Team:Slovenia/Notebook/Protocols
Protocols
For successful execution of all experiments, described in the previous pages, we used the following protocols:
Inovation in measurement
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
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.
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.
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
(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.
(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 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.
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 ionophore A23187 (10 µM).
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).
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.
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.
(A) Schematic presentation of a cell with increased sensitivity to mechanical stimulation due to expression of a mechanosensitive ion channel MscS and gas vesicle-forming proteins. Upon mechanical stimulation, calcium enters the cell via MscS channels, enabling activation of the calcium sensor and reconstitution of the split luciferase, (B) Images of petri dishes seeded with HEK293 cells, expressing the mechano-responsive calcium sensing system after mechanical stimulation .24 h after transfection, luciferin and CaCl2 were added to the media and the cells were stimulated by gentle drawing with a glass rod. Afterwards, camera images were taken in darkness with exposure time 30 s.
