Name: functional characterization of endogenously expressed human ryr1 variants
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Duration: 7 min 59 s
Description: Watch this Scientific Journal Video about Functional Characterization of Endogenously Expressed Human RYR1 Variants at JoVE.com

The work described in this manuscript was supported by grants from the Swiss National Science Foundation (SNF) and the Swiss Muscle Foundation.

The protocols described below comply with the ethics guidelines of the Ethikkommission Nordwest- und Zentralschweiz EKNZ.
1. Preparation of Epstein Barr immortalized B-lymphocyte cell lines11
<ol>
	<li>After informed consent, collect 30 mL of whole blood in EDTA-treated sterile tubes from the proband carrying a RYR1 mutation and from healthy family members with no mutation. 
	NOTE: Keep all solutions sterile and work in a tissue culture hood.</li>
	<li>Isolate mononuclear cells from whole blood by density gradient centrifugation media (e.g., Ficoll-Hypaque, .077 g/L).
	<ol>
		<li>Place 30 mL of sterile blood in a 50 mL conical sterile tube.</li>
		<li>Place the tip of a Pasteur pipette containing the density gradient centrifugation media at the bottom of tube and layer 20 mL sterile of density gradient centrifugation media solution slowly underneath the blood.</li>
		<li>Centrifuge for 30 min at 900 x g at 18&#176;-20&#176;C, with no break. 
		&#8203;NOTE: The mononuclear cell layer appears as a cloudy ring at the interphase between the density gradient centrifugation media layer and the top layer containing platelet enriched plasma.</li>
	</ol>
	</li>
	<li>With a sterile pipette gently remove the interphase layer containing mononuclear cells (approximately 3-5 mL) and transfer the solution to a clean 50 mL sterile conical tube.</li>
	<li>Add 20 mL of phosphate buffer saline (PBS) to rinse cells, centrifuge for 10 min at 600 x g at room temperature and resuspend the pellet in PBS. Repeat for a total of three times; this ensures that all the media is removed.</li>
	<li>After the last wash, resuspend cells in 1-2 mL of tissue culture medium (RPMI medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 100 units of penicillin and streptomycin). Place mononuclear cells (approximately 1 x 106 cells) in a T125 tissue culture flask containing 20 mL of tissue culture medium.</li>
	<li>Infect mononuclear cells with Epstein-Barr virus.
	<ol>
		<li>Use supernatants from B95.8 cell line cultures (containing 102-103 transforming units/mL stocked at -80 &#176;C) as a source of EBV.</li>
		<li>Resuspend 1 x 106 mononuclear cells from step 1.5 in 20 mL of tissue culture medium and expose them to 2 mL of supernatant from the B95.8 cell line in the presence of cyclosporin A (0.2 &#181;g/mL final concentration) for infection.</li>
	</ol>
	</li>
	<li>Place the flask in a 37 &#176;C cell culture incubator and allow the cells to grow. After one week change the culture medium. 
	NOTE: Once B-cells start proliferating they form recognizable clumps and grow rapidly so that the cultures can be expanded and frozen.</li>
	<li>Extract the genomic DNA from the EBV immortalized B-lymphocyte cell lines11 to confirm the presence or absence of the given mutation.</li>
</ol>
2. Intracellular Ca2+ measurements
NOTE: Changes in the intracellular calcium concentration of the EBV-transformed B-lymphocyte cell lines can be monitored in cell populations, with a spectrofluorometer equipped with a magnetic stirrer and cuvette holder set to 37 &#176;C. Alternatively Ca2+ changes can be monitored in single cells by fluorescence microscopy. In both cases cells are removed from the tissue culture flask, washed twice with Krebs Ringer&#39;s solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 20 mM HEPES, 1 mM NaHPO4, 5.5 mM glucose, pH 7.4 containing 1 mM CaCl2) and counted .
<ol>
	<li>For experiments in cell populations using a spectrofluorometer11,13,14
	<ol>
		<li>Resuspend cells at a final concentration of 1 x 107 cells/ mL in Krebs Ringer&#39;s solution and incubate at 37&#176;C for 30 min with a final concentration of 5 &#181;M Fura-2/AM.</li>
		<li>Centrifuge cells at 900 x g for 10 min and resuspend them in Krebs Ringer&#39;s solution at a concentration of 2 x 106 cells/mL.</li>
		<li>Measure fluorescence changes (ratio 340/380 nm) using a spectrofluorometer equipped with a magnetic stirrer set at maximal velocity and set to 37 &#176;C.</li>
		<li>Just before the experiment spin cells at 900 x g for 5 min in a microcentrifuge and quickly resuspend the pellet in 1.5 mL of Krebs Ringer&#39;s solution with 0.5 mM EGTA but no added Ca2+.</li>
		<li>Place cells in a 3 mL glass spectrofluorometer cuvette and record the fluorescence ratio (340 nm/380 nm excitation, 510 nm emission).</li>
		<li>Achieve a stable base line (approximately 30 seconds), add the selected concentration of RyR1 agonist (4-chloro-m-cresol, or 4-cmc) and record the calcium transient. 
		&#8203;NOTE: A 300 mM stock solution of 4-cmc made in DMSO is use used as a starting reagent. This solution can be made in advance, aliquoted and stored at -20 &#176;C for several months.</li>
		<li>Perform experiments for different 4-cmc concentrations. 
		NOTE: Include 75 &#181;M, 150 &#181;M, 300 &#181;M, 450 &#181;M, 600 &#181;M, 750 &#181;M to 1 mM to generate a dose response curve of agonist versus change in [Ca2+]. The different 4-cmc concentrations are obtained by adding the appropriate volume of 4-cmc from the stock solution, directly into the cuvette containing the Fura-2 loaded cells. For example, for a final concentration of 300 &#181;M 4-cmc, 1.5 &#181;L of the stock solution are added to the cuvette containing 1.5 mL of cells in Krebs Ringer&#39;s solution. For lower agonist concentrations, the 300 mM stock solution should be diluted to 75 mM with DMSO and the appropriate volume added to the cuvette containing 1.5 mL of cells in Krebs Ringer&#39;s solution.</li>
		<li>Add 400 nM thapsigargin to cells to calculate the total amount of Ca2+ present in intracellular stores. Record the peak Ca2+.</li>
		<li>Plot the peak calcium induced by a given 4-cmc concentration versus the peak calcium induced by thapsigargin, which is considered 100% and construct a 4-cmc dose response curve comparing cells from a proband and healthy relative</li>
	</ol>
	</li>
	<li>For experiments on single cells13
	<ol>
		<li>Dilute poly-L-lysine 1:10 in sterile H2O and pre-treat the glass coverslips for 30 min. Allow to air-dry under a sterile tissue culture hood.</li>
		<li>Re-suspend EBV-transformed B-lymphocytes to a final concentration of 1 x 106 cells/mL in Krebs Ringer&#39;s solution containing 1 mM CaCl2 and add a final concentration of 5 &#181;M Fura-2/AM.</li>
		<li>Place 1 mL of cells on the poly-L-lysine treated coverslips and incubate at 37 &#176;C in a humidified cell culture incubator for 30 min to allow the EBV cells to stick to the glass coverslip during loading.</li>
		<li>Place the coverslip in the perfusion chamber and start perfusion (at a rate of 2 mL/min) with Krebs Ringer&#39;s solution containing 1 mM Ca2+.</li>
		<li>Use an inverted fluorescent microscope (equipped with a 40x oil-immersion objective (0.17 numerical aperture), filters (BP 340/380, FT 425, BP 500/530) to record on-line measurements, with a software-controlled charge coupled device (CCD) camera attachment.</li>
		<li>Acquire Images at 1 s intervals at a fixed exposure time (100 ms for both 340- and 380-nm excitation wavelengths. Use imaging software to analyse changes in fluorescence. Measure the average pixel value for each cell at excitation wavelengths of 340 and 380 nm13.</li>
		<li>To achieve cell stimulation, use a cell perfusion stimulator with 12 valves and add different concentrations of 4-cmc. The flush valve contains Krebs Ringer&#39;s solution with no added Ca2+ plus 100 &#181;M La3+ to monitor calcium release from intracellular stores only.</li>
		<li>Construct a dose response curve of 4-cmc versus change in [Ca2+], as described above.</li>
	</ol>
	</li>
</ol>
3. Preparation of human myotubes from muscle biopsies10,12,15
NOTE: Different methods have been used by different laboratories to obtain satellite cell-derived myoblasts and myotubes. Below is the description of the method used in Basel.
<ol>
	<li>Rinse muscle biopsy with sterile PBS to remove excess blood and cut into small fragments of about 0.5-1 mm.</li>
	<li>Prepare 6 well tissue culture dishes with insert. Add 1.5 mL of human muscle growth medium to each well and 0.5 mL of human muscle growth medium to each insert. 
	NOTE: The growth medium is made up as follows: 500 mL of Dulbecco&#39;s modified Eagle&#39;s medium with high glucose, or DMEM (4.5 mg/mL), containing 10% horse serum, 5 ng/mL insulin, 3 mM glutamine, 600 ng/mL penicillin G and streptomycin, and 7 mM HEPES, pH 7.4. Commercially available skeletal muscle growth medium can also be used.</li>
	<li>Place 2-3 small muscle fragments into each insert (Figure 1A) and place culture dishes into the cell culture incubator (5% CO2, 37 &#176;C). After approximately 8-10 days satellite cells can be seen growing out of and surrounding the muscle biopsy, attached to the insert (Figure 1, B and C, arrows).</li>
	<li>Release a sufficient number of cells from the biopsy (after approximately 10-14 days), trypsinize as follows:
	<ol>
		<li>Remove all culture medium, rinse the cells once with 1 mL of PBS, add 0.5 mL of trypsin/EDTA solution (0.025% trypsin and 0.01% EDTA) and incubate at 37 &#176;C for 5 min.</li>
		<li>Add 1 mL of growth medium to the cells to neutralize the effect of trypsin and transfer the satellite cells into a new T25 cell culture flask; add 3 mL of growth medium and place the cells in a cell culture incubator (5% CO2, 37 &#176;C).</li>
		<li>Change the growth medium the next day to remove the EDTA and subsequently change the medium once per week.</li>
	</ol>
	</li>
	<li>When myoblasts are approximately 75% confluent, trypsinize and transfer them onto laminin-treated glass coverslip. 
	NOTE: As a proportion, cells growing in one T25 flask should be transferred onto one 43 mm diameter laminin-treated glass coverslip. The glass coverslip should be placed within a 60 mm diameter tissue culture plate containing 3 mL of growth medium.</li>
	<li>Grow cells on the glass coverslip in growth medium in a cell culture incubator (5% CO2, 37 &#176;C) changing the medium once per week. At 90% confluency, switch to differentiation medium made up as follows: high glucose DMEM (4.5 mg/mL), 0.5% bovine serum albumin, 10 ng/mL epidermal growth factor, 0.15 mg/mL creatine, 5 ng/mL insulin, 200 mM glutamine, 600 ng/mL penicillin G and streptomycin, and 7 mM HEPES, pH 7.4). Commercially available differentiation medium may also be used. Change the differentiation medium once per week.</li>
	<li>After 7-10 days in differentiation medium, multinucleated myotubes are visible. Assess for changes in [Ca2+] within one week, as described below.</li>
</ol>
4. [Ca2+]i ratio measurements determined with Fura-2
<ol>
	<li>Load glass coverslip grown myotubes with Fura-2/AM (final concentration of 5 &#181;M) diluted in DMEM for 30 min at 37 &#176;C. Briefly, remove the differentiation medium from the glass coverslip grown cells and add 2 mL fresh differentiation medium. Add 10 &#181;L of Fura-2 AM from a stock solution of 1 mM and incubate 30 min in a cell culture incubator (5% CO2, 37 &#176;C).</li>
	<li>Transfer glass coverslip to the perfusion chamber and rinse cells with Krebs Ringer&#39;s solution containing 2 mM CaCl2.</li>
	<li>Perform on-line [Ca2+] measurements as described above in the EBV single cell section with the use a 20x water immersion FLUAR objective (0.17 numerical aperture).</li>
</ol>

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The authors have nothing to disclose.

The protocols described in this paper have been successfully utilized by several laboratories to study the impact of RYR1 mutations on calcium homeostasis. The critical steps of the approaches outlined in this paper deal with sterility, cell culturing skills and techniques and availability of biological material. In principle, the use of EBV-immortalized B lymphocytes is simpler and allows one to generate cell lines containing mutant RyR1 channels. The cells can be frozen and stored in liquid nitrogen for many years and cultures can be re-started at any time. Additionally, one can chose whether to monitor calcium homeostasis in cell populations or at the single cell level. The former method is simpler, does not require a fluorescence microscope and allows the investigator to test cell lines generated from different individuals within a short period of time. The limitation being the velocity of cell growth and the availability of a fully equipped (heated and with magnetic stirrer) spectrofluorometer. As an alternative approach flow cytometry in combination with fluorescent calcium indicators can be used to measure calcium fluxes in EBV-immortalized B lymphocytes; in such a way changes in the intracellular calcium concentration can be determined19. If a fluorescent microscope, perfusion chamber and microperfusion system are available, then single cell imaging has the advantage of being more sensitive and giving more detailed information including cell to cell variability, kinetic analysis and identification of subcellular domains involved in calcium release. The latter approach is technically more challenging and requires more equipment.
There are multiple advantages to using EBV-immortalized B lymphocytes, including that other parameters aside [Ca2+]i homeostasis can be measured. For example 4-chloro-m-cresol induced acidification of B cells has been used to differentiate cells from control individuals from MHS patients20,21. Nevertheless one must keep in mind (i) that B cells do not express many of the proteins involved in skeletal muscle excitation contraction coupling that may indirectly influence calcium release, (ii) they are non-excitable cells thus cannot be activated physiologically by plasma membrane depolarization, and finally a report by Monnier et al. found that a mutation identified in a patient was not expressed in EBV-immortalized B cells because of a cryptic splice site22.
Patient derived primary muscle cell cultures have been used by several groups interested in studying the effect of mutations in different genes encoding proteins involved in calcium homeostasis10,23,24. These cells can be differentiated into multinucleated myotubes, respond to plasma membrane depolarization and can be assessed by electrophysiological means. In addition, they can be immortalized in order to obtain cell lines carrying RYR1 mutations25, though the procedure is far more complex than for B-lymphocytes. While it is also true that the muscle cells are slow growing and the time necessary from taking a biopsy to having a sufficient number of cells can be more than one month, it is also possible to store the small pieces of muscle biopsies in freezing medium in liquid nitrogen in order to obtain myoblasts years later. The long culturing times have the added risk of contamination by bacteria, yeasts or molds and as soon as a sufficiently large number of myoblasts have been obtained, it is important to freeze and store them in liquid nitrogen. Our laboratory has successfully applied the same technique outlined above for myotubes, to study calcium changes in human skin-derived fibroblasts transduced with myoD and differentiated into myotubes26. This was done to study the functional effect of RYR1 mutations when muscle biopsy-derived myoblasts were not available. As for B-lymphocytes, 4-chloro-m-cresol induced acidification of myotubes from patients with RYR1 mutations linked to MHS has been tested successfully27.
In conclusion, the use of biological material from patients to study the genotype-phenotype correlation has many advantages the drawback and can be used successfully to study the effect of mutations in RYR1. When using this approach however, it is important to keep in mind that mutations present in other genes may influence calcium homeostasis; therefore, the use of cells from different families harboring the same mutation as well as from family members not harboring the RYR1 mutation as controls, should be performed.

To date more than 700 RYR1 variants have been identified in the human population and linked to various neuromuscular disorders including malignant hyperthermia susceptibility (MHS), exercise induced rhabdomyolysis, central core disease (CCD), multi-minicore disease (MmD), centronuclear myopathy (CNM)1,2,3; nevertheless, studies to characterize their functional effects are lagging and only approximately 10% of mutations have been tested functionally. Different experimental approaches can be used to assess the impact of a given RyR1 variant, including transfection of heterologous cells such as HEK293 and COS-7 cells with plasmid encoding for the WT and mutant RYR1 cDNA4,5, transduction of dyspedic mouse fibroblasts with plasmids and vectors encoding for the WT and mutant RYR1 cDNA, followed by transduction with myo-D and differentiation into myotubes6, generation of transgenic animal models carrying mutant RyR1s7,8,9, characterization of cells from patients expressing the RYR1 variant endogenously10,11,12. Such methods have helped established how different mutations functionally impact the RyR1 Ca2+ channel.
Here, methods developed to assess the functional effects of RYR1 mutations are described. Various parameters of intracellular calcium homeostasis are investigated in human cells endogenously expressing the RyR1 calcium channel, including myotubes and Epstein Barr Virus (EBV) immortalized B-lymphocytes. Cells are obtained from patients, expanded in culture and loaded with ratiometric fluorescent calcium indictors such as Fura-2 or indo-1. Parameters which have been reported to be altered because of pathogenic RYR1 mutations including the resting [Ca2+], the sensitivity to different pharmacological agonists and the size of the intracellular Ca2+ stores are measured either at the single cell level, using fluorescence microscopy, or in cell populations using a fluorimeter. Results obtained in cells from mutation carriers are then compared to those obtained from healthy control family members. This approach has demonstrated that: (i) many mutations linked to MHS lead to an increase in the resting [Ca2+] and a shift to the left in the dose response curve to either KCl-induced depolarization or pharmacological RyR1 activation with 4-chloro-m-cresol10,11,12,13; (ii) mutations linked to CCD lead to a decrease in the peak [Ca2+] released by pharmacological activation of the RyR1 and decreased size if the intracellular Ca2+ stores12,13,14,15; (iii) some variants do not impact Ca2+ homeostasis13. Advantages of this experimental approach are: the RyR1 protein is not over-expressed and physiological levels are present, cells can be immortalized (both muscle cells and B-lymphocytes) providing cell lines containing mutations. Some disadvantages relate to the fact that patients may carry mutations in more than one gene encoding proteins involved in calcium homeostasis and/or excitation contraction coupling (ECC) and this may complicate experimental conclusions. For example, two JP-45 variants were identified in the MHS and control population and their presence were shown to impact the sensitivity of the dihydropyridine receptor (DHPR) to activation16. Patients need to be available, biological material needs to be freshly collected and ethical permits need to be obtained from the local ethical boards.

<table><tbody><tr><td>4-chloro-m-cresol</td><td>Fluka</td><td>24940</td><td></td></tr><tr><td>Blood collection tubes</td><td>Sarstedt</td><td>172202</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>caffeine</td><td>Merk</td><td>102584</td><td></td></tr><tr><td>Cascade 125+ CCD camera</td><td>Photometrics</td><td></td><td></td></tr><tr><td>Cascade 128+ CCD</td><td>Photometrics</td><td></td><td></td></tr><tr><td>Creatine</td><td>Sigma-Aldrich</td><td>C-3630</td><td></td></tr><tr><td>DMEM</td><td>ThermoFisher Scientific</td><td>11965092</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>41639</td><td></td></tr><tr><td>EGTA</td><td>Fluka</td><td>3778</td><td></td></tr><tr><td>Epidermal Growth Factor (EGF)</td><td>Sigma-Aldrich</td><td>E9644</td><td></td></tr><tr><td>Ficoll Paque</td><td>Cytiva</td><td>17144002</td><td></td></tr><tr><td>Foetal calf serum</td><td>ThermoFisher Scientific</td><td>26140079</td><td></td></tr><tr><td>Fura-2/AM</td><td>Invitrogen Life Sciences</td><td>F1201</td><td></td></tr><tr><td>Glutamax</td><td>Thermo Fisher Scientific</td><td>35050061</td><td></td></tr><tr><td>HEPES</td><td>ThermoFisher Scientific</td><td>15630049</td><td></td></tr><tr><td>Horse serum</td><td>Thermo Fisher Scientific</td><td>16050122</td><td></td></tr><tr><td>Insulin</td><td>ThermoFisher Scientific</td><td>A11382II</td><td></td></tr><tr><td>Ionomycin</td><td>Sigma</td><td>I0634</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Laminin</td><td>ThermoFisher Scientific</td><td>23017015</td><td></td></tr><tr><td>Lanthanum</td><td>Fluka</td><td>61490</td><td></td></tr><tr><td>Microperfusion system</td><td>ALA-Scientific</td><td>DAD VM 12 valve manifold</td><td></td></tr><tr><td>Origin Software</td><td>OriginLab Corp</td><td>Software</td><td></td></tr><tr><td>Pennicillin/Streptomycin</td><td>Gibco Life Sciences</td><td>15140-122</td><td></td></tr><tr><td>Perfusion chamber POC-R</td><td>Pecon</td><td>000000-1116-079</td><td></td></tr><tr><td>poly-L-lysine</td><td>Sigma-Aldrich</td><td>P8920</td><td></td></tr><tr><td>RPMI</td><td>ThermoFisher Scientific</td><td>21875091</td><td></td></tr><tr><td>Spectrofluorimeter</td><td>Perkin Elmer</td><td>LS50</td><td></td></tr><tr><td>Thapsigargin</td><td>Calbiochem</td><td>586005</td><td></td></tr><tr><td>Tissue culture dishes</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>Tissue culture flask</td><td>Falcon</td><td>353107</td><td></td></tr><tr><td>Tissue culture inserts</td><td>Falcon</td><td>353090</td><td></td></tr><tr><td>Trypsin/EDTA solution</td><td>ThermoFisher Scientific</td><td>25300054</td><td></td></tr><tr><td>Visiview</td><td>Visitron Systems GmbH</td><td>Software</td><td></td></tr><tr><td>Zeiss Axiovert S100 TV microscope</td><td>Carl Zeiss AG</td><td></td><td></td></tr><tr><td>Zeiss glass coverslips</td><td>Carl Zeiss AG</td><td>0727-016</td><td></td></tr></tbody></table>

functional characterization of endogenously expressed human ryr1 variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

[Ca2+]i measurements in populations of EBV-immortalized B lymphocytes 
Primary B-lymphocytes express the RyR1 isoform that functions as a Ca2+ release channel during B cell antigen receptor stimulated signaling processes17. Immortalization of B-cells with EBV, a procedure routinely used by geneticists to obtain cell lines containing genomic information of patients, provides the advantage of generating cell lines that express mutant RyR1 Ca2+ channels in patients harboring RYR1 mutations11,13. [Ca2+]i changes brought about by the addition of specific RyR1 agonists such as 4-chloro-m-cresol18 and caffeine can be easily monitored in order to establish whether a given RYR1 mutation alters the sensitivity to an agonist, the amount of calcium released, the resting [Ca2+], or other parameters that have shown to be impacted by mutations. [Ca2+]i changes can be monitored either in populations of Fura-2 loaded cells in suspension with a spectrofluorometer or on groups of cells attached to glass coverslips and examined by epifluorescence. Figure 2 shows a representative experiment carried out on cell suspensions. Immediately before being placed in the cuvette, cells were spun to remove Fura-2 that may have leaked out; cells were then resuspended to a final concentration of 1 x 106 cells/mL in warm (37&#176;C) Krebs Ringer&#39;s solution containing no additional Ca2+ plus 0.5 mM EGTA and placed in the spectrofluorometer. The magnetic stirrer was switched on to position 4 (the highest position) to keep cells in suspension and fluorescence was recorded. After a stable trace was obtained, the selected agonist was added (in Figure 2A this was 300 &#181;M 4-chloro-m-cresol) leading to a rapid Ca2+ increase which then slowly declined back to resting levels. The transient nature of the change in fluorescence is important as it indicates (i) that it is not an artefact caused for by the addition of a fluorescent or quenching compound, (ii) that it is not due to calcium binding to extracellular Fura-2 and (iii) that the cells are healthy and can actively remove calcium from their cytoplasm.
The same experiment needs to be repeated several times to be analyzed statistically. For each cell line and each day, the experiments are carried out and the total amount of rapidly releasable calcium in the stores needs to be determined by adding the SERCA inhibitor thapsigargin11,13,14. As shown in Figure&#160;2B, the addition of 400 nM thapsigargin causes a large calcium transient reaching a peak fluorescence value of 2.4 arbitrary units (a.u.); thus the total amount of calcium that can be released from the intracellular stores of the EBV-immortalized B- cells shown in Figure 2B equals 2.4 a.u. (thapsigargin peak) - 1.45 a.u. (resting ratio) or 0.95 This fluorescence value was considered 100% when constructing the dose response curve shown in Figure&#160;2C.
Single cell [Ca2+]i measurements in EBV-immortalized B lymphocytes 
This second approach relies on the availability of a fluorescence microscope and microperfusion set up allowing the stimulation of a single cell or small groups of cells with a given concentration of agonist and the simultaneous recording of the fluorescence changes. The syringes of the microperfusion system are loaded with different concentrations of the selected RyR1 agonist (either caffeine or 4-chloro-m-cresol) which will be used to generate dose response curves. In the example shown in Figure 3, cells were stimulated with 0.5-10 mM caffeine dissolved in Krebs Ringer&#39;s solution containing no added calcium plus 100 &#181;M La3+ in order to monitor Ca2+ release from intracellular stores. Glass coverslips on which Fura-2 loaded EBV-immortalized B lymphocytes were allowed to attach are placed in the perfusion chamber and perfused with Krebs Ringer&#39;s solution containing 1 mM Ca2+. Most of the cells will have adhered to the poly-L-lysine treated coverslip and small groups of cells should be identified and checked for Fura-2 loading. The tip of the perfusion system is placed close to the cells to bathe them (and not all the cells on the coverslip) with caffeine. Normally cells are stimulated starting from the lowest to the highest concentration of caffeine; for each concentration, a new cell or group of cells are selected. Ratiometric fluorescence measurements (excitation at 340 nm and 380 nm, emission at 510 nm) are recorded every second for up to 2 min. A few images are obtained before perfusion in order to obtain a steady baseline, subsequently cell perfusion is initiated, first by flushing cells with a solution of Krebs Ringer&#39;s solution containing 100 &#181;M La3+ for 5 seconds. This should not result in a change in florescence and is a control to assure that the cell(s) stick to the coverslip throughout the experiment; subsequently a solution containing the selected agonist concentration is flushed over the cell(s). In the example shown in Figure&#160;3A, the cells were stimulated with 5 mM caffeine for 20 seconds. The arrow shown indicates when the caffeine valve was opened and this results in an immediate increase in the 340/380 nm fluorescent ratio. After 20 seconds, the caffeine valve closes, and cells are flushed with Krebs Ringer&#39;s solution containing 100 &#181;M La3+; fluorescence is recorded until the baseline is reached. For each caffeine concentration the &#916;F, that is the caffeine induced peak 340/380 nm fluorescence - the initial resting 340/380 nm fluorescence is calculated and used to construct a dose response curve as shown in Figure&#160;3B. Mean &#916;F from 5-10 cells is averaged for each caffeine concentration. The amount of calcium in intracellular stores can also be monitored. In this case, cells are rinsed with Krebs Ringer&#39;s solution containing 0.5 mM EGTA and a solution of 1 &#181;M thapsigargin, 1 &#181;M ionomycin and 0.5 mM EGTA is added by hand to the cells (not through the microperfusion system as ionomycin sticks to the tubing and cannot be washed off) and the fluorescence is recorded for approximately 4-5 min. To calculate the &#916;F, a region of interest (ROI) outlining a cell is obtained and the changes in fluorescence within the ROI are calculated using an imaging software.
In summary, when using EBV immortalized B-cells to measure the sensitivity of the RyR1 to a specific agonist, repeated experiments should be performed on cells from one individual, on different days. For measurements on cell populations, the status of the intracellular calcium stores needs to be assessed and the EC50 to agonist induced calcium release is plotted relative to the total amount of calcium that can be released from the stores. One advantage of using this approach is that the [Ca2+] response of millions of cells is averaged; additionally, no microperfusion system and fluorescent microscopes are necessary. The single cell method allows the use of a smaller number of cells and the on-line visualization of changes in [Ca2+] of selected cells.
Single cell [Ca2+]i measurements in human satellite cell derived myotubes 
Glass coverslip grown and differentiated myotubes are loaded with Fura-2, transferred to the perfusion chamber and bathed in Krebs Ringer&#39;s solution containing 2 mM Ca2+ as described above for EBV cells. The syringes of the perfusion system are filled with the selected agonist and myotubes are stimulated as described above. Since not all cells on the coverslip are multinucleated myotubes, it is important to select the appropriate cell(s) that will be measured. Small groups of myotubes can be stimulated simultaneously. In the example shown in Figure&#160;4, a single myotube was flushed with a solution containing KCl, different concentrations of 4-chloro-m-cresol and finally caffeine, however, normally dose response curves to agonists are constructed, as indicated in the previous section, in order to compare the agonist sensitivity of cells from different individuals12,15,16. KCl is used as a way to depolarize the plasma membrane. In skeletal, muscle plasma membrane depolarization is sensed by the voltage sensing DHPR which thereby undergoes a conformational change leading to activation and opening of the RyR1. On the other hand, 4-chloro-m-cresol and caffeine are direct pharmacological activators of the RyR1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62196/62196fig01.jpg" /> 
Figure 1: Generation of primary human muscle biopsy-derived myoblast cultures. (A) Small fragments of muscle (arrows) are placed in inserts containing 0.5 mL growth medium. After 7-14 days satellite cells can be seen (small arrows) adjacent to the muscle tissue and growing on the bottom of the insert. Image taken through a 10x objective (B) and 20x objective (C). The grey boxes in panel A were used to cover the identity of the patient. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62196/62196fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62196/62196fig02.jpg" /> 
Figure 2: Calcium release experiments in EBV immortalized B lymphocytes. Ratiometric [Ca2+]i measurements in a population of Fura-2 loaded cells in suspension. A) The addition of 300 &#181;M 4-chloro-m-cresol (arrow) causes an immediate increase in the cytoplasmic [Ca2+], which subsequently decays back to resting levels within 500 seconds. In this example the &#916;F induced by the addition of 300 &#181;M 4-chloro-m-cresol is 1.7 fluorescence units- 1.4 fluorescence units = 0.3 fluorescence units. B) The addition of the SERCA inhibitor thapsigargin (400 nM, arrow) causes a larger increase in the Fura-2 fluorescence ratio which peaks at 2.4 fluorescence units, and subsequently decays to resting levels at 1.45 fluorescence units. The thapsigargin induced [Ca2+] transient represents the total amount of rapidly releasable calcium in the intracellular stores present in the cell population. The peak transient obtained (2.4-1.45= 0.95 units) is used to calculate the percentage of calcium released by a given concentration of 4-chloro-m-cresol. C) Representative dose response curves correlating the &#916;F as a percentage of the total amount of rapidly releasable calcium in intracellular stores. For 300 &#181;M 4-chloro-m-cresol this value is 0.3/0.95x100=31.6%. Each symbol represents the mean&#177; SEM% of 5-10 values from EBV immortalized cells from a control (closed circles, dotted line) and an MHS individual (closed squares, continuous line) carrying a RYR1 mutation. The curves were generated using a sigmoidal dose-response curve function. Panels A and B are adapted from Girard et al.11. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62196/62196fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62196/62196fig03.jpg" /> 
Figure 3: Caffeine-induced Ca2+ release in individual EBV-immortalized B lymphocytes from a control individual. A) Top panels, time lapse images&#160;(A) Phase-contrast; (B-E), single-cell [Ca2+]I measurements of fura-2-loaded EBV-immortalized lymphocytes: (B) t=0, (C) t=36 s, (D) t=50 s and (E) t=77 s after the application of 5 mM caffeine. Cells were individually stimulated by the addition of caffeine diluted in Krebs-Ringer solution. Scale bar=10 &#181;m. Bottom panel,&#160;representative trace obtained after stimulation of a single cell with 5 mM caffeine (arrow). B) Dose response curves showing the caffeine-dependent change in [Ca2+]i, expressed as change in fluorescence ratio (peak ratio 340/380 nm&#8722;resting ratio 340/380 nm). Each point represents the mean &#177; SEM of the change in fluorescence of 4-15 cells. The curves were generated using a sigmoidal dose-response curve function. Closed squares, dotted line, control cells; closed triangles, continuous line, MHS individual carrying a RYR1 mutation. This figure is an adaptation from Ducreux et al.13. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62196/62196fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62196/62196fig04.jpg" /> 
Figure 4: Calcium release stimulated by KCl, 4-chloro-m-cresol and caffeine in human myotubes from a control individual. Left panels: A) phase contrast (B-H), single cell intracellular Ca2+ measurements of Fura-2-loaded human myotubes. B) resting [Ca2+]i ; C) t=2 s after the application of 150 mM KCl. D) t= 2 s after the application of 150 &#181;M 4-chloro-m-cresol; E) t=2 s after the application of 300 &#181;M 4-chloro-m-cresol; F) t=2 s after the application of 600 &#181;M 4-chloro-m-cresol; G) t=2 s after the application of 10 mM caffeine; H) t=20 s after the application of caffeine. Right panel: Plot of time (s) versus fluorescence ratio (340/380 nm) in the stimulated cell. Myotubes were individually stimulated by addition of the agonist in Krebs-Ringer buffer containing 100 &#181;M La3+, thus the increase in [Ca2+]i represents only release of calcium from intracellular stores. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62196/62196fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Functional Characterization of Endogenously Expressed Human RYR1 Variants at JoVE.com

Functional Characterization of Endogenously Expressed Human RYR1 Variants

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

functional-characterization-endogenously-expressed-human-ryr1

Universidad San Sebastian

Research

JoVE Journal

Medicine

2.4K Views.  Basel University Hospital. Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described. 

Video: Functional Characterization of Endogenously Expressed Human RYR1 Variants

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Genetics

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Cancer Research

Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is pathogenic, one must query many databases to determine whether the gene of interest is linked to a genetic disease, whether the specific variant has been reported before, and what functional data is available in model organism databases that may provide clues about the gene&#8217;s function in human. MARRVEL (Model organism Aggregated Resources for Rare Variant ExpLoration) is a one-stop data collection tool for human genes and variants and their orthologous genes in seven model organisms including in mouse, rat, zebrafish, fruit fly, nematode worm, fission yeast, and budding yeast. In this Protocol, we provide an overview of what MARRVEL can be used for and discuss how different datasets can be used to assess whether a variant of unknown significance (VUS) in a known disease-causing gene or a variant in a gene of uncertain significance (GUS) may be pathogenic. This protocol will guide a user through searching multiple human databases simultaneously starting with a human gene with or without a variant of interest. We also discuss how to utilize data from OMIM, ExAC/gnomAD, ClinVar, Geno2MP, DGV and DECHIPHER. Moreover, we illustrate how to interpret a list of ortholog candidate genes, expression patterns, and GO terms in model organisms associated with each human gene. Furthermore, we discuss the value protein structural domain annotations provided and explain how to use the multiple species protein alignment feature to assess whether a variant of interest affects an evolutionarily conserved domain or amino acid. Finally, we will discuss three different use-cases of this website. MARRVEL is an easily accessible open access website designed for both clinical and basic researchers and serves as a starting point to design experiments for functional studies.

Here, we present a protocol to access and analyze many human and model organism databases efficiently. This protocol demonstrates the use of MARRVEL to analyze candidate disease-causing variants identified from next-generation sequencing efforts.

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is ...

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest control. To date, several diamide insecticides targeting pest RyRs have been commercialized, which generate annual revenue of 2 billion U.S. dollars. But comprehension of the mode of action of RyR-targeting insecticides is limited by the lack of structural information regarding insect RyR. This in turn restricts understanding of the development of insecticide resistance in pests. The diamondback moth (DBM) is a devastating pest destroying cruciferous crops worldwide, which has also been reported to show resistance to diamide insecticides. Therefore, it is of great practical importance to develop novel insecticides targeting the DBM RyR, especially targeting a region different from the traditional diamide binding site. Here, we present a protocol to structurally characterize the N-terminal domain of RyR from DBM. The x-ray crystal structure was solved by molecular replacement at a resolution of 2.84 &#197;, which shows a beta-trefoil folding motif and a flanking alpha helix. This protocol can be adapted for the expression, purification and structural characterization of other domains or proteins in general.

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

In this article, we describe the protocols of protein expression, purification, crystallization and structure determination of the N-terminal domain of&#160;ryanodine receptor from diamondback moth (Plutella xylostella).

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest ...

Biochemistry

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Chemistry

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

Developmental Biology

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Biology

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

The increased use of sequencing in medicine has identified millions of coding variants in the human genome. Many of these variants occur in genes associated with neurodevelopmental disorders, but the functional significance of the vast majority of variants remains unknown. The present protocol describes the study of variants for Ube3a, a gene that encodes an E3 ubiquitin ligase linked to both autism and Angelman syndrome. Duplication or triplication of Ube3a is strongly linked to autism, whereas its deletion causes Angelman syndrome. Thus, understanding the valence of changes in UBE3A protein activity is important for clinical outcomes. Here, a rapid, cell-based method that pairs Ube3a variants with a Wnt pathway reporter is described. This simple assay is scalable and can be used to determine the valence and magnitude of activity changes in any Ube3a variant. Moreover, the facility of this method allows the generation of a wealth of structure-function information, which can be used to gain deep insights into the enzymatic mechanisms of UBE3A.

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

A simple and scalable method was developed to assess the functional significance of missense variants in Ube3a, a gene whose loss and gain of function are linked to both Angelman syndrome and autism spectrum disorder.

The increased use of sequencing in medicine has identified millions of coding variants in the human genome. Many of these variants occur in genes ...

Neuroscience

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

Summary

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Chapters in this video

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Yeast As a Chassis for Developing Functional Assays to Study Human P53

Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study