Difference between revisions of "Team:Slovenia/Implementation/ProteaseInducible secretion"

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<b>Idea</b>
 
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<h1 class = "ui centered dividing header"><span class="section">nbsp;</span>Protease-based inducible secretion</h1>
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<p>Fast sensing and fast signaling pathway were developed using protease-based pathway. In the final step for the construction of rapidly responding cells we wanted to
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implement a fast output that would not require a slow transcription/translation biosynthesis of new proteins. We decided to engineer a system capable of regulated secretion
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of a protein using genetically encoded components.</p>
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<p>To achieve a fast regulated cellular response resulting in the release of a protein, we decided to mimic the release of insulin from beta cells where the protein of
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interest is pre-formed and present in the cell in secretory granules. In contrast to the specialized storage and release mechanism of insulin from beta cells we wanted to
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develop a more general and modular solution by making use of components already existing in different types of cells. Additionally, there should be minimal leakage from the
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protein depot in the uninduced state and after induction secretion from the cell should be fast.</p>
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Further explanation ...
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<p> Not many systems for the inducible release of proteins have been engineered to date. In one of the few examples Rivera et al. developed a system where the protein of interest
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was fused to a conditional aggregation domain (CAD). <x-ref>Rivera2000</x-ref>. These domains form aggregates in the endoplasmic reticulum (ER) that are too large to exit the ER.
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After the addition of a small synthetic molecule, the CADs start to disaggregate and the protein of interest can be secreted. In the second example Chen et al. introduced a
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light-triggered secretion system. They also based their system on conditional aggregation; however they used the plant photoreceptor UVR8 which forms photolabile homodimers to make
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aggregates on the ER membrane. Upon light excitation the aggregates made by UVR8 started to disaggregate and were transported from the ER to the plasma membrane, but have not been
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observed in the cell supernatant. <x-ref>Chen2013</x-ref>
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The weakness of the two described systems is that they both rely on the exogenous chemical or physical signals instead of using a biochemical signal to induce the secretion, which
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means that they can’t be integrated into the signaling system that’s senses the cellular state. In order to better respond to the state of the cell or a logic circuit inside a cell
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we decided to develop an inducible secretion system based on the biochemical signal.
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<p><br />Many proteins that reside on the membrane or in the lumen of the ER contain short peptide signals. Proteins present in the lumen of the ER contain a KDEL C-terminal sequence
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(Lys-Asp-Glu-Leu) while type I transmembrane (TM) proteins contain a dilysine (KKXX) motif on their C-terminus (cytosolic side). <x-ref> Munro1987, Jackson1990,
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Stornaiuolo2003</x-ref>. The mechanism that allows these proteins to stay in the ER is more retrieval than retention. However we decided to use the term retention for
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description of this process. ER luminal proteins interact with the KDEL receptor, a transmembrane ER resident protein. The cytosolic part of the KDEL receptor interacts with
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coat proteins I (COP I) which coat vesicles and are responsible for transporting proteins from the cis end of the Golgi apparatus (cis-GA) back to the ER. The KKXX motif present
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on type I TM proteins can directly interact with the COP I for retrieval. <x-ref> Stornaiuolo2003, Letourneur1994 </x-ref>.</p>
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<h1 class = "ui centered dividing header"><span class="section">nbsp;</span>Calcium-depended mediator</h1>
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<p>Our idea was that if we proteolytically remove the retention signal, the protein of interest would no longer be retrieved back to the ER and could be secreted from the cell.
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<div class = "ui segment">
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To achieve this we designed two types of secretory reporters, one type based on the luminal retention using KDEL sequence and the other based on the transmembrane retention with
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<p style = “background-color: #ff6666;”>A new split calcium sensing/reporting system based on split firefly luciferase linked to M13 and calmodulin was designed that
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a KKMP sequence. In each case, the retention sequence was preceded by a TEVp cleavage site to allow for inducible secretion, which could be replaced by any other peptide target
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is able to report the increase of the cytosolic calcium ions induced by mechanoreceptor stimulation by emitted light.</p>
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of our orthogonal protease set.</p>
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<h4>Secretion from the ER lumen</h4><br/>
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<p>To achieve and detect the inducible secretion from the ER lumen, we created two reporter constructs with a cleavable KDEL sequence targeted to the ER lumen: SEAPKDEL and
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TagRFPKDEL. Those proteins contained a protease target motif between the reporter domain and the KDEL domain, aimed to enable protein secretion after the proteolytic cleavage.
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We used a TEVp variant (erTEVp) for all of our experiments with luminal retention.</p>
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Further explanation ...
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<h3>Motivation</h3><br/>
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In order to rely on TEVp cleavage in the ER lumen, we had to take some additional considerations into account. Cesaratto et al.</x-ref>Cesaratto2015</x-ref> reported that the wild
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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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type TEV protease is not active in the lumen of ER. They designed a TEV protease variant active in the endoplasmic reticulum by preventing two major types of post-translational
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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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modifications: N-glycosylation and cysteine oxidation. To avoid these inhibiting modifications, mutations N23Q, C130S and N171T were made. To ensure correct localization and
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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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accumulation of this TEVp variant inside the endoplasmic reticulum, we also attached a signal sequence at the N-terminus and KDEL at the C-terminus of the protein.
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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 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.
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</p>
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<div class="ui segment">
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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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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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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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reconstitution of split proteins <x-ref>Whitaker2010</x-ref>.
 
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<h4>Results</h4><br/>
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Further explanation ...
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</div>
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<div style= "float:right;">  
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<div class="content">
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<figure data-ref="1">
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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
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<img class="ui medium image" src="https://static.igem.org/mediawiki/2016/9/9e/T--Slovenia--6.2.1.png" >
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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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<figcaption><b>Cleavage with ER-residing protease (erTEV) facilitates secretion of reporter from cells.</b><br/>  
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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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(A) Scheme of the reporter with cleavable KDEL retention signal and protease target motif.
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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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(B) The reporter with the KDEL retention signal was localized in the ER. HEK293T cells were transfected with the indicated reporters and in (C) also with erTEVp.  
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</p>
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Localization was detected with confocal microscopy. (C)The reporter was detected in the medium of cells only when cotransfected with erTEVp. HEK293T cells were transfected  
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with the indicated constructs. Reporters were detected with WB in the concentrated medium.
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<div style = "float:left">  
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</figure>
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<figure data-ref="1">
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<img class="ui medium image" src="https://static.igem.org/mediawiki/2016/c/cf/T--Slovenia--3.6.1.png" >
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<p>When the TagRFP<sup>KDEL</sup> reporter (<ref>1</ref>A) was expressed in the ER without an active erTEVp we confirmed its localization in the ER with confocal microscopy
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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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(<ref>1</ref>B). Additionally, we could not detect any TagRFP in the cell medium with Western blotting. When erTEVp was present and active in the ER, the KDEL sequence was
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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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removed from the reporter and the protein was secreted from the cell, which we detected with Western blot (<ref>1</ref>C), demonstrating that proteolytic activity in the ER can
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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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regulate protein secretion.
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binding.</figcaption>
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</p>
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</figure>
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</div>
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<div style = "float:left;">  
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<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
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<figure data-ref="2">
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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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<img class="ui medium image" src="https://static.igem.org/mediawiki/2016/6/61/T--Slovenia--6.2.2.png" >
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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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<figcaption><b>Secretion of the SEAP reporter from ER lumen by cleavage with ER-resident protease.</b><br/>  
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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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HEK293T cells were transfected with indicated reporter and erTEVp. Increased SEAP activity was detected in the medium of cells
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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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expressing both reporter and erTEVp protease.</figcaption></figure>
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</p>
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</div>  
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<p style="clear:right">Using SEAP<sup>KDEL</sup> we were able to confirm that the reporter is not present in the cell medium without coexpression of erTEVp. When erTEVp was cotransfected with  
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<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
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the reporter, we detected a large increase in enzymatic activity in the medium (<ref>2</ref>).
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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
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</p>
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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
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</div>
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(<ref>2</ref>B).
 +
</p>
 +
<div style = "float:right;">
 +
<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  
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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
 +
and anti-Myc antibodies and localization was confirmed on the confocal microscope.</figcaption>
 +
</figure>
 +
</div>
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<div style="clear:both" align = "left">
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<figure data-ref="5">
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<img class="ui medium image" 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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</figure>
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</div>
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<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
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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>.
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We prepared the single mutant E104Q and the double mutant E31Q, E104Q split calcium sensors and tested the systems on HEK293T cells (<ref>3</ref>).
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</p>
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<div style = "float:right;">  
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<figure data-ref="3">
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<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
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activation.</b><br/>
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(A) Schemes of split calcium sensors corresponding to the graphs below. (B) Mutations within calmodulin of the split calcium sensing systems changed
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the response to calcium. HEK293T cells were transfected with split calcium sensors (50 ng). 24 h after transfection, luciferase activities were measured
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immediately after addition of calcium ionophore A23187 (10 µM ).</figcaption>
 +
</figure>
 +
</div>
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<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)
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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.
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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>4</ref>).
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The response of the reported depended on the ratio, favoring an excess of the nLuc:M13, with the highest ratio close to 10.
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</p>
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<p>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
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difference between stimulated and unstimulated cells, already few minutes after the stimulation and could be used for real time monitoring.
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</p>
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<div style = "float:left;">  
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<figure data-ref="4">
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<img onclick="resize(this);" class="ui medium image" src="https://static.igem.org/mediawiki/2016/5/50/T--Slovenia--3.6.3.png" >
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<figcaption><b>Response of split calcium sensor depending on the ration of both protein components.</b><br/> Fold activation of split calcium sensor depended
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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
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luciferase activities were measured immediately after the addition of a ionophore A23187 (10 µM).</figcaption>
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</figure>
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</div>
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</div>
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<h4>Secretion from the ER membrane</h4><br/>
 
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<p>The second approach to regulate protein secretion from the ER by protease was to used KKMP ER retention peptide linked to the transmembrane protein with a protease target
 
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motif on the cytoplasmic side, N-terminal to the KKMP peptide. A transmembrane (TM) domain from the B-cell receptor
 
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(<a href="http://parts.igem.org/Part:BBa_K157010">Bba_K157010</a>) was used for the integration of target proteins in the ER membrane. Similar as described above, two reporter
 
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constructs with SEAP and TagRFP (SEAP:TM<sup>KKMP</sup> and TagRFP:TM<sup>KKMP</sup>) were designed and the constructs also contained a signal sequence at their N-terminus and a
 
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proteolytically cleavable ER retention signal at their C-terminus. In case of the transmembrane targeted reporters we used the KKMP retention signal preceded by 3 copies of the
 
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TEVp cleavage site on the cytosolic side of the membrane.</p>
 
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<p>Additionally, either one or four furin cleavage sites were inserted between the protein of interest on the luminal side of the ER, which enable cleavage of the reporter
 
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protein from the membrane, but this could occur only after the KKMP had been removed and the protein could enter the trans-GA. Furin is a native cellular endoprotease that is
 
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active only in the trans-GA.</x-ref>Henrich2003</x-ref> This allowed us to design our constructs so that they are cleaved off of the membrane without any modified scar sequences
 
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attached to them.</p>
 
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<h4>Results</h4><br/>
 
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<div style="float:left" align = "left;">
 
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<figure data-ref="3">
 
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<img onclick="resize(this);" class="ui medium image" src="https://static.igem.org/mediawiki/2016/b/b4/T--Slovenia--6.2.3.png" >
 
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<figcaption><b>Localization of protease-responsive reporters on ER depending on the proteolysis. </b><br/>
 
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(A) The transmembrane reporter without the KKMP retention signal was localized both on the ER and plasma membrane. (B) The transmembrane reporter with the KKMP retention
 
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signal was localized exclusively on the ER membrane. (C) After cleavage of the KKMP retention signal, the transmembrane reporter translocated to the plasma membrane. HEK293T
 
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cells were transfected with the indicated reporters and in (C) also with TEVp. Localization was detected with confocal microscopy. Each image is accompanied with a scheme of
 
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the transfected construct. (D) Glycosylated reporter was detected in the medium of cells transfected with the transmembrane reporter without the KKMP retention signal.
 
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HEK293T cells were transfected with the indicated constructs. Reporters were detected with WB in the concentrated medium. In lane 2, sample was incubated with N-glycosidase
 
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F.
 
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</figcaption>
 
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</figure>
 
 
</div>
 
</div>
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<p>Localization of the TagRFP:TM<sup>KKMP</sup> reporter was confirmed by the confocal microscopy. We used a control reporter without the KKMP retention signal (TagRFP:TM)
 
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which we detected both on the ER and the plasma membrane (<ref>3</ref>A). In case of the present KKMP retention signal, the reporter was detected only on the ER
 
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(<ref>3</ref>B). When TagRFP:TM<sup>KKMP</sup> was coexpressed with TEVp, localization of the reporter was similar to the localization of the positive control (TagRFP:TM)
 
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on the plasma membrane and the ER (<ref>3</ref>C).</p>
 
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<p>A band with a slightly larger apparent size than the expected size of TagRFP (28 kDa) was detected by western blotting in cells transfected with TagRFP:TM. We showed that the
 
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unexpected difference in size was due to glycosylation, as we detected the protein at the expected size after deglycosylation of the medium sample with N-glycosidase F. We were
 
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unable to detect a corresponding band in the medium of cells transfected with TagRFP:TM<sup>KKMP</sup> in the absence of the protease.</p>
 
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<p>Together, these results confirm that localization and secretion of the protein reporter with the transmembrane domain depends on the presence and proteolysis of the KKMP
 
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retention signal and that proteolysis can be used to induce secretion of already synthesized protein.</p>
 
 
 
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<div style="float:right"  align = "right">
 
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<figure data-ref="4">
 
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<img class="ui medium image" src="https://static.igem.org/mediawiki/2016/2/2f/T--Slovenia--6.2.4.png" >
 
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<figcaption><b>Inducible secretion of reporter localized on ER membrane.</b><br/> 
 
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SEAP activity was increased in the medium of cells induced with rapamycin. (B) Scheme of the transmembrane reporter with cleavable KKMP retention signal and inducible protease.
 
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HEK293T cells were transfected with the indicated reporter and rapamycin inducible split proteases. Uncleaved proteases were used as positive control.</figcaption>
 
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</figure>
 
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</div>
 
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<p style="clear:left">Finally, we cotransfected cells with SEAP:TM<sup>KKMP</sup> and rapamycin-inducible split TEVp. We detected increased levels of the SEAP enzymatic activity in the medium of
 
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cells stimulated with rapamycin, which was dose dependent with respect to the amount of the transfected reporter-coding plasmid (<ref>4</ref>). These results confirm that
 
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secretion of a target protein can be made inducible by an externally supplied signal, processed through our split protease system.
 
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Revision as of 00:08, 16 October 2016

Home

Project Achievements Medal requirements Idea

nbsp;Calcium-depended mediator

A new split calcium sensing/reporting system based on split firefly luciferase linked to M13 and calmodulin was designed that is able to report the increase of the cytosolic calcium ions induced by mechanoreceptor stimulation by emitted light.

Motivation


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 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 initiation of the signaling pathway. The ideal calcium sensor should be inactive at intracellular concentration of calcium and have a high response to calcium concentrations above physiological levels and should be detected by a quick and easy readout. For our intended application, the calcium sensor should also have 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 split protein fragments together and reconstitute the protein’s activity.

A wide pallet of genetically encoded calcium sensors are used for mapping intracellular calcium concentration Whitaker2010, including calmodulin, troponin C and aequorin Wilms2014. These reporters are based on different mechanisms of detection. From this abundant collection we chose the 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 a FRET pair 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, thus producing FRET Whitaker2010.


A luminescence based calcium sensor fLuc2.12 has high activity already at the resting levels of calcium.
HEK293T cells were transfected with 50 ng fLuc2.12. 24 h after transfection luciferase activities were measured immediately after addition of calcium ionophore A23187 (10 µM). Scheme: The chimeric protein M13-calmodulin fused to N- and C- fragments of split luciferase changes conformation upon calcium binding.

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 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 already in resting cells (1). We hypothesized the activation was a consequence of a 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, but lower leakage in comparison to a single molecule sensor has been reported by Miyawaki et al. Miyawaki1997.

Based on the inspection of the 3D structure of the CaM-M13 complex (PDB code: 2BBM), 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 2A. When transfected into HEK293T cells the sensor was expressed in the cytosol (2B).

Split calcium sensor is expressed in the cytosol.
(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.
Detection of calcium influx by the split calcium sensing reporter.

The split luciferase was tested on live cells (3) 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 Evans2009. We prepared the single mutant E104Q and the double mutant E31Q, E104Q split calcium sensors and tested the systems on HEK293T cells (3).

Split calcium sensing reporter with a single mutation E104Q within calmodulin demonstrated the highest signal-to-noise ratio for calcium activation.
(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 ).

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 (4). The response of the reported depended on the ratio, favoring an excess of the nLuc:M13, with the highest ratio close to 10.

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 difference between stimulated and unstimulated cells, already few minutes after the stimulation and could be used for real time monitoring.

Response of split calcium sensor depending on the ration of both protein components.
Fold activation of 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 luciferase activities were measured immediately after the addition of a ionophore A23187 (10 µM).