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

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     <title>Ca-dependent mediator</title>
 
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                         <b>Gas vesicles</b>
 
                         <b>Gas vesicles</b>
 
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                        <b>Ca-dependent mediator</b>
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                    </a>
 
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                         <b>Motivation</b>
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                         <b>Introduction</b>
 
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                             <h1 class = "ui left dividing header"><span id = "intro" class="section">&nbsp;</span>Ca-dependent reporter and mediator</h1>
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                             <h1 class = "ui left dividing header"><span id = "intro" class="section colorize">&nbsp;</span>Ca-dependent reporter and mediator</h1>
 
                             <div class = "ui segment" style = "background-color: #ebc7c7; ">
 
                             <div class = "ui segment" style = "background-color: #ebc7c7; ">
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                                 <p><b><ul><li>A new split calcium sensing/reporting system based on split firefly luciferase linked to M13 and calmodulin was designed that
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                                 <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.
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                                    is able to report the increase of the cytosolic calcium ions induced by mechanoreceptor stimulation by emitted light.</ul></b></p>
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<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>
 
                             </div>
 
                         </div>
 
                         </div>
 
                         <div class = "ui segment">
 
                         <div class = "ui segment">
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                             <h3><span id = "mot" class = "section"> &nbsp; </span></h3>
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                             <h3><span id = "mot" class = "section colorize"> &nbsp; </span></h3>
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                             <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
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                             <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
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                                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
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                                 initiation of the signaling pathway. The ideal calcium sensor should be inactive at resting intracellular concentrations of calcium, have a high response to calcium
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                                 initiation of the signaling pathway. The ideal calcium sensor should be inactive at intracellular concentration of calcium and have a high response to calcium
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                                 concentrations above physiological levels and be detected by a quick and easy readout. For our intended application, the calcium sensor should also have
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                                 concentrations above physiological levels and should be detected by a quick and easy readout. For our intended application, the calcium sensor should also have
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                                 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
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                                 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
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                                 split protein fragments together and reconstitute the protein’s activity.
 
                                 split protein fragments together and reconstitute the protein’s activity.
 
                             </p>
 
                             </p>
 
                         </div>
 
                         </div>
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                         <h1><span class="section"> &nbsp; </span>Results</h1>
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                         <h1><span class="section colorize"> &nbsp; </span>Results</h1>
  
 
                         <div class="ui segment">
 
                         <div class="ui segment">
 
                             <div>
 
                             <div>
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                                 <h3><span id = "cam" class="section">&nbsp;</span>CaMeleon based reporters</h3>
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                                 <h3><span id = "cam" class="section colorize">&nbsp;</span>CaMeleon based reporters</h3>
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                                 <p>A wide pallet of genetically encoded calcium sensors are used for mapping intracellular calcium concentration <x-ref>Whitaker2010</x-ref>,
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                                 <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>,
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                                     including calmodulin, troponin C and aequorin <x-ref>Wilms2014</x-ref>. These reporters are based on different mechanisms of detection. From this abundant
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                                     including calmodulin, troponin C and aequorin<x-ref>Wilms2014</x-ref>. From this abundant
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                                     collection we chose the calmodulin (CaM)-based calcium sensors (in particular CaMeleons), since their mechanism is based on a large conformational change, allowing
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                                     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 <x-ref>Whitaker2010</x-ref>.
 
                                     reconstitution of split proteins <x-ref>Whitaker2010</x-ref>.
 
                                 </p>
 
                                 </p>
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                                         <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
 
                                         <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
 
                                             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
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                                             (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
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                                             (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
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                                             M13 domain, bringing the two fluorescent proteins closer to each other, thus producing FRET <x-ref>Whitaker2010</x-ref>.
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                                             M13 domain, bringing the two fluorescent proteins closer to each other, resulting in the FRET effect<x-ref>Whitaker2010</x-ref>.
 
                                         </p>
 
                                         </p>
 
                                     </div>
 
                                     </div>
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                                     <figure data-ref="1">
 
                                     <figure data-ref="1">
 
                                         <img src="https://static.igem.org/mediawiki/2016/c/cf/T--Slovenia--3.6.1.png" >
 
                                         <img src="https://static.igem.org/mediawiki/2016/c/cf/T--Slovenia--3.6.1.png" >
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                                         <figcaption><b>A luminescence based calcium sensor fLuc2.12 has high activity already at the resting levels of calcium.</b><br/>
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                                         <figcaption><b>A luminescence based calcium sensor fLuc2.12 is highly active already at the resting levels of calcium.</b><br/>
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                                             <p style="text-align:justify">HEK293T cells were transfected with 50 ng fLuc2.12. 24 h after transfection luciferase activities were measured immediately after addition of calcium
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                                             <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
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                                                ionophore A23187 (10 µM). Scheme: The chimeric protein M13-calmodulin fused to N- and C- fragments of split luciferase changes conformation upon calcium
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                                                 binding.
 
                                                 binding.
 
                                             </p>
 
                                             </p>
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                                     </figure>
 
                                     </figure>
 
                                 </div>
 
                                 </div>
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                                 <p>We replaced the FRET pair of CaMeleon 2.12 by a split firefly luciferase (fLuc) as it provides a distinct signal even in small amounts and has a remarkable
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                                 <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
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                                     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
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                                     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
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                                     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
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                                     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
 
                                     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
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                                     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>.
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                                     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>
 
                                 </p>
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                                 <h3 style="clear:both"><span id = "split" class="section">&nbsp;</span>Split calcium sensor</h3>
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</div>
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                                 <p style="clear:both">An inspection of the crystal structure of CaM-M13 complex in its closed state (PDB code: 2BBM) suggested that reporter
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<div>
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                                     proteins can be directly fused to the termini of these two interacting proteins (<ref>1</ref>).</p>
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                                 <h3 style="clear:both"><span id = "split" class="section colorize">&nbsp;</span>Split calcium sensor</h3>
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                                 <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
 +
                                     proteins could be directly fused to the termini of these two interacting proteins (<ref>2</ref>).</p>
 
                                 </p>
 
                                 </p>
  
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                                     <figure data-ref="2">
 
                                     <figure data-ref="2">
 
                                         <img src="https://static.igem.org/mediawiki/2016/3/38/T--Slovenia--3.4.6.png" >
 
                                         <img src="https://static.igem.org/mediawiki/2016/3/38/T--Slovenia--3.4.6.png" >
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                                         <figcaption><b>Calmodulin and M13 peptide complex upon calcium binding.</b><br/>
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                                         <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).
 
                                             <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).
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                                                 M13 peptide is in color scale from red to yellow (N-term to C-term), while CaM is in color scale from blue to cyan (N-term to C-term),
+
                                                 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).
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                                                 Calcium is in green, the side chains of the mutated residues (E31Q and E104Q) are visible. C-terminal of Calmodulin (red)
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                                                 Calcium is shown in green, and the side chains of residues E31Q and E104Q are visible. The C-terminus of calmodulin (red)
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                                                 and N-terminal of the peptide M13 (blue) are brought together in the closed state.
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                                                 and the N-terminus of the M13 peptide (blue) are brought together in the closed state.
 
                                             </p>
 
                                             </p>
 
                                         </figcaption>
 
                                         </figcaption>
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                                 <p>We therefore decided to fuse 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>2.1</ref>A. When transfected into HEK293T cells the sensor was expressed in the cytosol
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                                 <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).
 
                                     (<ref>2.1</ref>B).
 
                                 </p>
 
                                 </p>
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                                     <figure data-ref="2.1">
 
                                     <figure data-ref="2.1">
 
                                         <img src="https://static.igem.org/mediawiki/2016/9/9c/T--Slovenia--3.6.1-2x.png" >
 
                                         <img src="https://static.igem.org/mediawiki/2016/9/9c/T--Slovenia--3.6.1-2x.png" >
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                                         <figcaption><b>Split calcium sensor is expressed in the cytosol.</b><br/>
+
                                         <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
 
                                             <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
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                                                 firefly luciferase. Free calcium ions trigger binding of M13 to calmodulin and formation of active luciferase. (B) Split calcium sensor is expressed in
+
                                                 firefly luciferase. Free calcium ions trigger binding of M13 to calmodulin and the formation of active luciferase. (B) Split calcium sensor is expressed in
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                                                 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
+
                                                 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
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                                                 and anti-Myc antibodies and localization was confirmed on the confocal microscope.
+
                                                 and anti-Myc antibodies and localization was confirmed with confocal microscopy.
 
                                             </p>
 
                                             </p>
 
                                         </figcaption>
 
                                         </figcaption>
 
                                     </figure>
 
                                     </figure>
 
                                 </div>
 
                                 </div>
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                            </div>
 
 
                             <div>
 
                             <div>
  
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                                     <figure data-ref="4">
 
                                     <figure data-ref="4">
 
                                         <img src="https://static.igem.org/mediawiki/2016/0/01/T--Slovenia--S.3.6.1.png" >
 
                                         <img src="https://static.igem.org/mediawiki/2016/0/01/T--Slovenia--S.3.6.1.png" >
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                                         <figcaption><b> Detection of calcium influx by the split calcium sensing reporter.</b> </figcaption>
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                                         <figcaption><b> Detection of calcium influx by the split calcium sensor.</b> </figcaption>
 
                                     </figure>
 
                                     </figure>
 
                                 </div>
 
                                 </div>
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                                 <p>The split luciferase was tested on live cells (<ref>4</ref>) but the ratio of the outputs from stimulated and non-stimulated cells remained low,
+
                                 <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 activation of the sensor was still detectable at the cytoplasmic concentration of calcium. In order to decrease the activation of the sensor at resting
+
                                     because the sensor was still highly activated at the resting cell calcium concentration. 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>.
+
                                     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 E104Q and the double mutant E31Q, E104Q split calcium sensors and tested the systems on HEK293T cells (<ref>5</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>
  
−
                                 <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)
+
                                 <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>).
−
                                    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 the two calcium sensor constructs  affects the fold change between the signal of non-stimulated and stimulated cells.
−
                                     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 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>).
−
                                     The response of the reported depended on the ratio, favoring an excess of the nLuc:M13, with the highest ratio close to 10 (<ref>6</ref>).
+
 
                                 </p>
 
                                 </p>
  
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                                     <figure data-ref="5">
 
                                     <figure data-ref="5">
 
                                         <img src="https://static.igem.org/mediawiki/2016/4/4a/T--Slovenia--3.6.2.png">
 
                                         <img 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
+
                                         <figcaption><b>The split calcium sensor with a single mutation E104Q within calmodulin demonstrated the highest signal-to-noise ratio.</b><br/>
−
                                            activation.</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
−
                                             <p style="text-align:justify">(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 50ng of both plasmids encoding split calcium sensors. 24 hours after transfection, luciferase activitiy was measured
−
                                                 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 10 µM calcium ionophore A23187.
−
                                                 immediately after addition of calcium ionophore A23187 (10 µM ).
+
 
                                             </p>
 
                                             </p>
 
                                         </figcaption>
 
                                         </figcaption>
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                                     <figure data-ref="6">
 
                                     <figure data-ref="6">
 
                                         <img src="https://static.igem.org/mediawiki/2016/5/50/T--Slovenia--3.6.3.png" >
 
                                         <img src="https://static.igem.org/mediawiki/2016/5/50/T--Slovenia--3.6.3.png" >
−
                                         <figcaption><b>Response of split calcium sensor depending on the ratio of both protein components.</b><br/>
+
                                         <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 split calcium sensor depended
+
                                             <p style="text-align:justify">Fold activation of the split calcium sensor depended
−
                                                 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
+
                                                 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 activities were measured immediately after the addition of a ionophore A23187 (10 µM).
+
                                                 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>
 
                                             </p>
 
                                         </figcaption>
 
                                         </figcaption>
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                                 </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 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
−
                                     difference between stimulated and unstimulated cells, already few minutes after the stimulation and could be used for real time monitoring.
+
                                     differentiation between stimulated and unstimulated cells only a few minutes after stimulation and could be used for real time monitoring.
 
                                 </p>
 
                                 </p>
−
<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 receptors </a>channels and different means of stimulation. In addition to ionophore stimulation, it also responds to stimulation by ultrasound and direct contact that underlies the <a href=" https://2016.igem.org/Team:Slovenia/Proof "> sense of touch </a>.</p>
+
 
 
 
<div style="float:right; width:50%">
 
<div style="float:right; width:50%">
−
<figure data-ref="5">
+
<figure data-ref="9">
 
<img src="https://static.igem.org/mediawiki/2016/4/4e/T--Slovenia--m1-m2.png">
 
<img src="https://static.igem.org/mediawiki/2016/4/4e/T--Slovenia--m1-m2.png">
−
<figcaption><b>Split calcium sensor responds to utrasound stimulation</b><br/> HEK293 cells were transfected with MscS mechanosensitive channels and split calcium sensor (CaM(E104Q):cLuc and nLuc:M13) 24 h prior to stimulation. 30 minutes before the ultrasound stimulation we added media with 1 mM luciferin and 4 mM CaCl2 <sup>2+</sup> and response was measured via bioluminescence imaging.</figcaption>
+
<figcaption>
 +
<b>The split calcium sensor responds to ultrasound stimulation.</b><br/>
 +
<p style="text-align:justify">
 +
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>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 receptors </a>channels and different means of stimulation. In addition to ionophore stimulation, it also responds to stimulation by ultrasound and direct contact that underlies the <a href=" https://2016.igem.org/Team:Slovenia/Proof "> sense of touch </a>.</p>
+
<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>
 
 
−
<p>Potential use of calcium sensor as a measurement device was tested. In addition to the split calcium sensor, HEK293 cells were transfected with MscS (<a href="http://parts.igem.org/Part:BBa_K1965000">BBa_K1965000</a>), (<ref>2</ref>A) and were stimulated by ultrasound. Increase of intracellular level of Ca<sup>2+</sup> caused reconstitution of split luciferase. Consequently, we were able to observe increase in luminiscence in less than one minute in the cells expressing MscS and split calcium sensor, proving that the designed split calcium sensor is able of detecting calcium influx caused by ultrasound stimulation. (<ref>5</ref>).</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>
 
<p style="clear:both"></p>
  
 
                             </div>
 
                             </div>
 +
</div>
 
                         </div>
 
                         </div>
−
                         <h1 class="ui left dividing header"><span id="ref-title" class="section">&nbsp;</span>References
+
                         <h3 class="ui left dividing header"><span id="ref-title" class="section colorize">&nbsp;</span>References
−
                         </h1>
+
                         </h3>
 
                         <div class="ui segment citing" id="references"></div>
 
                         <div class="ui segment citing" id="references"></div>
 
                     </div>
 
                     </div>
 +
                </div>
 
                 </div>
 
                 </div>
 
             </div>
 
             </div>

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