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Revision as of 00:19, 19 October 2016
Ca-dependent reporter and mediator
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.
Results
CaMeleon based reporters
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 CaMeleon 2.12 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.
Split calcium sensor
An inspection of the crystal structure of CaM-M13 complex in its closed state (PDB code: 2BBM) suggested that reporter proteins can be directly fused to the termini of these two interacting proteins (1).
The complex CaM-M13 in its closed state upon calcium binding (PDB 2BBM) (left) and rotated by 90° (right). 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), Calcium is in green, the side chains of the mutated residues (E31Q and E104Q) are visible. C-terminal of Calmodulin (red) and N-terminal of the peptide M13 (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) 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 2.1A. When transfected into HEK293T cells the sensor was expressed in the cytosol (2.1B).
(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 (4) 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
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 (6).
(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 ).
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).
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.
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 2+ and response was measured via bioluminescence imaging.
The split calcium sensor is compatible with different constructs such as designed mechano-responsive receptors channels and different means of stimulation. In addition to ionophore stimulation, it also responds to stimulation by ultrasound and direct contact that underlies the sense of touch .
Potential use of calcium sensor as a measurement device was tested. In addition to the split calcium sensor, HEK293 cells were transfected with MscS (BBa_K1965000), (2A) and were stimulated by ultrasound. Increase of intracellular level of Ca2+ 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. (5).
References
