Engineering Synthetic Proteins to Generate Ca2+ Signals in

Nov 29, 2016 - The versatility of Ca2+ signals allows it to regulate diverse cellular processes such as migration, apoptosis, motility and exocytosis...
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Engineering Synthetic Proteins to Generate Ca2+ Signals in Mammalian Cells Anam Qudrat† and Kevin Truong*,†,‡ †

Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario M5S 3G9, Canada ‡ Edward S. Rogers, Sr. Department of Electrical and Computer Engineering, University of Toronto, 10 King’s College Circle, Toronto, Ontario M5S 3G4, Canada S Supporting Information *

ABSTRACT: The versatility of Ca2+ signals allows it to regulate diverse cellular processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g., VEGFR2), Ca2+ signals are generated upon binding their ligand(s) (e.g., VEGF-A). Here, we employed a design strategy to engineer proteins that generate a Ca2+ signal upon binding various extracellular stimuli by creating fusions of protein domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. To test the strategy, we created chimeric proteins that generate Ca2+ signals upon stimulation with various extracellular stimuli (e.g., rapamycin, EDTA or extracellular free Ca2+). By coupling these chimeric proteins that generate Ca2+ signals with proteins that respond to Ca2+ signals, we rewired, for example, dynamic cellular blebbing to increases in extracellular free Ca2+. Thus, using this design strategy, it is possible to engineer proteins to generate a Ca2+ signal to rewire a wide range of extracellular stimuli to a wide range of Ca2+-activated processes. KEYWORDS: chimeras, Ca2+ signaling, VEGFR2, synthetic biology, protein engineering, transmembrane proteins, plasma membrane

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directed by blue light.6 While there has been much development of synthetic proteins that generate Ca2+ upon stimulation with light,8−10 the ability to generate Ca2+ using a variety of extracellular stimuli would expand the versatility of the Ca2+ signal in engineering synthetic biosystems that respond to their environment. Cells respond to changes in their environment through protein receptors on the plasma membrane (PM) that in some cases transduce an extracellular stimuli into an intracellular Ca2+ signal. In particular, the vascular endothelial growth factor receptor 2 (VEGFR2), a tyrosine kinase receptor, dimerizes upon VEGF-A binding, in a process that ultimately generates a Ca2+ signal.11 Dimerization triggers autophosphorylation of specific tyrosine residues on the cytoplasmic tail that facilitate the docking of SH2-binding domains to recruit phospholipase C (PLC). PLC then catalyzes the hydrolysis of phosphatidy-

he versatility of the Ca2+ signal arises from its use as a second messenger (or intermediate signal) to regulate diverse processes such as motility, apoptosis, transcription and exocytosis.1 By constructing networks consisting of proteins that generate Ca2+ signals and proteins that respond to Ca2+ signals, in theory, almost any cellular process can be rewired via a Ca2+ signal. Many natural proteins generate Ca2+ signals such as channelrhodopsin-2 (ChR2) and nicotinic acetylcholine receptor upon stimulation by blue light2 and acetylcholine,3 respectively. Likewise, many natural proteins respond to Ca2+ signals with different enzymatic activities such as calpain with protease activity4 and calcineurin with serine/threonine phosphatase activity.5 To expand the range of cellular activities that directly respond to Ca2+ signals, synthetic proteins have been engineered to be Ca2+-activated such as Rac16 (named Racer), Cdc426 and RhoA7 (named CaRQ) to control the formation of lamellipodia, filopodia and cellular blebs, respectively. As an example of rewiring using the Ca2+ signal, a cell expressing both Racer and ChR2 allowed migration © XXXX American Chemical Society

Received: October 21, 2016 Published: November 29, 2016 A

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Figure 1. Ecad12 chimera triggers a Ca2+ signal with high μM extracellular free Ca2+. (A) Schematic figure illustrating the mechanism of action of the Ecad12rec chimera. (B) HEK293 cells cotransfected with the green Ca2+ sensor GCaMP2 and the Ecad12rec construct show cytoplasmic and protein trafficking distribution, respectively. Images are false colored: Venus, green; Cerulean, cyan. Scale bar is 10 μm. (C) Ca2+ trace observed with the addition of 1 mM of CaCl2 shows an abrupt rise and a subsequent slower decline. (D) Box plot showing range of signal duration. Bar shows standard deviation. (E) Percentage of cells responding to a minimal concentration (i.e., 500 μM) of the stimulus (i.e., CaCl2). Samples compared with Student’s t test. Star indicates significance; p-value = 0.01. Error bars show standard deviation. (F) Multiple peaks after recurrent stimulation with an additional 500 μM CaCl2 added. All experiments were repeated at least 3 times with at least 3 cells per experiment.

extracellular media,12 FKBP12 (FK506 binding protein) and FRB heterodimerize when stimulated with rapamycin,13 the stromal interaction molecule 1 (STIM1) oligomerizes when stimulated with the EDTA Ca2+ chelator which decreases free Ca2+ in the extracellular media,14 and calmodulin (CaM) and myosin light chain kinase peptide (MLCKp) heterodimerize when stimulated with low μM increases in free Ca2+ in the extracellular media.15 As expected, these synthetic proteins responded to their respective extracellular stimuli by generating a Ca2+ signal. Further, when the Ecad12 chimeric protein was rewired with CaRQ,7 it generated cell blebbing upon stimulation with free Ca2+ in the extracellular media.

linositol 4, 5-bisphosphate (PIP2) to IP3 which binds to the IP3 receptor, releasing Ca2+ from the endoplasmic reticulum (ER).11 Initially, intracellular Ca2+ concentration rises by Ca2+ release from the ER and then subsequently by store-operated Ca2+ entry from the extracellular environment. To expand the extracellular stimuli that generate a Ca2+ signal, we investigated the protein design strategy of fusing extracellular domains, known to oligomerize upon stimuli binding, with the cytoplasmic tail of VEGFR2 known to generate Ca2+ signals. To demonstrate the feasibility of this design, we tested protein fusions of the cytoplasmic tail of VEGFR2 with four protein systems known to oligomerize: the first and second repeat of E-cadherin (Ecad12) dimerizes when stimulated with high μM increases in free Ca2+in the B

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Figure 2. FRB and FKBP12 chimera system triggers a Ca2+ signal with rapamycin. (A) Schematic figure illustrating the mechanism of action of the FRBrec/FKBP12rec chimeras. (B) HEK293 cells cotransfected with the green Ca2+ sensor GCaMP2 and the FRBrec/FKBP12rec constructs show cytoplasmic and protein trafficking distribution, respectively. Images are false colored: Venus, green; Cerulean, cyan; mRFP, red. Scale bar is 10 μm. (C) Ca2+ trace observed with the addition of 1 μM rapamycin shows an abrupt rise and a subsequent slower decline. (D) Box plot showing range of signal duration. Bar shows standard deviation. (E) Percentage of cells responding to a minimal concentration of the stimulus (i.e., rapamycin). Samples compared with Student’s t test. Star indicates significance; p-value = 0.03. Error bars show standard deviation. (F) Multiple peaks after recurrent stimulation with an additional 1 μM rapamycin added. All experiments were repeated at least 3 times with at least 3 cells per experiment.

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RESULTS AND DISCUSSION

HEK293 cells. While GCaMP2 was localized to the cytoplasm, Ecad12rec did not have an apparent PM localization (e.g., sharp outline of cell periphery) but instead appeared to be mostly trafficking through the cell (e.g., ER, golgi, nuclear envelope and moving vesicles) (Figure 1B). Further, Ecad12rec localization appeared similar to VEGFR2-Venus and both proteins appeared different than a red fluorescent protein labeled to the PM by the PM-targeting peptide of Lyn kinase (1MGCIKSKGKDSA12) (LynCherry) (Figure S1A and B, Table S1). Since the Ecad12rec contains the cytoplasmic tail of VEGFR2 associated with endocytic processes,19,20 it appears similar to VEGFR2. Due to the endocytic processes of VEGFR2, it is difficult to find any studies that show clear labeling of the PM. In studies of VEGFR2, the final destination of VEGFR2 is shown by treatment with cycloheximide (CHX) and chlor-

Ecad12 Chimera Triggers a Ca Signal with High μM Extracellular Free Ca2+. The Ecad12 chimera generates a Ca2+ signal upon stimulation with high μM extracellular Ca2+ concentration increases. The chimera named Ecad12rec was constructed by the tandem fusion of the leader sequence found in immunoglobulin kappa (IgK) (i.e., 1METDTLLLWVLLLWVPGSTGD21), Ecad12, Cerulean,16 the transmembrane domain of VEGFR2 and the cytoplasmic tail of VEGFR2 (Figure 1A). Homodimer formation of Ecad12 is dependent on Ca2+ which forms a weak homodimer where three Ca2+ binding sites are located in the linker region; the cis-dimers have low binding affinity (Kd = 720 μM).12,17,18 Ecad12rec was cotransfected with the green Ca2+ sensor GCaMP2 in 2+

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Figure 3. STIM1 chimera triggers a Ca2+ signal with EDTA. (A) Schematic figure illustrating the mechanism of action of the STIM1rec chimera. (B) HEK293 cells cotransfected with the red Ca2+ sensor R-GECO1 and the STIM1rec construct show cytoplasmic and protein trafficking distribution, respectively. Images are false colored: mRFP, red; Venus, green. Scale bar is 10 μm. (C) Ca2+ trace observed with the graduated addition of 300 μM EDTA shows an abrupt rise and a subsequent slower decline. (D) Box plot showing range of signal duration. Bar shows standard deviation. (E) Percentage of cells responding to a minimal amount of the stimulus (i.e., EDTA). Samples compared with Student’s t test. Star indicates significance; p-value = 0.01. Error bars show standard deviation. (F) Multiple peaks after recurrent stimulation with an additional 300 μM EDTA added. All experiments were repeated at least 3 times with at least 3 cells per experiment.

In Ca2+-free PBS media, CaCl2 was gradually added with consecutive bolus (10 μM) additions to the extracellular medium about once every minute. Although this stimulus had no effect on GCaMP2 alone (Figure S3C) (n = 3), an intracellular Ca2+ signal lasting about 92.8 ± 15.5 s was seen in the presence of Ecad12rec (n = 3) (Figure 1C and D, Video S1). Since extracellular Ca2+ cannot diffuse across the PM, some Ecad12rec must be on the PM (albeit a low amount) to transduce the extracellular Ca2+ stimulus to an intracellular Ca2+ signal. Relatively consistent with cis-dimerization affinity, the minimum amount of CaCl2 required to trigger a Ca2+ signal

oquine (CLQ) to inhibit protein synthesis and lysosomal degradation, respectively.19,20 Likewise, the same experiment was performed on VEGFR2-Venus and Ecad12rec. After treatment with 10 μg/mL CHX, both Ecad12rec and VEGFR2 had moving endosomes (Figure S1A and B, Table S1). To show that the final destination of Ecad12rec is the lysosome, we compared it to LAMP1 (lysosomal associated membrane protein 1) labeled with RFP. After treatment with 10 μg/mL CHX and 10 μM CLQ, Ecad12rec colocalized with LAMP1-RFP (Figure S1C and Table S1). D

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HEK293 cells. As expected, GCaMP2 was cytoplasmic (Figure 2B). Like Ecad12rec, the FKBP12rec and FRBrec did not have an apparent PM localization but instead appeared to be mostly trafficking through the cell (Figure 2B). While the addition of [1 μM]f rapamycin had no effect on GCaMP2 alone (Figure S3C) (n = 3), a Ca2+ signal was seen in the presence of the FKBP12rec and FRBrec system (Figure 2C). The Ca2+ signal had a sharp peak and a gradual fall, lasting for 42 ± 9.7 s (n = 3) (Figure 2C and D). While this duration is markedly lower compared to the Ecad12rec system, it is not statistically significant compared with the other chimeric systems. Since these experiments were conducted in Ca2+-free media, there was no possible contribution of extracellular Ca2+ influx to the Ca2+ signal thus producing a shorter signal. The average cell response was 47 ± 31.5% of the cells responding (n = 3, p = 0.03) (Figure 2E). Subsequent addition of rapamycin up to [2 μM]f generated an additional Ca2+ signal of the same duration but with reduced intensity, showing this system has adaptation (Figure 2F) (i.e., it responds to further changes in the extracellular free Ca2+) (n = 3). Further, when cells were treated with 10 nM thapsigargin, an initial signal showing Ca2+ exit from the ER was followed by a null signal when stimulated with 1 μM rapamycin (Figure S7), indicating that the Ca2+ originates from the ER. As a negative control, when the VEGFR2tail alone was cotransfected with GCaMP2 and stimulated with [1 μM]f rapamycin, the cells showed no Ca2+ signal (Figure S4B) (n = 3). Thus, rapamycin triggered a Ca2+ signal in cells transfected with the FRBrec and FKBP12rec system. STIM1 Chimera Triggers a Ca2+ Signal with EDTA. The STIM1 chimera generates a Ca2+ signal when stimulated with decreases in free Ca2+ in the extracellular media. The chimera named STIM1rec was constructed by the tandem fusion of the luminal domain of STIM1, the transmembrane domain of VEGFR2, the cytoplasmic tail of VEGFR2 and Venus26 (Figure 3A). When Ca2+ stores are depleted in the ER, stromal interaction molecule 1 (STIM1) oligomerizes and binds the Orai1 Ca2+ channels on the PM to trigger store-operated Ca2+ entry (SOCE).9 The Ca2+-binding affinity of STIM1 is low, ranging from 200 to 600 μM.14 The STIM1rec was cotransfected with the red Ca2+ indicator R-GECO1 in HEK293 cells. As expected, R-GECO1 was cytoplasmic (Figure 3B). Like Ecad12rec, STIM1rec did not have an apparent PM localization but instead appeared to be mostly trafficking through the cell (Figure 3B). In PBS media containing [1 mM]f CaCl2, the EDTA Ca2+ chelator was gradually added to decrease the free Ca2+ concentration. While this decrease had no effect on R-GECO1 alone (Figure S8C) (n = 3), a Ca2+ signal lasting for 77.2 ± 17.9 s was seen in the presence of STIM1rec (n = 3) (Figure 3C and D). Relatively consistent with the STIM1 Ca2+-binding affinity, the minimum amount of free Ca2+ required to trigger a Ca2+ signal was ∼700 μM (with EDTA [300 μM]f) with 56 ± 25.5% of the cells showing a signal (n = 3, p = 0.01) (Figure 3E). Subsequent decrease in free Ca2+ to ∼400 μM (with the addition of EDTA up to [600 μM]f) generated an additional Ca2+ signal of the same duration but with reduced intensity, showing this system has adaptation (Figure 3F) (i.e., it responds to further changes in the extracellular free Ca2+) (n = 3). When the VEGFR2tail alone was cotransfected with R-GECO1 and stimulated with [300 μM]f EDTA, the cells showed no Ca2+ signal (Figure S4C) (n = 3). Thus, decreasing extracellular Ca2+ triggered a Ca2+ signal in cells transfected with STIM1rec. E

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Figure 4. CaM and MLCKp chimera system triggers a Ca2+ signal with low μM extracellular free Ca2+. (A) Schematic figure illustrating the mechanism of action of the MLCKrec/CaMrec chimeras. (B) HEK293 cells cotransfected with the green Ca2+ sensor GCaMP2 and the MLCKrec/ CaMrec constructs show cytoplasmic and protein trafficking distribution, respectively. Images are false colored: Venus, green; Cerulean, cyan; mRFP, red. Scale bar is 10 μm. (C) Ca2+ trace observed with the addition of 100 μM of CaCl2 shows an abrupt rise and a subsequent slower decline. (D) Box plot showing range of signal duration. Bar shows standard deviation. (E) Percentage of cells responding to a minimal amount of the stimulus (i.e., CaCl2). Samples compared with Student’s t test. Star indicates significance; p-value = 0.02. Error bars show standard deviation. (F) Multiple peaks after recurrent stimulation with an additional 100 μM CaCl2 added. All experiments were repeated at least times with at least 3 cells per experiment.

CaM and MLCKp Chimera System Triggers a Ca2+ Signal with Low μM Extracellular Free Ca2+. The CaM and MLCKp chimera system generates a Ca2+ signal upon stimulation with low μM extracellular free Ca2+ increases. The chimeras named CaMrec and MLCKrec were created with the tandem fusion of the IgK leader sequence, CaM or MLCKp, Cerulean16 or mRFP,25 the transmembrane domain of VEGFR2 and the cytoplasmic tail of VEGFR2 (Figure 4A). Upon Ca2+ increases, CaM initially binds Ca2+ and then strongly binds CaM-binding peptides such as MLCKp (Kd ranges from 0.1−20 nM).15 This association is limited by CaM affinity to Ca2+ which has a Kd ranging from 0.5 to 5 μM.27 The CaMrec and MLCKrec were cotransfected with the green Ca2+

indicator GCaMP2 in HEK293 cells. Like Ecad12rec, the CaMrec and MLCKrec did not have an apparent PM localization but instead appeared to be mostly trafficking through the cell (Figure 4B). In PBS media containing [100 μM]f EDTA, CaCl2 was gradually added with consecutive bolus (10 μM) additions. Although this stimulus had no effect on GCaMP2 alone (Figure S3C) (n = 3), a Ca2+ signal lasting for 59.5 ± 20.6 s was seen in the presence of the CaMrec and MLCKrec system (n = 3) (Figure 4C and D). Relatively consistent with the CaM Ca2+-binding affinity, the minimum amount of CaCl2 required to trigger a Ca2+ signal was [100 μM]f (corresponding to free Ca2+ of ∼2.2 μM) with 44 ± 26.8% cells responding (n = 3, p = 0.02) (Figure 4E). F

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Figure 5. A synthetic circuit with Ecad12rec and CaRQ triggers dynamic blebbing. (A) Schematic figure illustrating the mechanism of action of the Ecad12rec chimera. (B) HEK293 cells cotransfected with Ecad12rec and the CaRQ constructs both show cytoplasmic distribution. Images taken at 5 min intervals. White arrow shows protruding bleb at 10 min. Insets highlight blebbing region. Images are false colored: Venus, green. Scale bar is 10 μm. Experiments were repeated at least 3 times with at least 3 cells per experiment.

Subsequent addition of CaCl2 up to [200 μM]f generated an additional Ca2+ signal of the same duration but with reduced intensity, showing this system has adaptation (Figure 4F) (i.e., it responds to further changes in the extracellular free Ca2+) (n = 3). When the VEGFR2tail alone was cotransfected with GCaMP2 and stimulated with [100 μM]f CaCl2, the cells showed no response (Figure S4D) (n = 3). Thus, low μM increases of extracellular free Ca2+ triggered a Ca2+ signal in cells transfected with the CaMrec and MLCKrec system. A Synthetic Circuit with Ecad12rec and CaRQ Triggers Dynamic Blebbing. To show that we can rewire these synthetic proteins to control cellular activity, we generated a synthetic circuit with Ecad12rec and CaRQ.7 CaRQ is an engineered RhoA protein that was made Ca2+-activated by targeted fusion with CaM and CaM-binding peptides.7 Upon the addition of [1 mM]f CaCl2, as expected, cells expressing Ecad12rec and CaRQ showed dynamic blebbing (n = 3) (Figure 5A). CaRQ was localized to the PM and showed dynamic blebbing for a duration of 26.6 ± 4.6 min (n = 3) (Figure 5B). The area of the blebs averaged 36.0 ± 19.1 μm2. As control, when these cells incubated with [1 μM]f Y-27632 (ROCKI inhibitor), dynamic blebbing was abolished. Thus, increases in extracellular Ca2+ triggered dynamic blebbing in cells transfected with the Ecad12rec and CaRQ system. Currently, the Ecad12rec system responds to physiological changes in Ca2+ in the mM range, but Ecad12rec can be mutated at its Ca2+ binding site to reduce its Ca2+ affinity as has been previously done.28 With reduced Ca2+ affinity, the Ecad12rec and CaRQ system could potentially be used to engineer a cell that migrates to abnormal Ca2+ deposits found in chronic kidney disease and atherosclerosis.29 The property of adaptivity shown by the synthetic proteins could play an important role for chemotaxis to the source of the ligand. As the ligand binds to the synthetic proteins on the cell, it stimulates CaRQ-mediated blebbing. Since the blebs need to occur successively for meaningful cell migration, adaptivity offers a potential mechanism. Adaptivity could be a way for the

cell to generate more Ca2+ signals and thus, bleb successively toward the source of the ligand.

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CONCLUSION We have demonstrated a design strategy to engineer proteins that generate a Ca2+ signal upon addition of various extracellular stimuli. As long as the stimulus triggers a dimerization of at least two protein domains, their fusion with the cytoplasmic tail of VEGFR2 will generate a Ca2+ signal. Applying this strategy, Ca2+ signals were observed by FRB and FKBP12 dimerization upon rapamycin binding, by STIM1 oligomerization upon decreasing free extracellular Ca2+, by CaM and MLCKp dimerization upon increasing free extracellular Ca2+ in the low μM range and by Ecad12 dimerization upon increasing free extracellular Ca2+ in the high μM range. Further, the Ecad12rec chimera was coupled to CaRQ in a synthetic circuit that triggered blebbing in mammalian cells upon increasing free extracellular Ca2+. This strategy can be further applied to generate other systems that are known to dimerize (e.g., cytokine receptors, receptor tyrosine kinases and G protein-coupled receptors that are activated by cytokines, growth factors and chemokines, respectively) to generate a Ca2+ signal that can rewire a wide range of extracellular stimuli to a wide range of Ca2+-activated processes. In particular, the Ecad12rec and STIM1rec proteins were designed to generate intracellular Ca2+ signals when there is an increase and decrease of free extracellular Ca2+, respectively, which could have interesting application in cellbased theory. By incorporating mutants that decrease Ca2+ affinity,28 Ecad12rec could detect abnormally high Ca2+ concentrations found at Ca2+ deposits in chronic kidney disease and atherosclerosis.29 In contrast, STIM1rec could detect abnormally low Ca2+ concentration found at local sites of disruptions of the blood brain barrier in neurodegenerative diseases.30,31 Low extracellular Ca2+ concentration increases endothelial permeability because the cell−cell adhesion proteins that maintain adherens and tight junctions depend G

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concentrations, cells were pretreated with PBS (+ [100 μM]f EDTA) to chelate residual free Ca2+ from the growth media. The amount of free Ca2+ was estimated by the Maxchelator program.37 For inhibition experiments, cells were preincubated in PBS for half an hour prior to stimulation with the respective drug (i.e., [100 nM]f staurosporine (Sigma), [1 μM]f Y-27632 (Sigma) or [10 nM]f thapsigargin (Sigma)). Signal Processing. The Ca2+ signal was measured using the Live Intensity Plot plugin for ImageJ.36 Specifically, for each image stack, the Z-axis profile was plotted for a region of interest (ROI) and using the straight-line tool, the length of the signal was manually measured from the rise of the peak to the basal level. Likewise, an ROI was used to plot FRET (Forster Resonance Energy Transfer) intensities YFP and CFP channels separately, before calculating the YFP:CFP ratios. The intensity values were then exported and normalized between 0 and 1, x−x using the formula xmax = x − min and reported as arbitrary x

on physiological levels of extracellular Ca2+concentration.30,31 Further, by coupling Ecad12rec and STIM1rec with CaRQ, it may be possible to design cells that migrate to these disease sites. Once at the disease sites, the cells could be further engineered to locally release therapeutics such as monoclonal antibodies.

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MATERIALS AND METHODS Plasmid Construction. Using our subcloning methodology,32 cassettes were created for FRB, FKBP12, the leader sequence for IgK, the transmembrane and cytoplasmic tail of VEGFR2, the luminal domain of STIM1, the first and repeat of E-cadherin (named Ecad12), CaM and MLCKp. The chimeric proteins of FRBrec, FKBP12rec, STIM1rec, MLCKrec, CaMrec and Ecad12rec were assembled following the subcloning methodology as described, previously.32,33 The plasmid for mammalian expression of CaRQ and VEGFR2-Venus were used from our previously work.7,34 The CMV-R-GECO1 and CMV-GCaMP2 plasmids for mammalian expression of RGECO1 and GCaMP2 were gifts from Robert Campbell and Karel Svoboda (Addgene plasmid #32444 and #18927), respectively. LAMP1-RFP was a gift from Walther Mothes (Addgene plasmid #1817). CYPHR, DAGR and CKAR were gifts from Alexandra Newton (Addgene plasmid #14864, #14865 and #14860 respectively). PICCHU was a gift from Michiyuki Matsuda. All plasmids were transformed in E. coli DH5-α strain and was isolated using the Mini-prep kit (Invitrogen). Cell Culture and Transfection. HEK293 cells were maintained in Dulbecco’s Modified Eagle’s Medium containing 25 mM D-glucose, 1 mM sodium pyruvate and 4 mM Lglutamine (Invitrogen, Carlsbad, CA) supplemented with 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich, St. Louis, MO) in T25 flasks (37 °C and 5% CO2). Cells were passaged at 95% confluency using 0.05% TrypLE with Phenol Red (Invitrogen) and seeded onto 24-well Multiwell Plates (Falcon, Corning, NY) at a dilution of 1:20. Cells were transiently transfected using Lipofectamine 3000 according to manufacturer’s protocols (Invitrogen). Post-transfection, cells were treated with 0.05% TrypLE with Phenol Red (Invitrogen) and plated in 96-well tissue culture plates (Sarstedt, Numbrecht, Germany) at a dilution of 1:4 for imaging. Cycloheximide Chase Assay. 24 h post-transfection, HEK293 cells were incubated with 10 μg/mL of cyclohexamide (CHX) (Sigma-Aldrich, St. Louis, MO) and imaged after 1 h. To inhibit lysosomal degradation, the cells were treated with 10 μM chloroquine (CLQ) (Sigma) before being imaged. Illumination and Imaging. Imaging was performed using an inverted IX81 microscope with Lambda DG4 xenon lamp source and QuantEM 512SC CCD camera with a 10× objective (Olympus). Filter excitation (EX) and emission (EM) bandpass specifications were as follows (in nm): CFP (EX: 438/24, EM: 482/32), YFP (EX: 500/24, EM: 542/27), RFP (EX: 580/20, EM: 630/60) (Semrock). For FRET imaging, YFP emission was collected from CFP excitation and compared against CFP emission from CFP excitation. Image acquisition and analysis was done with ImageJ and μManager software.35,36 Cell Stimulation. All stimulation experiments were performed 24 h post transfection of the chimeric system and the Ca2+ indicator. Cells were washed with Ca2+-free PBS prior to stimulus addition (i.e., CaCl2 (Sigma), EDTA (Sigma), rapamycin (Sigma)) and stimuli were added in consecutive bolus (10 μM) additions. To achieve low extracellular free Ca2+

max

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units (a.u.). Statistical Analysis. Unless otherwise stated, the data are expressed as mean ± s.d. All data with normal distribution and similar variance were analyzed for statistical significance using two-tailed, unpaired Student’s t-test, unless otherwise stated. A one-factor ANOVA with a Tukey−Kramer posthoc test was performed for multiple group comparisons. p-values < 0.05 were considered statistically significant. All experiments were repeated 3 times with at least 3−6 cells analyzed per experiment. Data were analyzed using Real Statistics Resource Pack for Excel (Microsoft, Redmond, WA).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.6b00310. Supporting Figures S1−S8 and Table S1. (PDF) Supporting Video S1. Ecad12rec cells respond to extracellular Ca2+. Upon the addition of 1 mM CaCl2, HEK293 cells transfected with Ecad12rec and the red Ca2+ sensor, Lyn-RGECO1 show Ca2+ transients. Scale bar is 100 μm. The experiment was repeated at least 3 times. (AVI)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 416-978-7772. Fax: 416-978-4317 164. ORCID

Kevin Truong: 0000-0002-9520-2144 Author Contributions

AQ conducted the experiments, analyzed the data and wrote the manuscript. KT conceived the initial idea and helped write the manuscript. All authors have read and approved the final manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by grants from the Canadian Cancer Society Research Institute (#701936) and NSERC (#0532214). Seema Nagaraj subcloned the plasmids. H

DOI: 10.1021/acssynbio.6b00310 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Research Article

ACS Synthetic Biology

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DOI: 10.1021/acssynbio.6b00310 ACS Synth. Biol. XXXX, XXX, XXX−XXX