In Cellulo Mapping of Subcellular Localized Bilirubin - ACS Chemical

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In Cellulo Mapping of Subcellular Localized Bilirubin Jong-Seok Park, Eunju Nam, Hye-Kyeong Lee, Mi Hee Lim, and Hyun-Woo Rhee ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00017 • Publication Date (Web): 27 May 2016 Downloaded from http://pubs.acs.org on June 2, 2016

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ACS Chemical Biology

In Cellulo Mapping of Subcellular Localized Bilirubin Jong-Seok Park, Eunju Nam, Hye-Kyeong Lee, Mi Hee Lim* and Hyun-Woo Rhee* Department of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulju-gun, Ulsan, 44919, Republic of Korea.

ABSTRACT: Bilirubin (BR) is a de novo synthesized metabolite of human cells. However, subcellular localization of BR in the different organelles of human cells has been largely unknown. Here, utilizing UnaG as a genetically encoded fluorescent BR sensor, we report the existence of relatively BR-enriched and BR–depleted microspaces in various cellular organelles of live cells. Our studies indicate that (i) cytoplasmic facing membrane of endoplasmic reticulum (ER) and the nucleus are relatively BR-enriched spaces; (ii) mitochondrial intermembrane space and ER lumen are relatively BR-depleted spaces. Thus, we demonstrate a relationship between such asymmetrical BR distribution in the ER membrane and the BR metabolic pathway. Furthermore, our results suggest plausible BR-transport and BR-regulating machineries in other cellular compartments, including the nucleus and mitochondria.

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Introduction Bilirubin (BR) is a de novo synthesized lipidic metabolite of mammals and a significant amount of BR (ca. 250–400 mg) is produced daily in the human body.1 BR is mainly generated through heme degradation pathways that consist of heme oxygenase (HO) and biliverdin reductase(BVR), which are localized at the endoplasmic reticulum (ER) membrane and in the cytoplasm, respectively.2 BR is a potent oxidant scavenger that can protect cells from a 10,000-fold excess of hydrogen peroxide(H2O2)2, 3. Likewise, BR protects biomolecules such as lipids from oxidative stress and several studies have shown that it may protect the immunological system.4 For example, BR protects albumin-bound linoleic acid from peroxyl radical induced oxidation in normal human plasma.4 This indicates that BR plays an important role as a physiological antioxidant in human body. However, BR accumulation in the human body associated with a failure of its clearance causes fatal diseases such as hyperbilirubinemia, chronic jaundice, encephalopathy, Crigler-Najjar syndrome and Dublin-Johnson syndromes.1 Currently, the biological and pathological roles of BR are largely unknown because the basic information, such as its subcellular distribution remains elusive. Several methods have been developed for BR detection in vitro using various analytical instruments5-7; however, they cannot be employed for BR detection in cellulo. Very recently, based on the significant increase in the two-photon fluorescence of the BR dimer, Sun and coworkers successfully detected differentiated BR levels in developed tumors and undeveloped tumor tissue.8 In that study, the authors focused on measuring the total BR dimer levels in different tissues, and not on the subcellular BR distribution in live cells.8 Only one report exists that attempts to measure subcellular bilirubin levels by serial organelle purifications of the cellular organelle 2 ACS Paragon Plus Environment

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fractions, of rat brain.9 In this report, BR concentrations measured in myelin and cytosol fractions were higher than in the microsome, nucleus, and mitochondrial fractions. Results of the previous report, however, may include false information because of contamination issues related to conventional organelle purification methods.10 In addition, this method did not distinguish subcompartmentally localized BR, at ER and the mitochondrion. Thus, the development of a new and accurate method is necessary for the detection of specific metabolites and understanding their physiological function. Fluorescence imaging with a genetically encoded sensor may be a most useful method for monitoring the distribution of metabolites of interest. Generally, genetically encoded sensors have five benefits: (i) available for spatiotemporal detection during various physiological events (ii) selective for a specific analyte, (iii) possibility of long term imaging (iv) in living cells, and (v) facile targeting of specific compartments. Several fluorescence imaging sensors for adenosine triphosphate (ATP)11, guanosine triphosphate (GTP)12, cyclic adenosine monophosphate (cAMP)13, hydrogen peroxide (H2O2)14, 15, hydrogen sulfide (H2S)16, metal ions (i.e., calcium17-19, copper20 and zinc ion21, 22), glutamate23 and glucose24, 25 have been designed. However, the variety of metabolites detected by those sensors is highly limited. In contrast to these previous works, herein, to the best of our knowledge, we present the first report of in vivo visualization of subcellular BR distribution using UnaG as a genetically encoded BR sensor. Recently, UnaG has been identified as a novel BR (IX-alpha) binding protein.26 Protein-bound BR is a green fluorophore for UnaG fluorescence (λex = 498 nm, λem = 527 nm, ε = 0.51) whereas nearly no fluorescence is associated with free BR and apo-UnaG (Figure 1).26 Because UnaG has a very specific selectivity and binding affinity (Kd = 98 pM) for BR but not for other BRrelated compounds, including ditaurobilirubin, urobilin, and biliverdin, purified UnaG has been 3 ACS Paragon Plus Environment


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employed to measure BR concentrations in human blood.26 However, to the best of our knowledge, UnaG has not been employed to measure subcellular localized BR in live cells. In this study, we targeted UnaG to various subcellular compartments by genetically tagging it with signal peptides and attempted relative quantification of local BR levels in living cells. We demonstrated that relatively “BR-rich” and “BR-depleted” cellular organelles are present in live mammalian cells, which has not been reported until now. We also demonstrated that the asymmetrical BR distribution at ER membrane is associated with BR metabolic pathways. Moreover, we found that local BR levels are tightly controlled in mitochondria and our results suggest the existence of active BR nuclear transporters.

Results and Discussion Different UnaG fluorescence intensities in different cellular organelles. Through fusion of various well-known targeting signal sequences and domains, UnaG was transiently expressed and targeted to several cellular organelles in living mammalian cells. As shown in Figure 2, most subcellularly targeted UnaG constructs showed considerable green fluorescence, which was not observed in untransfected cells. The fluorescence showed distinct patterns in each targeted compartment, indicating that UnaG fusion proteins were well-expressed and were properly transported into the specific organelles (Figure 2). We used HEK-293T cells, s frequently employed human cell culture line derived from the kidney. Using the HEK-293T cell line, we were able to separate strongly fluorescent constructs from weakly fluorescent constructs. The former included UnaG-NES (cytoplasm), UnaG-NLS (nucleus), UnaG-Sec61B (rough ER membrane cytosol face), and UnaG-PM (plasma membrane). The latter included Sec61B-UnaG (rough ER membrane luminal face), and Sco1-UnaG and LACTB-UnaG (both in the mitochondri4 ACS Paragon Plus Environment

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al intermembrane space, IMS). Sec61 complex is an ER membrane protein translocator (aka translocon) and N-terminus of Sec61 beta(Sec61B) is exposed to the cytosol, whereas its C-terminus is exposed to the ER lumen.35 Because of the possibility of decreased specificity of BR binding to UnaG in different subcellular microenvironments, UnaG fluorescence intensity in live cells could not be directly interpreted to signify endogenous BR levels in each organelle without assaying UnaG activity and its expression in that organelle. To assess this, we added exogenous BR (10 μM) to the growth medium and we found that it enhanced green fluorescence of Sco1-UnaG and Sec61B-UnaG in the mitochondria and ER of living cells, respectively, after 1 min incubation (Figure 2). We also checked whether their expression pattern matches the fluorescence pattern of organelle marker proteins (i.e., Matrix-BFP for mitochondria, Cyt-ERM-BFP for ER) (Figure S1). This assay verified that UnaG in the IMS and ER lumen was functional. We found that other organelle-targeted UnaG fusions also displayed increased fluorescence after exogenous BR addition, indicating that UnaG is generally functional under various conditions in cellular compartments in living cells. Next, we attempted to observe distinct UnaG fluorescence intensities of various organelles in other human cell lines. Detection of subcellular UnaG fluorescence was carried out in Neuro-2a (N2a; neuroblastoma cell line) and U2-OS (osteosarcoma cell line) cells originating from the brain and the bone, respectively. As shown in Figure S2, the majority of N2a and U2-OS cells transfected with UnaG constructs displayed green fluorescence, except for one construct, Sco1UnaG. This particular IMS construct showed a similarly very weak fluorescence in HEK-293T cells. In addition, Sec61B-UnaG exhibited weak fluorescence in the three cell lines. UnaG-Sec61B and UnaG-NLS constructs showed the strongest fluorescence in all cell lines examined. Moreover, UnaG fluorescence enhancement by exogenous BR (10 μM) was also reproducibly observed in 5 ACS Paragon Plus Environment


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N2a and U2-OS cells. Thus, we concluded that different UnaG fluorescence intensities in different subcellular compartments are not an artifact of a single cell type, but are a consistent feature of different mammalian cell lines.

Identification of relatively BR-enriched and BR-depeleted microspaces Because of the possibility that certain recombinant UnaG constructs were expressed at low levels or were actively degraded, similarly to other recombinant proteins, we used immunofluorescence imaging to verify that all UnaG construct expression levels fell within a similar range (detailed procedures can be found in Experimental Section). As depicted in Figures S3–5, all UnaG constructs were well-expressed and they were in a similar range of expression. Furthermore, we also observed that BR binding was not compromised during the fixation and permeabilization steps because of the very high binding affinity (vide supra) and because the remaining fluorescence of holo-UnaG in the fixed cells followed the same tendency as fluorescence of holoUnaG in live cells. This remaining holo-UnaG fluorescence is very useful in relative quantifications of BR levels by pixel-wise fluorescent scatter plot analysis and box plot analysis with immunofluorescence intensity. Because there is no commercially available primary antibody for UnaG, we designed all our UnaG construct possess the Flag peptide sequence (DYDDDDK) for the immunofluorescence assay with the mouse anti-Flag antibody. This anti-Flag antibody can be detected by secondary Alexa Fluor 568-goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA; 1:1000 dilution) and this fluorescent intensity can be observed in AF568 channel. For pixel-wise image analysis, we converted the gray scale image to read-pixel intensity using ImageJ software (National Institutes of Health, Bethesda, MD, USA), following which, the intensities assigned to each pixel (at least 6 ACS Paragon Plus Environment

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2000) were collected. For each sub-compartmental UnaG image, red fluorescence intensity (AF568 channel) and green fluorescence intensity (AF488 channel) of each pixel was visualized in a scatter plot (Figure S6), and fluorescence intensity (FI) pixel ratio (green FI/red FI) was regarded as the ratio of holo-UnaG/total UnaG. Ratio distribution was analyzed by a box plot (Figure 3). Consequently, the relative local BR concentration (holo-UnaG/total UnaG) was quantified in each compartment. Nucleus and ER cytosolic membrane space ratios had the highest mean values (i.e., UnaG-NLS: 2.63 ± 0.40; UnaG-Sec61B: 2.20 ± 0.54 in HEK-293T cells, Table S2), and IMS and ER luminal space ratios had the lowest mean values (i.e., Sec61B-UnaG: 0.50 ± 0.11; Sco1UnaG: 0.08 ± 0.02 in HEK-293T cells, Table S2). This ratio is a relative value that could change depending on the fluorescence microscopy laser intensities, detector sensitivity, etc., however, the relative tendency (UnaG-NLS > UnaG-Sec61B > Matrix-UnaG ≈ UnaG-KDEL ≈ UnaG-NES > Sec61B-UnaG ≈ Sco1-UnaG) did not change between experiments with the same optical settings of the microscope. Proposed BR distribution in mammalian cell is shown in Figure 6A.

Subcellular BR distributions related to the BR metabolic pathway. Our results indicated that the local BR concentration was relatively high in the ER cytosolic membrane and nucleus but very low in the ER lumen and IMS. The high local BR concentration at the ER cytosolic membrane may be associated with BR biosynthetic machinery composed of two enzymes, heme oxygenase 1 (HO-1) and biliverdin reductase.2, 27 The catalytic domain of HO-1 is localized at the ER cytosolic membrane and biliverdin reductase is localized in the cytosol.2, 27 We hypothesized that highly lipophilicity of biliverdin and BR28 may result in their accumulation at their site of origin at the ER cytosolic membrane.

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To confirm our hypothesis, we tested sub-cellular targeted UnaG in amyloid beta precursor protein (APP)-overexpressing N2a (N2a-APP) and N2a cells. Snyder and co-workers showed that APP overexpression in neuronal cells affects total BR levels because the overexpressed APP physically interacts with HO-1 and decreases HO-1 activity in the neurons .27 In their study, they found that the total BR level decreased as APP expression increased, as measured by liquid chromatography. Using our genetically-encoded sensor, we attempted to observe the changes of subcellular compartmental BR level by APP expression. As shown in Figure 4A, the fluorescence of all UnaG constructs was higher in N2a cells compared with N2a-APP cells. Both cell lines were expressing APP but the level of APP expression was different, as measured by anti-APP antibody immunofluorescence. All the UnaG constructs examined had higher florescence intensity ratio values (AF488 FI/AF568 FI) in normal N2a cells than in N2a-APP cells (Figure 4B and Figure S7). Our results support the previous observation that decreasing HO-1 by overexpressing APP affects global BR levels. We also confirmed that APP localized to the ER (Figure S8), which supports the notion that its inhibition of BR biosynthesis occurs at the ER membrane. It is noteworthy that Taketani and co-workers reported that total intracellular BR levels decreased upon HO inhibitor treatment29, 30 and our results are broadly consistent with their findings.

Local BR levels in ER lumen regulated by UDP-glucuronosyltransferase We postulated that low BR concentrations in ER lumen may be caused by the conjugation of BR to glucuronic acid by UDP-glucuronosyltransferase (UGT1A1) whose catalytic domain is localized in the ER lumen.31, 32 This conversion of BR to BR-glucuronide by UGT1A1 is crucial for the enhancement of BR solubility in aqueous solutions and BR-glucuronide is secreted by an active transport system.33 We postulated that this active UGT1A1 conversion may reduce the concentra8 ACS Paragon Plus Environment

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tion of free BR in the ER lumen and that weak fluorescence intensity of UnaG-KDEL and Sec61BUnaG constructs may be associated with the decreased local concentration of unconjugated BR in the ER lumen, on account of the high specificity of UnaG binding of unconjugated, free, BR.26 To determine whether enzymatic activity of UGT1A1 was responsible for the low BR levels in ER lumen, we measured UnaG-KDEL fluorescence before and after indinavir (IDV) treatment. IDV was originally developed as a viral protease inhibitor for human immunodeficiency virus (HIV); however, IDV is also known to efficiently inhibit UGT1A1 activity.34 IDV (100 μM) was used to treat HEK-293T cells that expressed subcellularly targeted UnaG for 12 h, according to a previously reported IDV treatment protocol.34 As shown in Figure 5, the fluorescence of UnaG-KDEL construct increased more noticeably in IDV-treated HEK-293T cells than in non- treated samples, suggesting increased unconjugated BR levels in the ER lumen following UGT1A1 inhibition. The average pixel fluorescence intensity ratio (AF488/AF568) of UnaG-KDEL increased from 0.90 ± 0.23 to 1.77 ± 0.29 (Table S2). A similarly enhanced UnaG fluorescence was observed for Sec61BUnaG construct, which faces the ER lumen. Interestingly, fluorescence intensity of UnaGs at the ER cytosolic membrane also increased upon IDV treatment. For example, the fluorescence of C1(1-29)-UnaG targeted to the smooth ER cytosolic face was greatly enhanced with IDV and the average pixel intensity ratio increased from 2.24 ± 0.47 to 3.65 ± 0.86 (Table S2). Based on these findings, we postulated that the accumulated unconjugated BR in the ER luminal membrane might be flipped and spread to the ER cytosolic membrane (Figure 6B). Interestingly, the fluorescence of UnaG-NLS was barely affected by IDV presence and the pixel intensity somewhat decreased from 2.63 ± 0.40 to 2.49 ± 0.30 (Table S2). The fluorescence of UnaG-PM fusion was slightly reduced during IDV treatment. This suggests that local BR levels in the nucleus and plasma membrane were not directly affected by UGT1A1 inhibition.

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Since immunostaining cannot be performed in live cells, we prepared mCherry-fused UnaG constructs to observe the effects of IDV treatment on BR levels in targeted compartments in live cells. As shown in Figure S9, we observed that the average pixel fluorescence intensity ratio of UnaG-mCherry-Sec61B increased from 2.27 to 3.72 by IDV treatment. This fold increase (163%) was similar to fold signal increase in our immunostaining experiments (149%). We also found that other constructs (e.g., UnaG-mCherry-KDEL, LACTB-UnaG-mCherry, Matrix-UnaG-mCherry) showed a moderately increased AF488/AF568 ratio, while the change was negligible for UnaGmCherry-NLS, which was also observed in immunostaining analysis (Table S3). Notably, UnaG fluorescence is known to be quite stable in physiological pH condition.26 We verified that UnaG fluorescence did not change under different pH-buffered conditions (pH 4–11) and various redox potentials (NADH/NAD+ = 0–0.16%)36, as measured in fixed cells bearing the UnaG-NLS construct(Figure S10). We also confirmed that UnaG expression in living cells negligibly alters intracellular level of reactive oxygen species (ROS) (Figure S11),

Conclusion In this study, we demonstrated the utility of UnaG as an in cellulo genetically encoded fluorescent sensor for detecting BR levels in subcellular organelles. By fusing it with various signal sequences, we were able to target UnaG to diverse cellular organelles. We discovered that IMS is a relatively BR-depleted microspace, while the ER cytosolic membrane and the nucleus are relatively BR-enriched spaces. Furthermore, we found that majority BR is synthesized at ER and our results suggest the existence of plausible BR-transport and BR-regulating systems in other subcellular compartments, including the nucleus and mitochondria.


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Experimental Section Plasmid vector construction. Bacterial codon optimized UnaG gene was synthesized by Bioneer (Daejon, Korea). Fusion constructs were generated in the pcDNA3.1(+) mammalian expression vector by cloning the PCR-amplified UnaG gene and various organelle targeting sequences: nucleus exclusion signal (NES)37, and nucleus localization signal (NLS).38 ER lumen-targeting IgK leader sequence and resting signal sequence (KDEL) were used to create ER lumen resting construct (UnaG-KDEL). The entire peptide sequence of ER translocon transmembrane protein Sec61B was used for construction of a rough ER cytosol-facing membrane anchoring construct (UnaG-Sec61B) by fusing UnaG to Sec61B N-terminus. Rough ER luminal-facing membrane anchoring construct (Sec61B-UnaG) was created by fusing UnaG to Sec61B C-terminus.39 Smooth ER cytosolic facing membrane anchoring domain (C1(1–29)) of cytochrome P4502C238 was used for C1(1-29)-UnaG construction. Mitochondrial matrix targeting sequence of COXVIII 40 was used in mitomatrix-UnaG (or matrix-UnaG) fusion and mitochondrial intermembrane space (IMS) targeting sequence of LACTB

41

and whole peptide sequence of Sco142 was used for constructing

LACTB-UnaG and Sco1-UnaG, respectively. Plasma membrane (PM) anchoring domain

43

was

used to construct UnaG-PM. These signal sequences target different subcellular spaces as shown in Figure 1. Detailed sequence information and additional methods are described in the Supporting Information. All the plasmids reported here will be deposited at addgene (Cambridge, MA, USA). Mammalian cell culture and transfection. HEK-293T, U2-OS, and Neuro-2a (N2a) cells were obtained from ATCC (Manassas, VA, USA) (passages < 20). Cos-7 cells were purchased from Korean Cell Line Bank (Seoul, Korea). All cells were cultured in FBS-containing media. N2a cells stably overexpressing human APP were a generous gift from Professor Gopal Thinakaran (UniverACS Paragon Plus Environment

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sity of Chicago, USA). Cells were transfected at 60–80% confluence using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). After 12–18 h, UnaG fluorescence was measured. Fluorescence microscope imaging. For live cell imaging experiments (Figures 1, 2), the transfected cells which had been seeded onto fibronectin-coated round glass coverslips (Neuvitro Corporation, El Monte, CA, USA) were placed into DPBS and imaged using LSM780 Confocal Microscope (Zeiss, Solms, Germany). For the BR addition experiment, BR (10 μM in DPBS) was incubated for 1 min after the first image was taken and the same cell was imaged using the same imaging setup. For immunofluorescence measurements (Figures 3, 5), cells were fixed and permeabilized with 4% paraformaldehyde at room temperature for 15 min, and 0.4% Triton-X100 at room temperature for 1 h, respectively. Next, the cells were washed three times with Dulbecco’s phosphate-buffered saline (DPBS) and blocked for 1 h with 2% BSA in DPBS at room temperature. After blocking, the cells were incubated with primary antibodies, mouse anti-Flag antibody (Sigma Aldrich, St. Louis, MO, USA; 1:3000 dilution) and rabbit anti-APP antibody (Abcam, Cambridge, UK; cat#: 32136 1:3000 dilution) for Figure 5 only, for 1 h at room temperature. After washing four times (5 min each) with DPBS, the cells were incubated with secondary Alexa Fluor 568-goat antimouse IgG (Invitrogen, Carlsbad, CA, USA; 1:1000 dilution) and Alexa Fluor 647-goat anti-rabbit IgG (Invitrogen, 1:1000 dilution) for 30 min at room temperature. The cells were then washed four times (5 min each) with DPBS and maintained in DPBS on ice for imaging with LSM780 confocal microscope. Immunofluorescence image analysis. To extract relative local distributions of BR, we validated fluorescence intensity ratios between the expression level (anti-V5, AF568 channel) and fluorescence of holo-UnaG (AF488 channel) by analyzing immunofluorescence images. The AF488/AF568 ratio at each pixel could represent the ratio of holo-UnaG (BR-UnaG)/total UnaG at that pixel. To validate the expression level of total UnaG, we designed all our UnaG constructs ACS Paragon Plus Environment

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to incorporate the Flag peptide sequence (DYDDDDK) for immunofluorescence assaying with mouse anti-Flag antibody. This anti-Flag antibody can be detected by secondary Alexa Fluor 568goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA; 1:1000 dilution) and this fluorescent intensity can be observed in AF568 channel. To detect locally distributed BR, we measured holo-UnaG fluorescence, which was not compromised in fixed/permeabilized samples. This fluorescent intensity can be observed in AF488 channel. For every imaging experiment, we verified that there was no significant signal bleed-through between AF568 and AF488 channels in our microscope settings. Finally, 2000 pixels per image were randomly chosen and AF568 fluorescence intensity and AF488 fluorescence intensity at the same pixel position was extracted and analyzed by scatter plot analysis (Figure S6) and the AF488 FI/AF568 FI ratio was analyzed by box plot analysis (Figure 3). All images that were used in scatter plot analysis and box plot analysis are shown in Supporting Information. Indinavir treatment assay. For indinavir (IDV) treatment, HEK-293T cells from ATCC (Manassas, VA, USA) (passages < 20) were cultured in FBS-containing media. After transfection with the organelle constructs as described above, indinavir (100 µM) was treated in growth medium for 12 h. The cells were then washed four times (5 min each) with DPBS and immunofluorescence measurements were carried out as described above. Leica TCS SP8 STED 3X confocal microscopy was used for imaging.

ASSOCIATED CONTENT Supporting Information Detailed experimental information, Figures S1-11 and Tables S1-3. This material is available free of charge via the Internet at http://pubs.acs.org.


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Corresponding Author Hyun-Woo Rhee ([email protected]) and Mi-Hee Lim ([email protected]) Acknowledgement UnaG gene was a generous gift of Dr. Miyawaki (RIKEN, Japan) and codon-optimized UnaG was synthesized in Bioneer (Daejon, Korea). This work was supported by NRF (2013R1A1A1057597) and the 2016 UNIST research fund (1.160001.01). Instrumentation support was from Samsung Science and Technology Foundation (SSTF-BA1401-11).

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Figure Legends Figure 1. Detection of subcellularly localized bilirubin by UnaG. (A) Scheme of bilirubin detection by UnaG. (B) Targeted sub-cellular compartments of UnaG constructs and enzymes which are associated with bilirubin metabolism in this study. C1(1-29), cytoplasmic facing ER membrane-targeting domain. IMS, intermembrane space of the mitochondria. Two IMS-UnaG constructs, LACTB-UnaG and Sco1-UnaG, were tested in this study. PM, plasma membrane. NES, nuclear exclusion signal. NLS, nuclear localization signal. KDEL, ER lumen localization signal with N-terminal ER lumen-targeting sequence. HO-1, heme oxygenase 1. BVR, biliverdin reductase. UGT1A1, UDP-glucuronosyltransferase 1A. (C) Map of genetic constructs used in this study, targeting UnaG to different organelles.

Figure 2. In cellulo BR sensing in various organelles using UnaG in live HEK-293T cells. Images before exogenous BR treatment are shown (left) and images after BR (10 µM) treatment are shown (right). Scale bar = 20 µm.

Figure 3. Ratios of holo-UnaG (BR-UnaG) to total UnaG in different subcellular compartments in different cell types. (A) Scheme of pixel-wise fluorescence intensity analysis from the images. (B) Box plot representation of pixel-wise intensity ratio (AF488/AF568) in images of various subcellular compartments targeted UnaG in different mammalian cell lines (top: HEK293T cells; middle: U2-OS cells; bottom: N2a cells). Original images for this analysis are shown in Supporting Information.


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Figure 4. Detection of local BR level in various subcellular compartments of Neuro 2a and Neuro 2a-APP cell lines. (A) UnaG fluorescence (AF488 channel) and immunofluorescence (Flag: AF568 and APP: AF647 channel) in N2a and N2a-APP cell lines with various constructs targeting ER membrane facing cytosol (UnaG-Sec61B), ER lumen (KDEL), nucleus(NLS), mitochondria matrix(Matrix) and plasma membrane(PM). UnaG fluorescence was detected in AF488 channel and UnaG expression level was detected in AF568 channel using anti-Flag antibody and anti-mouse-AF568 antibodies. APP expression was assessed in AF647 channel using anti-APP antibody and anti-rabbit-AF647 antibodies. Higher laser intensity image of APP is optimized for endogenous APP levels in N2a cell line. The merge image shows that UnaG transfection did not alter APP expression levels in N2a and N2a-APP cell lines. All UnaG constructs were imaged using the same microscope imaging setup. Scale bar = 20 µm. (B) Box plot presentation of pixel-wise intensity ratio (AF488/AF568) for images shown in (A).

Figure 5. The effect of IDV treatment on subcellular BR level in Hek-293T cells (A) Confocal-microscopy images. UnaG fluorescence was detected in AF488 channel and UnaG expression was assessed in AF568 channel using anti-Flag and anti-mouse-AF568 antibodies. All immunofluorescence images were generated with LSM780 Confocal Microscope (Zeiss, Solms, Germany). All transfected cells were incubated with IDV overnight, at 37 °C in CO2 incubator. Scale bar = 100 µm. (B) Box plot representation of pixel-wise intensity ratio (AF488/AF568) of images shown in (A).

Figure 6. A proposed model of bilirubin distribution in living cells. (A) Proposed BR distribution in mammalian cells. (B) Asymmetric distribution of BR in ER membrane and the associated BR metabolic pathway. BV, biliverdin; BVR, biliverdin reductase. ACS Paragon Plus Environment

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Figure1


A

B (

=UnaG)

Mitochondria IMS

Tom20

UnaGSec61B

KDEL

PM Sec61BUnaG

Matrix UGT1A1 HO-1

BVR

NLS NES

Cytoplasm

Nucleus C

NES

Flag

UnaG-NES

PM targeting sequence UnaG-PM

UnaG

UnaG

NLS

Flag

UnaG-NLS

UnaG

Flag ERM targeting sequence Flag

ER lumen sequence KDEL UnaG-KDEL

C1(1-29)-UnaG

UnaG Flag

Sec61B-UnaG

Sec61B

UnaG

Flag

Flag UnaG-Sec61B

Mitomatrix targeting sequence Matrix-UnaG

UnaG

UnaG

Flag Tom20-UnaG

Tom20

Sec61B

IMS targeting sequence of LACTB LACTB-UnaG

UnaG

UnaG

Flag Sco1-UnaG

UnaG Flag


Sco1

UnaG Flag


Figure 2. Living cell

Living cell + Addition of BR

UnaG-PM

Sec61B-UnaG

UnaG-NLS

UnaG-Sec61B

TOM20-UnaG

C1(1-29)-UnaG

ScoI-UnaG

UnaG-KDEL

UnaG-NES

Living cell

Living cell + Addition of BR

Matrix-UnaG

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Figure 3. B

Flag-UnaG-Sec61B UnaG fluorescence (AF488 channel) Anti-Flag (AF568 channel)

Ratio (AF488/AF568)

A

HEK-293T (n=2000)

Pixel 1 Pixel 2

AF488

AF568

AF488/ AF568

623 253

125 802

4.9 0.31

Ratio (AF488/AF568)

Pixel 1 Pixel 2

U2-OS (n=2000)

Box Plot Analysis

Pixel 1→

Pixel 2→

Ratio (AF488/AF568)

…

Ratio (AF488/AF568)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


UnaG-Sec61B


N2a (n=2000)

A

Anti-Flag

Merge

NLS N2a N2a-APP

KDEL N2a N2a-APP

UnaG-Sec61B N2a N2a-APP

UnaG

Matrix N2a N2a-APP

B Ratio (AF488/AF568)

Figure 4

Anti-APP Laser intensity

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PM N2a N2a-APP

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N2a N2a APP

N2a N2a N2a N2a APP APP

UnaG-Sec61B UnaG-KDEL UnaG-NLS

N2a N2a N2a N2a APP APP

Matrix-UnaG


UnaG-PM

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Figure 5. A

+ IDV

No IDV UnaG

Anti-Flag Overlay

UnaG

No IDV

Anti-Flag Overlay

UnaG

Anti-Flag Overlay

+ IDV UnaG

Anti-Flag Overlay

NLS

UnaG- Sec61BC1(1-29) Sec61B UnaG

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NES ScoI

KDEL

PM

Matrix

TOM20

Ratio (AF488/AF568)

B

UnaG UnaG UnaG ScoI Matrix Tom20 UnaG C1(1-29) UnaG Sec61B -NLS -NES -PM -UnaG -UnaG -UnaG -KDEL -UnaG -Sec61B -UnaG

(n=9000) ACS Paragon Plus Environment


Figure 6 A Bilirubin-enriched Bilirubin normal Bilirubin-depleted

B heme BV Reductase UGT1A1

Cytosol

HO-1

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ER lumen

= bilirubin

= bilirubin glucuronide


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TOC Graphic 478x152mm (96 x 96 DPI)


In Cellulo Mapping of Subcellular Localized Bilirubin - ACS Chemical

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