Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Podsumowanie

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

Streszczenie

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Wprowadzenie

The endoplasmic reticulum (ER) functions in many cellular capacities including protein folding, protein secretion, lipid homeostasis, and intracellular signaling¹. Central to normal ER function is maintaining luminal calcium concentrations at ~5,000 times those found in the cytoplasm^2-4. This energy intensive process is regulated by the sarco/endoplasmic reticulum calcium ATPase (SERCA), a pump that moves calcium ions into the ER. Efflux of calcium from the ER is mediated primarily by the ryanodine (RyR) and inositol triphosphate (IP3R) receptors. Because many ER processes are dependent on calcium, disrupting the store can lead to ER stress and eventual cell death.

ER calcium dysregulation has been observed in diseases including cardiomyopathy, diabetes, Alzheimer’s, and Parkinson’s⁵. Owing to the progressive nature of these diseases, it has been challenging to delineate the cause-effect relationship between pathogenesis and alterations in the ER calcium store. A number of technologies have allowed for significant advances in our understanding of ER calcium dynamics, including dyes and genetically encoded calcium indicators (GECIs). Low affinity calcium dyes, which increase in fluorescence when bound to Ca²⁺, can be loaded into cells to examine subcellular compartments with high concentrations of calcium⁶. GECIs, such as D1ER and CatchER allow for monitoring of calcium fluctuations with more precise control of subcellular localization^7-9. Recently, another class of GECIs called calcium-measuring organelle-entrapped protein indicators (CEPIA) have been described¹⁰. A third approach combining genetics and small molecule chemistry is targeted-esterase dye loading (TED), which utilizes a genetically encoded carboxylesterase (targeted to the ER) with an ester-based calcium dye¹¹.

While the aforementioned approaches have inherent strengths and weaknesses, they can provide valuable insight into ER calcium dynamics through acute measurements of fluorescence. They are, however, not optimal for the longitudinal studies often required to investigate disease progression. With the goal of devising a method to monitor calcium dynamics over extended periods of time, we identified and developed a protein modification to create the secreted ER calcium monitoring proteins (SERCaMPs)¹².

SERCaMP circumvents several limitations associated with other methodologies, by providing a minimally invasive approach to repeatedly interrogate the ER calcium store. We have previously demonstrated that the carboxy-terminal peptide ASARTDL (alanine-serine-alanine-arginine-threonine-aspartic acid-leucine) is sufficient to promote ER retention; however, under conditions that cause decreases in ER calcium, the peptide sequence is no longer able to retain ER localization and the protein is secreted¹³. The basis of the SERCaMP technology is the appendage of ASARTDL to the carboxy-terminus of a secreted protein (e.g. Gaussia luciferase, or GLuc) such that secretion is triggered by ER calcium depletion, thus creating a robust reporter of ER calcium dysregulation¹². The expression of GLuc-SERCaMP via transgenic methods enables biological fluids including cell culture medium and plasma to be analyzed for changes in GLuc activity as an indicator of ER calcium homeostasis. The method has applications for the longitudinal study of progressive alterations in the ER calcium store both in vitro and in vivo. The following protocol is written as a general outline for using GLuc-based SERCaMP to study ER calcium homeostasis, but the protocol can serve as a guide for alternative reporter SERCaMPs.

Protokół

1. In Vitro Assay: Detecting SERCaMP Release from a Stable SH-SY5Y Cell Line

1. Plate SH-SY5Y-GLuc-ASARTDL (SERCaMP) in tissue culture treated plates at 150,000 cells per cm² of surface area. For 96 well plates, for example, seed 50,000 cells per well (Figure 1A). Grow SH-SY5Y cells in DMEM (high glucose, GlutaMAX, pyruvate) + 10% bovine growth serum + 1x penicillin/streptomycin.
   1. Passage cells up to 15 times (Figure 1B). Higher passage number has not been tested.
   2. Return cells to humidified incubator at 37 °C with 5.5% CO₂ and incubate overnight.
2. Before experimental treatment(s), collect a baseline sample for each well by transferring 5 µl of culture supernatant to an opaque walled 96 well plate. Before collecting, gently tap the plate on all sides and swirl to avoid gradient effects. Seal the opaque plate with an adhesive sealing sheet and store at 4 °C until time of enzymatic assay.
   Note: Secreted GLuc-SERCaMP is very stable in cell culture medium. We previously reported approximately 5-10% of GLuc activity was lost after a 72 hr incubation at 37 °C (incubated on SH-SY5Y cells)¹².
3. Treat cells as desired to examine ER calcium depletion (e.g. environmental, pharmacologic, genetic manipulations). Avoid long exposure to ambient environment (outside of the controlled incubator) as pH changes will rapidly occur at atmospheric CO₂. Add HEPES to the culture medium at 20 mM, to stabilize pH¹⁴, if prolonged manipulations are required. Avoid exposure to light when using HEPES in culture medium¹⁵.
   1. Positive control: Treat cells with 100 nM thapsigargin (Tg) or 50 µM cyclopiazonic acid (CPA) for 4-6 hr for experiments in SH-SY5Y cells. Maximal response in other cell lines may require longer incubations or different concentrations of compounds.
   2. Negative control: Collect culture medium from parental SH-SY5Y cells. In our experience, the background luminescence for this assay is less than 0.05% of basal secretion from stable SH-SY5Y-GLuc-SERCaMP cells.
4. Collect and store 5 µl samples of conditioned media at the desired timepoints, as outlined in step 1.2.
5. Assess enzymatic activity in the medium as outlined in Section 5.
6. Examine intracellular GLuc by immunoblot or luminescence assay.
   1. For immunoblot: lyse cells in modified RIPA buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 0.25% sodium deoxycholate, 1 mM EDTA, 1% NP40, and protease inhibitors. Separate on SDS-polyacrylamide gel and transfer to 0.20 µm PVDF membrane. Incubate membrane with GLuc antibody (1:2,000 dilution) overnight at 4 °C. Perform secondary antibody incubation and membrane washes as per user-defined protocol for detection reagent of choice.
   2. For luminescence assay (Figure 2): perform the following steps on ice:
      1. Remove all medium from wells of the tissue culture plate and rinse with 200 µl of cold PBS.
      2. Add 75 µl of lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40 and protease inhibitors to each well.
      3. Rotate the plate on an orbital shaker (120 rpm) at 4 °C for 20 min.
      4. Gently pipette the lysate up and down using a multichannel pipettor, avoiding air bubbles. Transfer 5 µl of lysate to an opaque plate and assess luminescence as outlined in Section 5.

2. In Vitro Assay: Transient Transfection of Immortalized Cells with SERCaMP

Note: We have observed that transient transfection procedures can induce cellular stress and blunt the subsequent GLuc-SERCaMP response. The following approach was developed to minimize transfection effects in SH-SY5Y cells. Optimization of this procedure (including choice of transfection reagent) for alternate cell lines may be required. When possible, it is recommended to use stable cell lines or viral transduction methods outlined in Sections 1 and 3 respectively.

1. Plate SH-SY5Y cells in a 100 mm dish at 4 x 10⁶ cells. This dish will be sufficient to re-seed approximately 300 wells (96 well plate format) following the transfection procedure.
2. Transfect cells with Xfect reagent using 20 µg of plasmid DNA (encoding GLuc-SERCaMP) and 6 µl of Xfect reagent. Scale DNA and Xfect reagent accordingly for larger or smaller culture vessels.
3. Return cells to incubator for 48 hr.
4. Trypsinize cells and reseed to 96 well plates at 60,000 cells per well. Follow subsequent steps outlined in Section 1.

3. In Vitro Assay: Viral Vector-mediated Expression of GLuc-SERCaMP

Note: Adeno-associated viral (AAV) vector packaging¹⁶ and purification¹² and lentivirus production¹³ have been previously reported.

1. Titer GLuc-SERCaMP AAV using the following primers and probes: forward primer, 5′-CACGCCCAAGATGAAGAAGT-3′; reverse primer, 5′-GAACCCAGGAATCTCAGGAATG-3′; probe (5′-6-FAM/3′-BHQ-1 labeled), 5′-TACGAAGGCGACAAAGAGTCCGC-3’ for quantitative PCR.
   1. Prepare the standard curve by linearizing a plasmid containing GLuc and purifying the DNA using a spin-column (e.g. Machery-Nagel NucleoSpin Gel and PCR cleanup). Quantitate the DNA by separating on an agarose gel alongside a mass ladder. Prepare 1:10 dilutions of DNA from 100 pg/ml to 10 fg/ml in PBS. Calculate the copies/ml of GLuc plasmid DNA at each of the concentrations based on the molecular weight of the plasmid.
   2. Dilute the AAV stocks in PBS (appropriate dilution will be dependent on parameters of viral preparation and determined empirically).
   3. Run the PCR reaction in a real-time PCR system using the following conditions: 95 °C × 5 min, 94 °C × 20 sec, and 60 °C × 1 min for 41 cycles. Calculate copies/mL using the standard curve.
2. Titer lentivirus with a p24 Lenti-X rapid titer kit. Thaw lentiviral aliquots on ice and dilute the p24 control protein in SH-SY5Y medium.
   1. Dilute the lentivirus such that it will fall on the standard curve (e.g. 1:20,000 or 1:100,000). The dilution factor required will vary based on the lentiviral preparation parameters and must be determined empirically. Follow the manufacturer’s instructions for all subsequent steps.
3. Plate cells to 96 well plates. For SH-SY5Y cells, plating procedures outlined in Section 1 can be followed. For rat primary cortical neurons, cells should be isolated and seeded on appropriately coated plates as previously described¹⁶. Maintain rat primary cortical neurons in Neurobasal medium supplemented with 1x B27 and 500 µM L-glutamine. Perform half medium exchanges every other day. 
4. Transduce the cells with virus. The goal of viral transduction is to achieve low level expression, as Gaussia luciferase provides robust signal.
   1. AAV transduction SH-SY5Y cells: Transduce the day following plating. Dilute AAV to 6.0 x 10⁷ vg/µl in AAV dilution buffer (PBS + 0.5mM MgCl₂). Add 5 µl of diluted AAV (3.0 x 10⁸ vg; MOI of approximately 6,000) per well of 96 well plate (Figure 3A). Use 10% bleach to inactivate viral vector waste.
   2. AAV transduction of rat primary cortical neurons: Transduce 6-8 days after plating. Dilute AAV to 4.0 x 10⁶ vg/µl in AAV dilution buffer. Add 5 µl of diluted AAV (2.0 x 10⁷ vg; MOI of approximately 350) per well of 96 well plate (Figure 3B). The optimal MOI should be determined empirically for other cell types. Use 10% bleach to inactivate viral vector waste.
   3. Lentiviral transduction of SH-SY5Y cells: Transduce the day following plating. Dilute lentivirus to 20 pg/µl p24 (concentration determined using titering kit) in Hank’s balanced salt solution. Add 5µl of the diluted virus per well (100 pg of p24, equivalent to 1,250,000 lentiviral particles, or ~1,250 IFUs) of a 96 well plate containing 100 µl volume. Scale accordingly for larger formats. Use 10% bleach to inactivate viral waste.
5. Collect a pre-treatment sample of medium and begin experimental treatments 48 hr (SH-SY5Y) or 5-7 days (rat primary cortical neurons) after transduction.

4. In Vivo SERCaMP Assay

Note: Before conducting any animal procedures be sure to obtain proper approval through your institution. All survival surgeries are to be done under sterile conditions with adequate anesthesia. All procedures described below have been approved and are in compliance with NIH ACUC guidelines.

1. Autoclave surgical instruments prior to start of surgery (121 °C, 30 min sterilization, 20 min dry time). Clean all instruments in an ultrasonicator immediately following all procedures and prior to autoclaving. Maintain sterile conditions during survival surgery. For surgeries requiring multiple animals, wipe surgical instruments with 70% alcohol, ultrasonicate and bead sterilize between rats. Refer to Materials List for necessary instruments.
2. Prepare rat for surgery. Here we use male Sprague-Dawley (180-200 g).
   1. Anesthetize rats using gas isoflurane for 3 min (4-5% Isoflurane delivered at 1,000 cc/min) followed by intraperitoneal injections of xylazine (8 mg/kg) and ketamine (80 mg/kg). Apply ophthalmic ointment to protect corneas from drying out. Begin surgery once the rat is deeply anesthetized as demonstrated by lack of withdrawal reflex following tail or foot pinch.
   2. Shave the region of abdomen slightly below the ribs (low thoracic region) to the mid abdominal region. Scrub the surgical area three times, alternating 70% alcohol and Betadine scrub. Place rat in supine position on sterile surgical area with sterile surgical drapes.
3. Dilute AAV-GLuc-SERCaMP to 7.6 x 10⁹ vg/ml (final concentration). Mix well by inverting the tube.
   Note: Range of viral concentration can vary (Figure 4).
   1. Pipet 105 µl of diluted AAV into a sterile dish. Use a 30-gauge needle to collect the virus into a syringe.
4. Perform surgery to expose the liver and inject AAV-GLuc-SERCaMP.
   1. Prior to making an incision in the abdomen, apply 0.25% bupivacaine to the incision area.  Using a scalpel, make a horizontal incision below rib cage (approximately 2-3 cm). Blunt dissect to separate connective tissue from hypodermis.
   2. Cut abdominal muscle, exposing the right medial lobe of liver.
      Note: additional lobes may be injected based on end application.
   3. Place animal under surgical microscope and adjust to get right medial lobe of liver in the field of view. Inject virus into parenchyma of the medial lobe; 3 separate sites, approximately 33 µl per site.
      Note: Leave needle in tissue for 5-10 sec following injection to ensure delivery of the entire injection volume.
   4. Suture the abdominal muscle and skin separately and add Neosporin using cotton tipped applicator. Place rat in a recovery chamber until consciousness is regained and rat is able to maintain upright position. Do not leave rat(s) unattended until it has regained sufficient consciousness to maintain sternal recumbency.   House singly until the incision has healed and sutures have been removed (7-14 days).  
      Note: As per NIH post-surgical guidelines, maintain surgical record.  Annotate cage card with procedure, date, experiment identifier, body weight, and day of surgery. Pain/distress, feces production, activity, and food and water consumption are to be monitored and recorded 3 days post- surgery. Post-operative analgesia is provided by adding acetaminophen to the drinking water (450 mg/100 cc) although we do not typically observe signs of pain or distress following surgery.
5. Begin tail blood collection 4-7 days after injection. Prepare blood collection tubes by labeling and pre-weighing. Add 50 µl heparin (1,000 U/ml) to each tube.
6. Place rat in isoflurane anesthesia chamber for 3 min (4-5% Isoflurane delivered at 1,000 cc/min). Remove rat from chamber and place in nose cone (2-3% Isoflurane delivered at 500 cc/min). Blood collection can begin once the rat is deeply anesthetized as demonstrated by lack of withdrawal reflex following tail pinch.
   1. Using sterile scissors cut tip of tail (1-2 mm) and collect blood drop-wise into pre-filled heparin tube. Collect blood until volume reaches greater than 2:1 blood to heparin ratio (>100 µl blood/50 µl heparin). Blood collection volumes can be adjusted based on experimental design. Use a wet cotton applicator to apply styptic powder to the tail to stop bleeding.
   2. Store blood tubes at 4 °C if collecting subsequent samples. Wipe scissors with ethanol pad and bead sterilize between collections.
   3. Weigh collection tubes and adjust with heparin to obtain 2:1 ratio (blood:heparin). This step will normalize the amount of heparin in each sample (Table 1).
   4. Centrifuge tubes at 2,000 x g for 5 min at 4 °C. Transfer the supernatant (plasma) to a fresh tube and store at -80 °C until time of luciferase assay (section 5).
      Note: Storage of samples at 4 °C up to 72 hr prior to enzymatic assay has minimal effect on luminescence (Figure 5A). Up to 3 freeze-thaw cycles of plasma samples have no effect on luminescence (Figure 5B).
7. Thapsigargin administration (positive control):
   1. Prepare thapsigargin by diluting in ethanol to a final concentration of 2.5 mg/ml. Inject thapsigargin at 1 mg/kg i.p. into the lower abdomen.
      Note: Thapsigargin increases thrombin-induced platelet coagulation¹⁷ and can make blood collection from tail more difficult.
8. Gaussia luciferase assay:
   1. Thaw the plasma samples on ice.
      Note: For longitudinal studies, thaw and perform luminescence assay for all timepoint samples on the same day (using single preparation of substrate).
   2. Transfer 10 µl of plasma to an opaque walled plate and measure enzymatic activity as described in section 5. Run 3-4 technical replicates of each plasma sample.
9. Euthanasia
   Note: The advantage of SERCaMP technology is the ability to longitudinally monitor ER calcium. Depending on experimental parameters and endpoint analyses, animals are to be euthanized by measures appropriate for endpoint analyses and in accordance with institutional ACUC guidelines.

5. Luminescence Assay

1. Prepare coelenterazine (CTZ) stock solutions by diluting the compound in acidified methanol (30 µl of 10 N HCl to 3 ml of methanol) to 20 mM. Prepare single use aliquots and store at -80 °C.
2. Prepare the working substrate on the day of assay.
   1. For in vitro assays, dilute coelenterazine to 8 µM in PBS, e.g. add 10 µl of 20 mM CTZ stock to 25 ml of PBS (Figure 6A, B).
   2. For in vivo assays, dilute coelenterazine to 100 µM in PBS, 500 mM ascorbic acid, 5 mM NaCl.
3. Incubate prepared CTZ for at least 30 min at room temperature before beginning the assay.
   Note: This step is often included in Gaussia luciferase assays as there are reports of rapid substrate decay during first 30 min after preparing. We have not observed this effect in our system (Figure 6C); however, we often include the incubation step as we have found no negative impact on the assay.
4. Use a plate reader that is capable of monitoring bioluminescence and equipped with a substrate injector. Prime the lines with substrate.
   Note: The initial substrate through the lines can be prone to degradation. To avoid artificially low readings for early samples, inject 20-30 empty wells (loading an empty plate on the reader) before measuring experimental samples.
5. Inject 100 µl of substrate into well, shake medium speed for 5 sec, and measure light emission. For the Biotek Synergy II plate reader, integrate light emission over 0.5 sec (in vitro samples) and 5 sec (in vivo samples) for the read step. Optimize plate reader parameters for ideal assay performance.
   Note: Gaussia luciferase exhibits flash kinetics with rapid signal decay (compared to glow kinetics observed with other luciferases). The flash kinetics of GLuc is influenced by serum in cell culture medium (Figure 7A), highlighting the importance of controlling for medium composition across samples. Due to the rapid decay of luminescence after adding substrate (flash kinetics), the time between inject and read steps must be uniform for all samples. The plate reader should be set to inject substrate to a well, wait a fixed time (e.g. we use a 5 sec shake step), and read that well. Adding substrate to an entire plate before reading poses a significant challenge unless substrate can be added to all wells simultaneously. If an injector is not available, the issue can be partially circumvented by incubating the plate for 10 min between substrate addition and measurement (thus avoiding the steep part of the decay curve) (Figure 7B).
6. Repeat the injection, wait time, and read steps for each well on the plate.

Wyniki

The GLuc-SERCaMP method allows for assessment of ER calcium homeostasis by sampling extracellular fluids. Several controls can be included in the experimental design to enhance interpretation of results. First, use of a constitutively secreted reporter (e.g. GLuc without the C-terminal ASARTDL or “GLuc-No Tag”) can be employed to assess the effects of experimental treatments on the secretory pathway (global cellular secretion) and transgene expression. For instance, an increase in the extracellular l...

Dyskusje

This protocol highlights the in vitro and in vivo utility of GLuc-SERCaMP to monitor depletion of ER calcium. Although the protein modification to generate SERCaMP appears to generalize to other reporter proteins¹², we chose Gaussia luciferase for its robust (200-1,000 fold greater) bioluminescence compared to other luciferases¹⁸. We demonstrate detectable thapsigargin-induced GLuc-SERCaMP release across a 100-fold dose range of GLuc-SERCaMP virus delivered to primary rat ...

Materiały

Name	Company	Catalog Number	Comments
1.5 ml tubes	Fisher	02-682-550
10% NP-40 solution	Pierce	28324	for intracellular GLuc assays
1 ml luer-lok syringes	Fisher	14-823-30
200 microliter filter tips	Rainin	RT-L200F
3-0 surgical sutures	Fisher	NC9598192
30 G needles	Fisher Scientific	14-821-13A
Adhesive microplate sealing sheets	Thermo	AB-0558
Alcohol prep pads	Fisher	22-246-073
Anesthesia Auto Flow System	E-Z Anesthesia	EZ-AF9000
Animal recovery chamber	Lyon Vet	ICU-912-004
B27 supplement	Life Technologies	17504-044
Betadine solution	Fisher	NC9386574
Bleach	Clorox	n/a
Bovine growth serum	Thermo	SH30541.03
Coelenterazine, Native	Regis Technologies	1-361204-200
Cotton tipped applicators	Puritan	806-WC
Cutting needles 3/8 circle sutures	WPI	501803
Digital ultrasconic cleaner	Fisher Scientific	FS60D
DMEM high glucose, GlutaMAX, pyruvate	Life Technologies	10569-010
DNA mass ladder	Life Technologies	10496-016
Gaussia luciferase (recombinant protein)	Nanolight	321-100
Gaussia luciferase antibody (for WB, ICC, or IHC)	New England Biolabs	E8023S	1:2,000 (WB)
Germinator 500	CellPoint Scientific	DS-401
Gluc assay plates (96 well, opaque)	Fisher	07-200-589
Hank's balanced salt solution	Life Technologies	14175-095
Heparin	Allmedtech	63323-276-02
Isoflurane	Butler Schein	29404
Ketamine	Henry Schein	995-2949
Kwik Stop Styptic powder	Butler Schein	5867
L-glutamine	Sigma	G8540
Methanol	Fisher	a452-4
Microfuge 22R Centrifuge	Bekman Colter	368831
Neosporin	Fisher	19-898-143
Neurobasal medium	Life Technologies	21103049
Nikon Stereoscope	Nikon	SMZ745T
Nucleospin Gel and PCR Cleanup	Machery-Nagel	740609
P200 pipet	Rainin	L-200XLS+
p24 Lenti-X rapid titer kit	Clontech	632200
PCR film seal	Fisher	AB0558
Penicillin/streptomycin	Life Technologies	15140-122
Protease inhibitor cocktail	Sigma	P8340
ReFresh Charcoal Filter canister	E-Z Anesthesia	EZ-258
Scalpel blades, #10	Fine Science tools Inc	10010-00
SD rats 150-200 g	Charles River	Rats	rats ordered at 150-200 g.  Surgery 5 days after arrival
Small animal ear tags	National Band and Tag co	1005-1
Sterile surgical drapes	Braintree Scientific	SP-MPS
Synergy 2 plate reader	BioTek	n/a
TaqMan Universal PCR Master Mix	Applied Biosystems	4304437
Thapsigargin	Sigma	T9033	harmful to human health
Virapower lentiviral packaging mix	Life Technologies	K4975-00
Xfect Transfection reagent	Clontech	631318
Xylazine	Valley Vet	468RX