Difference between revisions of "Team:Slovenia/Implementation/ProteaseInducible secretion"
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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 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>. | 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> | + | 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> | </p> | ||
<div style = "float:right;"> | <div style = "float:right;"> | ||
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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) | <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. | 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> | + | 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. | The response of the reported depended on the ratio, favoring an excess of the nLuc:M13, with the highest ratio close to 10. | ||
</p> | </p> | ||
Revision as of 00:12, 16 October 2016
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
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
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.
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).
(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.
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
(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 (5). 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.
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).
