For successful execution of all experiments, described in the previous pages, we used the following protocols:

• Cell cultures
• Cloning
• Mechanical stimulation
• Microscopy
• Preparation of lipid microbubbles
• Protein detection
• Reporter assays

• A new, simple and reliable approach for real time measurement of the intracellular calcium concentration was developed.

Our project required a sensitive and fast method for detecting cytosolic calcium concentrations. This measurement is a fast-relay system, has low response at the physiological cytosolic level of calcium and works in vivo in mammalian cells.

Sensor development and characterization

While calcium influx could be detected by exogenous fluorescent dyes such as Fura Red and Fluo-4, our project required a sensitive, 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 for the initiation of the signaling pathway. An 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 palette of genetically encoded calcium sensors have been 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 selected calmodulin (CaM)-based calcium sensors (in particular CaMeleons), since their mechanism is based on a large conformational change, allowing reconstitution of split proteins Whitaker2010.

We replaced the FRET pair of CaMeleon2.12 by a split firefly luciferase (fLuc) as it provides a distinct signal even at small amounts and has a high 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 used, 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, BBa_K1965016) 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 2A. When transfected into HEK293T cells the sensor was expressed in the cytosol (2B).

The split luciferase reporter 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 the resting levels of calcium we introduced two mutations E31Q, E104Q in the 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 both reporters on HEK293T cells (3).

Split calcium reporter with a single mutation (E104Q) introduced into calmodulin worked 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 calcium sensor. We additionally tested whether the ratio between the components of the calcium sensor affects the fold change between the 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.

This reporter (CaM(E104Q):cLuc + nLuc:M13) was later used to detect response of cells to activation of mechanoreceptors, where it enabled clear difference between stimulated and unstimulated cells, already few minutes after the stimulation and could be used for real time monitoring of mechanosensing.

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 (5).

As shown in (6, cells were able to convert the mechanical stimulus (in this case, touching with a glass rod: Touchpaint )into light signal by activation of the calcium sensor and reconstitution of the split luciferase.