
 
−
</div>
 
−
<h4 style="clear:both"><span class="section">&nbsp;</span>Split calcium sensor</h4>
 
−
<div style="float:right">
 
−
<figure data-ref="3">
 
−
<img class="ui medium image" src="https://static.igem.org/mediawiki/2016/0/01/T--Slovenia--S.3.6.1.png" >
 
−
  <figcaption><b> Detection of calcium influx by the split calcium sensing reporter.</b> </figcaption>
 
−
</figure>
 
−
</div>
 
−
<p>The split luciferase was tested on live cells (<ref>3</ref>) but the ratio of the outputs from stimulated and non-stimulated cells remained low,
 
−
because activation of the sensor was still detectable at the cytoplasmic concentration of calcium. In order to decrease the activation of the sensor at resting
 
−
levels of calcium we introduced two mutations E31Q, E104Q in EF hand motifs, reported previously to decrease the affinity of CaM to calcium <x-ref>Evans2009</x-ref>.
 
−
We prepared the single mutant E104Q and the double mutant E31Q, E104Q split calcium sensors and tested the systems on HEK293T cells (<ref>4</ref>).
 
−
</p>
 
−
<div style = "float:left;">
 
−
<figure data-ref="4">
 
−
<img onclick="resize(this);" class="ui medium image" src="https://static.igem.org/mediawiki/2016/4/4a/T--Slovenia--3.6.2.png">
 
−
<figcaption><b>Split calcium sensing reporter with a single mutation E104Q within calmodulin demonstrated the highest signal-to-noise ratio for calcium
 
−
activation.</b><br/>
 
−
(A) Schemes of split calcium sensors corresponding to the graphs below. (B) Mutations within calmodulin of the split calcium sensing systems changed
 
−
the response to calcium. HEK293T cells were transfected with split calcium sensors (50 ng). 24 h after transfection, luciferase activities were measured
 
−
immediately after addition of calcium ionophore A23187 (10 µM ).</figcaption>
 
−
</figure>
 
−
</div>
 
−
<p>Split calcium reporter with a single mutation (E104Q) introduced into calmodulin was proved to work best, whereas the sensor with two mutations (E31Q, E104Q)
 
−
generated a low signal. The sensor with E104Q mutation had the highest ratio of the stimulated vs. resting cells, therefore representing the best with calcium sensor.
 
−
We additionally tested whether the ratio between parts of the calcium sensor affects the fold change between signal of non-stimulated and stimulated cells (<ref>5</ref>).
 
−
The response of the reported depended on the ratio, favoring an excess of the nLuc:M13, with the highest ratio close to 10.
 
−
</p>
 
 
 
−
 
+
<div style="float:right; width:50%">
−
<div style = "float:right;">  
+
<figure data-ref="9">
−
<figure data-ref="5">
+
<img src="https://static.igem.org/mediawiki/2016/4/4e/T--Slovenia--m1-m2.png">
−
<img onclick="resize(this);" class="ui medium image" src="https://static.igem.org/mediawiki/2016/5/50/T--Slovenia--3.6.3.png" >
+
<figcaption>
−
<figcaption><b>Response of split calcium sensor depending on the ration of both protein components.</b><br/> Fold activation of split calcium sensor depended
+
<b>The split calcium sensor responds to ultrasound stimulation.</b><br/>
−
on ratio between the CaM:cLuc and nLuc:M13. HEK293T cells were transfected with split calcium sensor CaM:cLuc and nLuc:M13. 24 h after transfection
+
<p style="text-align:justify">
−
luciferase activities were measured immediately after the addition of a ionophore A23187 (10 µM).</figcaption>
+
HEK293 cells were transfected with plasmids encoding the MscS mechanoreceptor and the split calcium sensor (CaM<sup>E104Q</sup>:cLuc and nLuc:M13) 24 hours prior to stimulation. 30 minutes before the ultrasound stimulation, media with 1 mM luciferin and 4 mM CaCl2 was added and the response was measured via bioluminescence imaging.
 +
</p>
 +
</figcaption>
 
</figure>
 
</figure>
 
</div>
 
</div>
−
<p style="clear:both">Those reporters were later used to detect response of cells to activation of mechanoreceptors based on the ultrasound and mechanical stress, where they enables clean
+
<p>The split calcium sensor is compatible with different constructs, such as designed <a href=" https://2016.igem.org/Team:Slovenia/Mechanosensing/Mechanosensitive_channels "> mechano-responsive channels</a> and with different means of stimulation. In addition to ionophore stimulation, it also responds to stimulation by ultrasound and to direct contact that underlies the <a href=" https://2016.igem.org/Team:Slovenia/Proof "> sense of touch </a>.</p>
−
difference between stimulated and unstimulated cells, already few minutes after the stimulation and could be used for real time monitoring.
+
−
</p>
+
−
 
+
 
 
 +
<p>Potential use of our calcium sensor as a <a href="https://2016.igem.org/Team:Slovenia/Measurement">measurement device</a> was tested. In addition to the plasmids encoding the split calcium sensor, HEK293 cells were co-transfected with the MscS mechanoreceptor (<a href="http://parts.igem.org/Part:BBa_K1965000">BBa_K1965000</a>) plasmid (<ref>9</ref>) and stimulated by ultrasound. Increase of the intracellular level of Ca<sup>2+</sup> caused reconstitution of split luciferase. Consequently, we were able to observe an increase in luminiscence in less than one minute, confirming that the designed split calcium sensor is able of detecting calcium influx caused by ultrasound stimulation (<ref>9</ref>).</p>
 +
 +
<p style="clear:both"></p>
  
 +
                            </div>
 
</div>
 
</div>
−
+
                        </div>
−
+
                        <h3 class="ui left dividing header"><span id="ref-title" class="section colorize">&nbsp;</span>References
−
</div>
+
                        </h3>
−
+
                        <div class="ui segment citing" id="references"></div>
−
</div>
+
                    </div>
−
</div>
+
                </div>
−
</div>
+
                </div>
−
</div>
+
            </div>
−
</div>
+
        </div>
 +
    </div>
 +
</div>
 +
<div>
 +
    <a href="//igem.org/Main_Page">
 +
        <img border="0" alt="iGEM" src="//2016.igem.org/wiki/images/8/84/T--Slovenia--logo_250x250.png" width="5%" style = "position: fixed; bottom:0%; right:1%;">
 +
    </a>
 +
</div>
 
</body>
 
</body>
 
</html>
 
</html>

Latest revision as of 18:19, 19 October 2016

Ca-dependent mediator

Gas vesicles Ca-dependent mediator Achievements Introduction CaMeleon Split calcium sensors Protease signaling

 Ca-dependent reporter and mediator

  • A new split calcium sensing/reporter system based on split firefly luciferase fused to M13 and calmodulin was designed and tested.
  • The designed sensor was able to report the increase of the cytosolic calcium ions induced by mechanoreceptor stimulation via emitted light.

 

While calcium influx can be detected by exogenous fluorescent dyes such as the FuraRed and Fluo-4, we needed a genetically encoded calcium sensor for the purposes of our project. Our sensor should couple a change in calcium concentration to a biologically relevant output, such as the luciferase reporter or reconstitution of a split protease for the initiation of the signaling pathway. The ideal calcium sensor should be inactive at resting intracellular concentrations of calcium, have a high response to calcium concentrations above physiological levels and be detected by a quick and easy readout. For our intended application, the calcium sensor should also have the potential to act as a reconstitution mechanism for split proteins so that a conformational rearrangement in the presence of calcium would bring the two split protein fragments together and reconstitute the protein’s activity.

  Results

 CaMeleon based reporters

A wide pallet of genetically encoded calcium sensors, based on different mechanisms of detection, are used for mapping intracellular calcium concentrationWhitaker2010, including calmodulin, troponin C and aequorinWilms2014. From this abundant collection we chose calmodulin (CaM)-based calcium sensors (in particular CaMeleons), since their mechanism is based on a large conformational change, allowing reconstitution of split proteins Whitaker2010.

Further explanation ...

CaMeleons are based on a genetic fusion of a recombinant calcium binding protein with a pair of fluorescent proteins, forming a FRET (Förster resonance energy transfer) based sensor. Yellow CaMeleon 2.12 is a CaMeleon composed of calmodulin and a CaM-binding domain of the skeletal muscle myosin light chain kinase (M13), forming the backbone of the sensor, and the FRET pair EYFP-ECFP linked to the termini of the construct. The binding of calcium causes calmodulin to wrap around the M13 domain, bringing the two fluorescent proteins closer to each other, resulting in the FRET effectWhitaker2010.


A luminescence based calcium sensor fLuc2.12 is highly active already at the resting levels of calcium.

HEK293T cells were transfected with 50 ng of the plasmid encoding fLuc2.12. 24 hours after transfection luciferase activity was measured immediately after addition of 10 µM calcium ionophore A23187. Scheme: The fLuc2.12 chimeric protein (M13-calmodulin fused to N- and C- fragments of split luciferase) changes conformation upon calcium binding.

We replaced the FRET pair of Yellow CaMeleon 2.12 by a split firefly luciferase (fLuc), as split luciferase provides a distinct signal even in small amounts and has a remarkable signal-to-noise ratio. The new luciferase based calcium sensor was named fLuc2.12. The fLuc2.12 construct was tested on HEK293T cells, but we found that the sensor was active already in resting cells (1). We hypothesized that activation was a consequence of the close proximity of calmodulin and M13 in the fusion even in the absence of calcium binding. In order to resolve this problem we set out to test a similar sensor based on two separate molecules. Two-molecule-based CaM sensors have not been widely studied, however lower leakage in comparison to a single molecule FRET sensor has been reported by Miyawaki et al. Miyawaki1997.

 Split calcium sensor

An inspection of the crystal structure of the CaM-M13 complex in its closed state (PDB code: 2BBM) suggested that reporter proteins could be directly fused to the termini of these two interacting proteins (2).

The Calmodulin and M13 peptide complex upon calcium binding.

The complex CaM-M13 in its closed state upon calcium binding (PDB 2BBM) (left) and rotated by 90° (right). The M13 peptide is in color scale from red to yellow (N-term to C-term), while calmodulin is in color scale from blue to cyan (N-term to C-term). Calcium is shown in green, and the side chains of residues E31Q and E104Q are visible. The C-terminus of calmodulin (red) and the N-terminus of the M13 peptide (blue) are brought together in the closed state.

We therefore decided to fuse the N-terminal fragment of the split firefly luciferase to the N-terminus of M13 (nLuc:M13, BBa_K1965014) and the C-terminal fragment of the split firefly luciferase to the C-terminus of calmodulin (CaM:cLuc, BBa_K1965015). The split calcium sensor is represented in 2.1A. When transfected into HEK293T cells the sensor was expressed in the cytosol (2.1B).

Cytosolic expression of the split calcium sensor.

(A) Scheme of the function of the split calcium sensor. Calmodulin is fused to the C-terminal fragment and M13 is fused to N-terminal fragment of split firefly luciferase. Free calcium ions trigger binding of M13 to calmodulin and the formation of active luciferase. (B) Split calcium sensor is expressed in the cytosol. HEK293T cells were transfected with plasmids encoding nLuc:M13 and CaM:cLuc in the ratio 5:1. 24 hours after transfection cells were fixed, stained with anti-HA and anti-Myc antibodies and localization was confirmed with confocal microscopy.

Detection of calcium influx by the split calcium sensor.

The split calcium sensor was tested on live cells (4) but the ratio of the outputs from stimulated and non-stimulated cells remained low, because the sensor was still highly activated at the resting cell calcium concentration. In order to decrease the activation of the sensor at resting calcium levels, we introduced mutations E31Q and E104Q in the EF hand motifs, previously reported to decrease the affinity of calmodulin to calcium Evans2009. We prepared the single mutant CaME104Q and the double mutant CaME31Q, E104Q split calcium sensors and tested them on HEK293T cells (5).

Split calcium reporter with a single mutation (E104Q) exhibited significantly reduced activation at resting calcium levels and high activation when cells were induced with ionophore. However, the sensor with two mutations (E31Q, E104Q) generated a low signal at induction. The sensor with the E104Q mutation resulted in the highest signal-to-noise ratio, therefore representing the best calcium sensor (5). We additionally tested whether the ratio between the two calcium sensor constructs affects the fold change between the signal of non-stimulated and stimulated cells. The response of the calcium sensor depended on the ratio, favoring an excess of the nLuc:M13 construct, with the highest fold change close to 10-fold (6).

The split calcium sensor with a single mutation E104Q within calmodulin demonstrated the highest signal-to-noise ratio.

(A) Schemes of split calcium sensors corresponding to the graphs below. (B) Mutations within the calmodulin construct of the split calcium sensor affected the response to calcium. HEK293T cells were transfected with 50ng of both plasmids encoding split calcium sensors. 24 hours after transfection, luciferase activitiy was measured immediately after addition of 10 µM calcium ionophore A23187.

Response of the split calcium sensor depending on the ratio of both protein components.

Fold activation of the split calcium sensor depended on ratio between both components. HEK293T cells were transfected with plasmids encoding the split calcium sensor CaME104Q:cLuc and nLuc:M13. 24 h after transfection luciferase activity was measured immediately after the addition of 10 µM calcium ionophore A23187. The dots represent fold increase, which were obtained by division of value with corresponding mock.

Our newly designed calcium sensor was later used to detect activation of mechanoreceptors based on ultrasound stimulation and mechanical stress, where it enabled a clear differentiation between stimulated and unstimulated cells only a few minutes after stimulation and could be used for real time monitoring.

The split calcium sensor responds to ultrasound stimulation.

HEK293 cells were transfected with plasmids encoding the MscS mechanoreceptor and the split calcium sensor (CaME104Q:cLuc and nLuc:M13) 24 hours prior to stimulation. 30 minutes before the ultrasound stimulation, media with 1 mM luciferin and 4 mM CaCl2 was added and the response was measured via bioluminescence imaging.

The split calcium sensor is compatible with different constructs, such as designed mechano-responsive channels and with different means of stimulation. In addition to ionophore stimulation, it also responds to stimulation by ultrasound and to direct contact that underlies the sense of touch .

Potential use of our calcium sensor as a measurement device was tested. In addition to the plasmids encoding the split calcium sensor, HEK293 cells were co-transfected with the MscS mechanoreceptor (BBa_K1965000) plasmid (9) and stimulated by ultrasound. Increase of the intracellular level of Ca2+ caused reconstitution of split luciferase. Consequently, we were able to observe an increase in luminiscence in less than one minute, confirming that the designed split calcium sensor is able of detecting calcium influx caused by ultrasound stimulation (9).

 References

iGEM