Calcium indicator experiments based on the fusion of calmodulin and fluorescent protein
Calcium indicator experiments based on the fusion of calmodulin and fluorescent protein
Calmodulin (CaM) is a ubiquitously distributed protein involved in calcium-involved signaling. Upon Ca2+ influx, CaM acquires a strong affinity for binding various intracellular proteins bearing one or more CaM recognition sequences, initiating or terminating Ca2+-regulated signaling crosstalk. The source of this experiment is "A Guide to Modern Protein Engineering Experiments" [Germany] K.M. Arndt, K.M. Miller, eds.
Operation method
Calcium indicators based on the fusion of calmodulin and fluorescent proteins
Materials and Instruments
PRSETB Bacterial Expression Plasmid HeLa Cell Line Move The methods described in this paper cover the cloning and expression of chameleons in bacteria and eukaryotic cells (see 4.3.1 ), the biochemical and biophysical characterization of chameleons (see 4.3.2 ), and the use of chameleons for in vivo Ca2+ imaging by FRET techniques (see 4.3.3 ). For more product details, please visit Aladdin Scientific website.
Phenylmethyl transverse acyl fluoride EGTA buffer Calcium chloride buffer
Ultrasonic generator Fluorescence spectrometer
3.1 Cloning of Chameleon
Calcium chameleons are assembled modularly using standard recombinant DNA methods [8] using 5 different components: CFP, YFP, N-CaM, C-CaM and CRS. CFP and YFP plasmids were available from Clontech; CaM was obtained from Dr. Ikum; and CRS was synthesized by oligonucleotide chain reaction. DNA manipulation for the production of calochromes is not described in detail here, as there is no single technique that fits all possibilities (see Note 4). After the calmodulin was made, the fusion fragment was cloned into the PRSETB plasmid to express enough protein to complete biochemical and biophysical characterization. We cloned the fusion fragments into the pcDNA3 plasmid (see Note 5) for expression in transient mammalian cells for in vivo Ca2+ imaging experiments.
3.2 Biochemical and biophysical characterization
Before the calcium chameleon can be used for in vivo Ca2+ imaging, it is purified (see 4.3.2.1 and Note 6 ) and in vivo fluorescence experiments are completed to determine its dynamic range (see 4.3.2.2 ) and calcium binding curve (see 4.3.2.3 ).
3.2.1 Calcium Chromophore Purification from Bacterial Cells
( 1 ) BL21 ( DE3 ) cells were transfected with a bacterial plasmid using standard molecular biology methods [8] .
( 2 ) Cells were plated on LB plates containing ampicillin and held at 37°C overnight.
( 3 ) Select a single colony and grow it at 37°C in 100 ml of LB culture medium containing 100 μmol/L ampicillin.
( 4 ) At 600 nm optical density of 0.7, induce with 0.5 mmol/L IPTG for 3 hours.
( 5 ) Centrifuge the cells at 3000 g for 30 min.
( 6 ) Cells were resuspended in 10 ml of 50 mmol/L HEPES, pH 7.4, containing 10% glycerol, 100 mmol/KCl, 1 mmol/L CaCl2 and 1 mmol/L phenylmethylsulfonyl fluoride.
( 7 ) The solution was sonicated with a 0.5-inch sonication head at maximum power, 10% duty cycle, four times for 4 min. between sonications, the solution was cooled down for 5 min.
( 8 ) Centrifuge cell debris at 30,000 g for 20 min.
( 9 ) Gently mix the supernatant with 1 ml of Ni-NTA agarose syrup at 4°C for 30 min.
( 10 ) Wash the column with 10 ml of 50 mmol/L HEPES containing 100 mmol/L potassium chloride and 5 mmol/L imidazole pH 7.4.
( 11 ) Elute with 1 ml of 50 mmol/L HEPES containing 100 mmol/L KCl and 100 mmol/L imidazole. It is not necessary to cut off the His-tag because it does not interfere with the fluorescent properties.
( 12 ) Dialyze the sample at 4°C with 2 L of 50 mmol/L HEPES containing 100 mmol/L KCl, pH 7.4.
3.2.2 Fluorescence spectroscopy
Changes in FRET efficiency (or, in the case of calcium chameleons, changes in Ca2+ concentration) can often be observed in terms of changes in the emission ratio (simply the peak intensity emitted by the acceptor divided by the peak intensity emitted by the donor).The dynamic range of the Ca2+ indicator is defined as the maximum ratio , Rmax , divided by the minimum ratio, Rmin. Larger dynamic ranges are more effective in in vivo Ca2+ indication.
( 1 ) Dilute the sample in a 1 ml tube of 50 mmol/L HEPES containing 100 mmol/L KCl, 20 μmol/L EGTA. Any dilution factor can be used as long as the emission signal remains measurable. Record the fluorescence emission spectrum from 450 to 570 nm under 433 nm excitation.
( 2 ) Record the fluorescence emission spectrum of a blank sample.
( 3 ) Subtract the recording in step (2) from the recording in step (1) to obtain the fluorescence spectrum of the calcium chameleon in the absence of Ca2+. Determine Rmin from this spectrum.
( 4 ) Repeat steps (1 ) to (3) in the presence of 1 mmol/L Ca2+ to determine the fluorescence spectrum of the calcium chameleon in the presence of Ca2+.
( 5 ) Figure 4.3 shows the fluorescence spectra of YC6.1 [ 5 ] before and after the addition of Ca2+, and the Rmax and Rmin are 1.1 and 2.4, respectively. 
3.2.3 Ca2+ binding properties
Ca2+ binding curves are used to assess the effective range of [ Ca2+ ] that can be measured by the Ca2+ indicator. Ca2+/EDTA and Ca2+/EGTA buffers are used as a concentration standard because at low [ Ca2+ ], Ca2+ contamination significantly distorts the blank [ Ca2+ ] level (see Note 7).
( 1 ) In a 1 ml tube, dilute the sample with EGTA. Record the fluorescence emission spectrum from 450 to 570 nm under 433 nm excitation. Determine the excitation ratio.
( 2 ) To obtain the Ca2+ binding curve, successive additions of calcium chloride solution were made to the sample and the emission ratio was determined. Since the experiments described in this chapter were done in EGTA and calcium chloride buffer at 20°C, the free calcium can be obtained by solving the following quadratic equation (see Ref. [ 9 ] for different conditions): 
( 3 ) To plot the Ca2+ binding curve, [ Ca2+ ] is plotted against the emission ratio. Figure 4.4 shows the Ca2+ binding curve for YC6.1. 
3.3 Imaging Ca2+ with a Calcium Chromatograph
This section describes simple experiments for Ca2+ imaging in HeLa cells; however, with minor corrections, this method can be applied to other adherent cells and physiological conditions. Cell culture preparation (see 4.3.3.1) and data acquisition (see 4.3.3.2) are described separately.
3.3.1 Cell culture preparation
( 1 ) Spread HeLa cells on a 35 mm diameter glass dish containing DMEM-10% FBS culture medium.
( 2 ) Conserve the cells at 37°C (5% carbon dioxide) until 50%~80% of the cells are confluent.
( 3 ) Using Genejuice ( Novagen), transfect the cells with a mammalian cell expression plasmid containing a calcium chameleon.
( 4 ) The transfection mix was removed after 6 h and replaced by 1.5 ml of DMEM-10%FBS culture medium.
( 5 ) The cells were incubated at 37°C (5% carbon dioxide) for 24 h. Cells were prepared for Ca2+ imaging.
4.3.3.2 Data Collection
Experiments should be done in a dark room to minimize background light. Figure 4.5 shows the microscopy equipment we used. 
( 1 ) Turn on the xenon lamp, microscope, filter converter and computer.
( 2 ) Remove the medium from the petri dish.
( 3 ) Wash with 1 ml HBSS ( + calcium chloride).
( 4 ) Add 1 ml fresh HBSS ( + calcium chloride).
( 5 ) Place the cells on the microscope stage.
( 6 ) Start the Meta Fluor software. The Meta Fluor software controls the shutter, filter converter and camera during data acquisition.
( 7 ) Pick YFP fluorescence with the eyepiece to find cells expressing calcium chameleons. Turn the filter indication to the U-WNIBA bandpass right-angle lens position and make the ND filter at 0.1% to 10% depending on the level of fluorescence emission.The U-WNIBA right-angle lens is useful for quickly spotting YFP-fluorescent cells using the eyepiece, while the 10% ND filter is used to prevent light fading.
( 8 ) Using a 40X oil objective, move the center of the microscopic field of view to a healthy-looking cell that exhibits strong cytoplasmic fluorescence. Figure 4.6 is an image showing the typical fluorescence distribution of healthy HeLa cells.
( 9 ) Capture images on a computer screen, frame by frame, using Meta Fluor, while adjusting the focus until the clearest image is obtained.
( 10 ) Turn the filter indicator to the CFP-YFP FRET rectangular lens position.
( 11 ) To monitor the peak excitation of CFP and YFP as a result of the peak excitation of CFP against time, the following data acquisition conditions were set: time interval greater than 10 s; exposure time 200 ms for both CFP and YFP; MetaFluor will show the change in emission ratio with time.
( 12 ) Select a region of interest on the CCD image. Usually this area covers the cells of interest. Then start data acquisition. The fluorescence emission intensity of CFP and YFP in the region of interest, together with their emission ratios, will be monitored in real time (see Note 8).
( 13 ) When the emission ratios have reached steady state, add 50 μl of 12 mmol/L histamine to the dish to achieve a final concentration of approximately 100 μmol/L. Be careful not to move the dish. Histamine binds to receptors on the plasma membrane, initiating a signaling cascade that causes Ca2+ to be released from the endoplasmic reticulum via the inosine-1,4,5-trisphosphate receptor [ 10, 11]. This causes a conformational change in the calcium chameleon, which is observed due to an increase in YFP emission intensity and a decrease in CFP intensity. Consequently, the emission ratio should increase. When the effect of histamine subsides, the emission ratio should decrease to the steady-state level. Figure 4.6 shows a screen shot of the Meta Fluor software during data acquisition. 
( 14 ) In order to correlate the emission ratio with the cytoplasmic body [ Ca2+ ], Rmin and Rmax must be measured in order to map the emission ratio to the Ca2+ binding curve. Add 50 μl of 20 μmol/L ionomycin to approximate a final concentration of 1 μmol/L. Ionomycin opens the channels at the plasma membrane to allow Ca2+ to percolate through. Due to saturation of the medium with calcium chloride, the emission ratio rises to Rmax. to determine Rmin, add 50 μl of 100 mmol/L EGTA and 600 μmol/L BAPTA-AM to approximate a final concentration of 5 mmol/L and 30 μmol/L, respectively. the emission ratio should drop to Rmin The emission ratio should drop to Rmin.
( 15 ) Stop data acquisition.