Team:Slovenia/Mechanosensing/CaDependent mediator
Ca-dependent reporter and mediator
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 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 the FRET pair EYFP-ECFP linked to the termini of the construct. The binding of calcium causes calmodulin to wrap around the
M13 domain, bringing the two fluorescent proteins closer to each other, resulting in the FRET effect
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.
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 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).
(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.
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
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).
(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.
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.
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
