Labeling Lysosomes and Tracking Lysosome-Dependent Apoptosis

31 May 2012 - ABSTRACT: In this study, we describe a new strategy for labeling and tracking lysosomes with a cell-permeable fluorescent activity-based...
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Labeling Lysosomes and Tracking Lysosome-Dependent Apoptosis with a Cell-Permeable Activity-Based Probe Fengkai Fan,†,‡ Si Nie,†,‡ Dongmei Yang,†,‡ Meijie Luo,†,‡ Hua Shi,†,‡ and Yu-Hui Zhang*,†,‡ †

Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, and ‡Key Laboratory of Biomedical Photonics of Ministry of Education, Department of Biomedical Engineering, Huazhong University of Science and Technology (HUST), 1037 Luoyu Road, Wuhan 430074, P. R. China S Supporting Information *

ABSTRACT: In this study, we describe a new strategy for labeling and tracking lysosomes with a cell-permeable fluorescent activity-based probe (CpFABP) that is covalently bound to select lysosomal proteins. Colocalization studies that utilized LysoTracker probes as standard lysosomal markers demonstrated that our novel probe is effective in specifically labeling lysosomes in various kinds of live cells. Furthermore, our studies revealed that this probe has the ability to label fixed cells, permeabilized cells, and NH4Cl-treated cells, unlike LysoTracker probes, which show ineffective labeling under the same conditions. Remarkably, when applied to monitor the process of lysosome-dependent apoptosis, our probe not only displayed the expected release of lysosomal cathepsins from lysosomes into the cytosol but also revealed additional information about the location of the cathepsins during apoptosis, which is undetectable by other chemical lysosome markers. These results suggest a wide array of promising applications for our probe and provide useful guidelines for its use as a lysosome marker in lysosome-related studies.

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contain two key elements: a reactive “warhead”, which covalently binds the active site of the targeted enzymes, and a reporter tag, such as a fluorophore or biotin, which allows the labeled enzymes to be identified, purified, and/or visualized. Recently, ABPs have been used widely for functional proteomic studies of various enzymes, such as metalloproteases, serine, and cysteine proteases.22 However, ABPs have not yet been utilized as markers for labeling and tracking intracellular organelles. In this study, we describe a novel strategy that employs cell-permeable fluorescent ABPs (CpFABPs) to label and track lysosomes. The CpFABPs have three components: (a) an epoxysuccinyl scaffold that has been reported to selectively form covalent bonds with cysteine cathepsins,23−25 (b) a fluorophore for visualizing the covalently bound enzymes, and (c) an arginine-rich cell-penetrating peptide (CPP) to engender the cellular permeability of the probe.26 We demonstrate that the CpFABPs may be used as highly selective and biocompatible lysosomal markers not only in various kinds of live cells but also in fixed cells, permeabilized cells, and NH4Cl-treated cells. In addition, the CpFABP was also shown to track lysosome-dependent apoptosis in real-time in live cells.

INTRODUCTION Lysosomes are dynamic, membrane-bound organelles that contain more than 50 acid hydrolases and a high proton concentration (pH ≤ 5).1 The primary function of lysosomes is to degrade endocytosed and intracellular material. However, increasing evidence has shown that lysosomes are also involved in other complex and layered activities, such as intracellular transport, cell antigen processing, and the initiation of apoptosis.1−4 Lysosomal dysfunction has been implicated in diverse diseases, including inflammation, tumor formation, and several neurodegenerative diseases.5−7 The ability to visualize lysosomes is critical for understanding intracellular metabolism8 and cell membrane recycling,9 and for evaluating drug and gene delivery systems.10,11 A variety of chemical markers for labeling lysosomes have been developed, and their labeling strategies can be divided into two main types. The first type of label exploits the acidic property of lysosomes and the tendency for weak basic amines to selectively accumulate in acidic organelles. Small-molecule markers, including DAMP,12 neutral red,13 acridine orange,14 and the LysoTracker probes,15 are examples of this labeling strategy. The other strategy utilizes large molecules, such as dextran labeled with a fluorophore,16,17 modified quantum dots,18 and nanoparticles (DSiNPs),19 and their ability to enter living cells through endocytosis.18−20 This ultimately leads to localization within the lysosome, as it is the final compartment of the degradative endocytotic pathway.21 Activity-based probes (ABPs) are small synthetic molecules that can profile the enzymatic activity in proteomes.22 ABPs © 2012 American Chemical Society

Received: March 20, 2012 Revised: May 7, 2012 Published: May 31, 2012 1309

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EXPERIMENTAL PROCEDURES Materials. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), horse serum (hyclone), glutamax, CO2-independent medium, and Opti-MEM I Reduced Serum Medium (Opti-MEM) were purchased from Gibco BRL. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich. Lipofectamine 2000 and LysoTracker DND 26 were purchased from Invitrogen. LysoTracker Red was obtained from Beyotime. Ammonium chloride (NH4Cl) and hydrogen peroxide (H2O2) were supplied by the Sinopharm Chemical Reagent Beijing Co. Ltd. Fmocamino acids and all other chemical reagents were obtained from GL Biochem. All starting materials were used without additional purification. Synthesis of the Probes. Probes CpFABP-G, CpFABP-R, 1, and 2 were synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) on 2-chlorotrityl chloride resin, with standard deprotection and coupling procedures. The probes were purified by preparative high-performance liquid chromatography (HPLC) to reach a purity greater than 90%, and the mass was confirmed by electrospray ionization (ESI) mass spectrometry (Figure S1 of the Supporting Information). Cell Culture. HeLa cells (human epitheloid cervix carcinoma cell line), MCF-7 cells (human breast adenocarcinoma cell line), Cos-7 cells (African Green Monkey fibroblast-like kidney cell line), and RAW 264.7 cells (mouse leukemic monocyte macrophage cell line) were cultured in DMEM supplemented with 10% (v/v) FBS. Cells were grown at 37 °C under CO2 (5%, v/v) for 2−3 days. Peritoneal macrophage cells were collected from C57 black mice 3 days after intraperitoneal injection of 1.5 mL 6% starch broth and suspended in RPMI-1640 medium containing 10% FBS, penicillin (100 IU/mL), and streptomycin (50 μg/mL). The cell suspensions were plated onto eight-well Lab-Tek chamber slides (Nunc). After incubation for 3 h at 37 °C under an atmosphere of 5% CO2, the cells were then washed gently twice to remove the nonadherent cells. Primary hippocampal neurons were obtained from embryos (E18) of pregnant mice. Hippocampi was dissected and treated with 0.125% trypsin at 37 °C for 10 min. The cells were dissociated by trituration and then suspended in DMEM containing 10% heat-inactivated horse serum and 2 mM glutamax. Then, the cells were seeded onto eight-well Lab-Tek chamber slides at a density of 2.0 × 104 cells/well. The chamber slides were precoated via a 12 h incubation in poly(L-lysine) followed by washing and an 8 h incubation with laminin. The cells were cultured at 37 °C under CO2 (5%, v/v) for 3 days. Confocal Laser Scanning Microscopy. HeLa cells, MCF7 cells, Cos-7 cells, or RAW 264.7 cells were seeded onto eightwell Lab-Tek chamber slides (Nunc) at a density of 1.0 × 104 cells/well in DMEM (300 μL) containing 10% FBS. The cells were then grown at 37 °C in CO2 (5%, v/v) overnight. They were washed with phosphate-buffered saline (PBS, pH 7.4) and were incubated with the indicated probes (10 μM, 300 μL) in PBS for 30 min. The medium was discarded, and the cells were rinsed with PBS. After a postincubation in DMEM (300 μL) containing 10% FBS for 0, 2, 4, 8, 12, or 23 h, the cells were analyzed with a confocal laser scanning microscope. To stain lysosomes, the cells were incubated with the indicated concentrations of LysoTracker Red or DND 26 (300 μL) for 30 min before imaging.

Cells were also stained with the indicated probes and LysoTracker Red or DND 26 (as mentioned previously) before NH4Cl treatment or fixation and permeabilization. The labeled cells were washed with PBS and exposed to 10 mM NH4Cl in PBS (300 μL) for 1 h at 37 °C with 5% CO2, or fixed by 4% paraformaldehyde in PBS (300 μL) at room temperature for 20 min, or fixed (4% paraformaldehyde) and then permeabilized by 0.2% Triton-X 100 in PBS (300 μL) at room temperature for 5 min. To track lysosome-dependent apoptosis, HeLa cells were either transfected or not transfected with cathepsin B-mLumin. They were then incubated with CpFABP-G (10 μM, 300 μL) in PBS for 30 min followed by a 4 h postincubation in DMEM (300 μL) containing 10% FBS, or they were labeled with LysoTracker Red or DND 26 (300 μL, 1 μM in PBS) for 30 min. The stained cells were either left untreated or treated with 1 mM H2O2 in a CO2-independent medium at 37 °C under 5% CO2 for 1.5 h and were analyzed using a confocal laser scanning microscope. Images were obtained every 5 min. To detect the probes, the fluorescence signals were detected using a Fluoview FV1000 (Olympus, Japan) confocal laser scanning microscope equipped with a 60× oil-immersion objective lens (NA 1.4) and a HeNe-G laser (Green CpFABPG channel: excitation (EX) 488 nm, emission (EM) 505− 555 nm; LysoTracker DND 26 channel: EX 488 nm, EM 505− 535 nm; Red CpFABP-R, LysoTracker Red, and cathepsin B-mLumin channel: EX 543 nm, EM 570−680 nm). All fluorescence images were analyzed and the background subtracted with Image J software. Pearson’s coefficient was quantified using the JACOP plugin from Image J.27 Direct Labeling of Proteins in Living Cells. RAW 264.7 cells were seeded onto 100 mm culture dishes (Corning) at a density of 1.0 × 107 cells/well in DMEM (8 mL) containing 10% FBS and grown at 37 °C in CO2 (5%) overnight. The cells were then incubated with CpFABP-G or probe 2 in PBS (5 mL, 10 μM) for 30 min. Subsequently, all media were replaced by DMEM containing 10% FBS. After a 5 h postincubation, the cells were harvested by trypsin digestion and washed with cold PBS. The cell pellets were incubated in RIPA buffer (Beyotime Inst. Biotech) containing 1× complete Mini Protease Inhibitor Cocktail (Roche Applied Science) and were sonicated with 100 × 1 s pulses (with a 2 s pause between pulses) in an icecold water bath. Subsequently, the cell lysates were centrifuged at 40 000 g for 20 min at 4 °C to remove cellular debris. The protein concentration of the supernatant was determined using Pierce’s Coomassie (Bradford) Assay. The supernatants (20 μg/lane) were added to 5× SDS sample buffer, denatured by boiling for 5 min, and resolved on a 13.5% SDS-PAGE gel. The gels were visualized using a Typhoon 9410 laser scanning system (Amersham Biosciences). In addition, some samples on the gel were transferred onto a polyvinylidenedifluoride membrane, and the membrane was then divided into several pieces. The membrane containing proteins between 26 kD and 43 kD was analyzed by Western blot with anticathepsin B polyclonal antibody (1:1000). The membrane was incubated with horseradish peroxidase (HRP)conjugated goat antirabbit secondary antibody (1:4000). The HRP activity was visualized using the enhanced chemiluminescence (ECL) system. The images were collected using a ChemiDoc XRS system (Bio-Rad). Competitive Labeling Study. RAW 264.7 cells were seeded onto 100 mm culture dishes (Corning) at a density of 1.0 × 107 cells/well in DMEM (8 mL) containing 10% FBS and 1310

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grown at 37 °C under CO2 (5%, v/v) overnight. The cells were harvested by trypsin digestion. Then, they were washed with cold PBS, and the cell pellets were lysed with 50 mM citrate phosphate buffer (pH 4.5) containing 1% CHAPS, 0.5% Triton X-100, and 5 mM DTT for 30 min on ice. Subsequently, the cell lysates were centrifuged at 17 000 g for 15 min at 4 °C to remove cellular debris. The supernatants were diluted in the lysis buffer to reach a final concentration of 10 μg/μL, and they were aliquoted and stored at −80 °C. The supernatants (8 μL, 10 μg/μL) were treated with the indicated concentrations of E-64 for 1 h and were incubated with CpFABP-G (final concentration of 10 μM) for an additional hour. Subsequently, the lysates were added to the 5× SDS sample buffer, denatured by boiling for 5 min, and resolved on a 13.5% SDS-PAGE gel. The results were visualized and recorded by the Typhoon 9410 laser scanning system (Amersham Biosciences). Cell Toxicity Analysis. The cytotoxicity of CpFABP-G was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HeLa cells were seeded into a 96-well plate at a density of 5000 cells/well in DMEM (150 μL) containing 10% FBS. The cells were then grown at 37 °C under CO2 (5%, v/v) overnight. The cells were incubated with 150 μL of CpFABP-G (5, 10, or 20 μM) in DMEM containing 10% FBS for 12 h. Then, 15 μL of the MTT solution (5 mg/mL in PBS) was added to each well. After 4 h, the medium was gently removed, and the formazan products were solubilized with 200 μL of dimethyl sulfoxide (DMSO) for 30 min. Absorbance was measured at 570 nm using the ELISA Reader GeniosPlus (Tecan). Cell viability was expressed as a ratio of the A570 of probe-treated cells to the untreated controls. All measurements were performed in triplicate. Plasmid Construction. Using the pDsRed-N1-cathepsin B template (a generous gift from Prof. Jianping Liu, Inst. of Genetics, Fudan University), a DNA fragment encoding cathepsin B with restriction sites for BamHI and XhoI was amplified by PCR. The primers used for the cathepsin B amplification were as follows: forward 5′-ACGCGGATCCCACCATGCTGCCTGCAAGCTTCGAT-3′ and reverse 5′-CGGTCGGAATTCCTCGAGATCGGTGCGTGGAATGCCAGCCACCACTTCTGATTCGA-3′. The coding sequence of mLumin was amplified according to methods recently reported28and contained restriction sites for XhoI and EcoRI. The mLumin forward primer was 5′-CGCACCGATCTAGAGCCGGTCGCCACCATGGTGTCTAAGGGCGAAGAGC-3′, and the reverse was 5′-ACCGGAATTCTTACTTGTACAGCTCGTCCATGCCATTAA-3′. After the products were purified on an agarose gel (1%), the cathepsin B sequence was digested with BamHI and XhoI. The mLumin DNA fragment was cut by XhoI and EcoRI. The resulting DNA fragments were inserted into the pCDNA 3.1 vector (Invitrogen) that had been digested by BamHI and EcoRI. The sequence of the pCDNA 3.1-cathersin BmLumin construct was confirmed by sequencing. Transfection. HeLa cells were seeded onto eight-well LabTek chamber slides at a density of 2 × 104 cells/well in DMEM (300 μL) containing 10% FBS. The cells were then grown at 37 °C with CO2 (5%, v/v) for 24 h. Then, all medium was replaced by fresh Opti-MEM (150 μL). Transient transfections were performed using the Lipofectamine 2000 reagent following the manufacturer’s instructions. After 24 h, the transfected cells were used for the labeling experiments.

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RESULTS Synthesis of CpFABPs. We synthesized the CpFABPs using standard solid-phase peptide synthesis (Figure 1).29,30

Figure 1. Structures of probes CpFABP-G, CpFABP-R, 1, and 2, FAM, 5(6)-carboxyfluorescein.

CpFABP-G and CpFABP-R contain different fluorophores (5(6)-carboxyfluorescein (FAM) in CpFABP-G and rhodamine B in CpFABP-R). The epoxysuccinyl scaffold (Figure 1) is generated from the commonly used cysteine cathepsin ABP DCG-04 and has been shown to selectively label numerous cysteine cathepsins in cell and tissue lysates, including cathepsins B, F, C, H, K, L, S, and Z, which are typically localized to lysosomal compartments.24,31 We employed a cell-penetrating octa-arginine peptide (rRrRrRRR, r: D-Arg, R: L-Arg) to enhance the cellular permeability of CpFABPs, which contain cellimpermeable fluorophore (e.g., FAM). We also generated probe 1, which does not contain a cell-penetrating peptide, and probe 2, which does not contain the reactive scaffold, as controls for CpFABP-G (Figure 1b). Characterization of CpFABPs for Labeling Lysosomes. The LysoTracker probes are the most popular fluorescent acidotropic probes for lysosome labeling and tracking due to their high selectivity and effective labeling of living cells.15 Therefore, we investigated the feasibility of using CpFABP-G and CpFABP-R as lysosome markers by performing a colocalization study employing the LysoTracker probes as the standard lysosome markers. After incubation with LysoTracker Red for 30 min or 2 h at 75 nM (a recommended working concentration in the manufacturer’s instructions), the cells could not be sufficiently stained; therefore, we increased the labeling concentration of the dye, as shown in previous studies (Figure S2 of the Supporting Information).19 Figure 2a indicates that, after incubation with living cells for 30 min, CpFABP-G entered the cells efficiently and exhibited a diffuse pattern with a small amount of vesicle staining. In contrast, no obvious fluorescent signal was detected for probe 1, the probe that lacked a cell-penetrating peptide (Figure 2b). These results suggest that the introduction of the cell-penetrating peptide significantly increases the cell permeability of CpFABP-G. An increase in postincubation time from 0 to 4 h (Figure 2a) revealed that the diffuse green fluorescence staining of 1311

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Figure 2. Feasibility of CpFABP-G as a lysosome marker. (a) Confocal microscopy of living HeLa cells that were incubated with CpFABP-G (green, 10 μM) for 30 min and postincubated in DMEM containing 10% FBS for 0, 2, 4, 8, 12, and 23 h, respectively. Lysosomes were labeled with LysoTracker Red (500 nM) for 30 min before imaging. (b) Confocal microscopy of living HeLa cells that were incubated with probe 1 (10 μM) for 30 min. (c) Confocal microscopy of living HeLa cells that were incubated with probe 2 (10 μM) for 30 min and postincubated in DMEM containing 10% FBS for 0 and 4 h, respectively. Scale bar: 10 μm. (d) Quantitative fluorescence colocalization analysis. The colocalization of CpFABP-G with LysoTracker Red was quantified by Pearson’s coefficients35 (mean ± SD, n = 6) that were obtained with Image J.

0.746 ± 0.053, 0.841 ± 0.007, 0.834 ± 0.098, and 0.832 ± 0.013 for Cos-7 cells, MCF-7 cells, Raw 264.7 cells, primary peritoneal macrophage cells, and primary neurons, respectively. The results indicate that CpFABP-G is an effective lysosomal marker in many cell types. We also measured cell viability using the MTT assay to test the cellular toxicity of CpFABP-G (Figure S4 of the Supporting Information). HeLa cells were incubated with CpFABP-G for 12 h at concentrations up to 20 μM, and no obvious cytotoxicity was observed, indicating that the probe is very biocompatible. As shown in Figure 3b, CpFABP-R, with the red fluorophore (rhodamine B), also colocalized well with the green LysoTracker DND 26 probe (Pearson’s coefficient = 0.749 ± 0.025), suggesting that altering the fluorophore that is attached to the probe does not influence its ability to label lysosomes. We used fluorescein and rhodamine B because they are the most commonly used and relatively inexpensive. Considering that various smallmolecule fluorophores with pH-insensitivity, high photostability, and different fluorescent colors, such as Alexa Fluor dyes,32 are commercially available and that CpFABP-G exhibited a long retention time (>23 h) in lysosomes (Figure 2a and d), a variety of the probes with an array of fluorophores are promising for multicolor labeling applications and long-term lysosomal tracking. Living cells were labeled with CpFABP-G or probe 2, lysed, and analyzed by SDS-PAGE and in-gel fluorescence scanning or Western blotting. Figure 4a shows that there was no obvious fluorescent staining on the SDS-PAGE gel for probe 2, the probe lacking a reactive warhead. In contrast, multiple proteases were labeled by CpFABP-G, and Western blot analysis using

CpFABP-G throughout the cytosol gradually gave way to increased staining in punctate vesicles. After 4 h postincubation, CpFABP-G colocalized with the LysoTracker Red (200 or 500 nM) probe extremely well (Figure 2d and Figure S2 of the Supporting Information),27 indicating that it may be used as a highly specific lysosomal marker in living cells. In contrast, probe 2, which lacked the reactive warhead (Figure 2c), maintained a diffuse staining pattern throughout the cytoplasm and nucleus during the postincubation from 0 to 4 h, suggesting that cellular clearance of the probe is not obvious within 4 h. The results also imply that the disappearance of the cytosolic diffuse staining of CpFABP-G is likely due to accumulation of the probe into lysosomes from the cytosol rather than due to exclusion from the cells. Moreover, prolonging the postincubation time to 8 and 12 h did not significantly increase the colocalization efficiency of CpFABP-G (Figure 2d). Therefore, the postincubation time of 4 h was utilized in further experiments. Increasing the concentration of CpFABP-G from 2.5 to 10 μM increased its colocalization efficiency, and no further obvious increment was observed at 15 μM (Figure S3 of the Supporting Information). It is worth noting that, even after 23 h postincubation, CpFABP-G maintained excellent colocalization with the LysoTracker Red (freshly applied to the cells before imaging) with a Pearson’s coefficient of 0.648 ± 0.022 (Figure 2a and d), indicating that it was retained in lysosomes for a long time (>23 h). We also tested the ability of CpFABP-G to label lysosomes in various kinds of cells. As shown in Figure 3a, excellent staining patterns of lysosomes have been achieved in all live cells that were tested, with Pearson’s coefficients of 0.804 ± 0.030, 1312

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Figure 3. Confocal microscopy images of (a) living Cos-7 cells, MCF-7 cells, Raw 264.7 cells, primary peritoneal macrophage cells, and primary neurons that were incubated with CpFABP-G (10 μM) for 30 min and postincubated in DMEM containing 10% FBS for 4 h and (b) living HeLa cells that were incubated with CpFABP-R (10 μM) for 30 min and postincubated in DMEM containing 10% FBS for 4 h. Lysosomes were labeled with LysoTracker Red or DND 26 (500 nM) for 30 min before imaging. Scale bar: 10 μm. Pearson’s coefficients (mean ± SD, n = 6) for Cos-7 cells, MCF-7 cells, Raw 264.7 cells, primary peritoneal macrophage cells, primary neurons, and HeLa cells 0.804 ± 0.030, 0.746 ± 0.053, 0.841 ± 0.007, 0.834 ± 0.098, 0.832 ± 0.013, and 0.749 ± 0.025, respectively.

Application of CpFABPs to Fixed Cells, Permeabilized Cells, and NH4Cl-Treated Cells. Cell fixation and permeabilization are often required to probe intracellular structures using immunocytochemistry or are used for better morphological preservation.35 Therefore, we investigated the ability of CpFABPs to label lysosomes following fixation and permeabilization. After fixation by 4% paraformaldehyde or fixation following permeabilization with 0.2% Triton X-100, the fluorescence of both LysoTracker Red (red, Figure 5a−c) and DND 26 (green, Figure S6 of the Supporting Information) completely disappeared within the cells. In contrast, fixation and permeabilization had little effect on the lysosome labeling pattern of CpFABP-G (Figure 5a−c) and CpFABP-R (Figure S6 of the Supporting Information). The absence of the fluorescence of the LysoTracker probes may be a result of cellular fixation and permeabilization-mediated destruction of the acidic environment of lysosomes, which prevents these weak basic probes from accumulating in the lysosomes. In contrast, CpFABP-G, which is pH-independent and labels lysosomes by binding the proteins inside them, maintained its ability to label lysosomes under the same conditions. Ammonium chloride (NH4Cl), a lysosomal inhibitor, is often employed in lysosome-related studies. It can neutralize the acidic environment in lysosomes and thus decrease normal lysosomal protease activities.36,37 Figure 5d shows that, after the cells were treated with NH4Cl, the red fluorescence of LysoTracker Red completely disappeared due to the neutralization of the acidic environment in lysosomes with NH4Cl. In contrast,

Figure 4. Analysis of labeled proteins by SDS-PAGE followed by in-gel fluorescence scanning or Western blotting. RAW 264.7 cells were incubated with CpFABP-G or probe 2 (10 μM) for 30 min. After 5 h postincubation in DMEM with 10% FBS, the cells were lysed. Samples were separated on a 13.5% SDS-PAGE gel and visualized by in-gel fluorescence scanning (a) or by Western blotting using anticathepsin B polyclonal antibody (b).

anticathepsin B polyclonal antibody indicated that one of the labeled proteins is possibly cathepsin B (Figure 4a and b). Pretreatment with E-64, a natural, potent, and irreversible inhibitor of cysteine cathepsins,33 competes specifically with the labeling by CpFABP-G in the lysates, suggesting that CpFABPG is specific for cysteine cathepsins, in good agreement with previous studies (Figure S5 of the Supporting Information).34 At this time, we are unable to identify each of the proteases labeled by CpFABP-G in living cells; however, the results indicate that the ability of CpFABP-G to label cysteine cathepsins, such as cathepsin B, is through the reactive warhead. 1313

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Figure 5. Confocal microscopy images of living HeLa cells (a) stained with CpFABP-G and LysoTracker Red, (b) stained with CpFABP-G and LysoTracker Red and then fixed with 4% paraformaldehyde, (c) stained with CpFABP-G and LysoTracker Red and then fixed with 4% paraformaldehyde following permeabilization with 0.2% Triton X-100, and (d) stained with CpFABP-G and LysoTracker Red and then treated with 50 mM NH4Cl for 1 h. Scale bar: 10 μm.

the treatment with NH4Cl had little effect on the lysosome labeling pattern of CpFABP-G. Taken together, these results suggest that CpFABPs may be employed for lysosome labeling in living cells, fixed cells, permeabilized cells, and NH4Cl-treated cells, demonstrating their potential for use in a wide array of applications. Application of CpFABP-G for Tracking LysosomeDependent Apoptosis. Apoptosis is a tightly regulated process of programmed cell death. Improper regulation of apoptosis has been implicated in various cancers, autoimmune diseases, and neurodegenerative disorders.7,38,39 Throughout the past decade, accumulating evidence has suggested that lysosomes play an important role in the regulation of cell death and that the release of lysosomal cathepsins, such as cathepsins D, B, and L, into the cytosol, is often involved in the initiation of apoptosis.1,3,40 Lysosomal markers, such as the LysoTracker probes, acridine orange, and fluorescence-labeled dextran, have been used to monitor lysosome-dependent apoptosis.17,40 They monitor apoptosis indirectly by tracking diffusion of the probe upon lysosomal membrane breakdown, rather than by visualizing the release of lysosomal cathepsins. A pepstatin A−BODIPY FL conjugate can directly track cathepsin release by binding with cathepsin D through pepstatin A; however, after pepstatin A−BODIPY FL−cathepsin D complexes are released into the cytosol, their green fluorescence disappears.41 Probes that are designed based on the substrates of cathepsins, such as the Magic Red cathepsin reagent, have also been employed to detect apoptosis.42 However, these substratebased probes provide a readout of enzyme activity but is not linked to the target of the protease; hence, the signals observed in living cells cannot be directly correlated with the localization of cathepsins. Because the epoxysuccinyl scaffold of CpFABP-G can selectively form a covalent bond with cysteine cathepsins, we investigated the feasibility of using CpFABP-G as a marker to track lysosome-dependent apoptosis. Although we have not currently identified all of the proteases labeled by CpFABP-G in living cells, Figure 4 reveals that CpFABP-G was indeed able to label cathepsin B. We used a cathepsin B-mLumin28 (a red fluorescent protein) fusion protein as a standard for cathepsin release because cathepsin B has been shown to be translocated from lysosomes into the cytosol during apoptosis.42,43 Figure 6a indicates that CpFABP-G colocalized extremely well with

Figure 6. Treatment with 1 mM H2O2 results in the partial release of cathepsin B from lysosomes. HeLa cells transiently transfected by cathepsin B-mLumin were stained with CpFABP-G (green, 10 μM) for 30 min and were postincubated for 4 h. The cells were then either left untreated (control) or treated with 1 mM H2O2 for 1.5 h. The cells were imaged with confocal laser scanning microscopy. Scale bar: 10 μm.

cathepsin B-mLumin in living cells, which further confirmed the high selectivity of CpFABP-G as a lysosomal marker. After H2O2-mediated induction of apoptosis,4,30,44 CpFABP-G and cathepsin B-mLumin were partially released from lysosomes into the cytosol and the high degree of colocalization was maintained. In contrast, the punctate labeling pattern of LysoTracker DND 26 showed no obvious change before and after the induction of apoptosis, and its colocalization with cathepsin B-mLumin in the cytosol was poor after the activation of apoptosis (Figure S7 of the Supporting Information). These results suggest that CpFABP-G may be used an excellent marker to track lysosome-dependent apoptosis. We applied CpFABP-G to monitor the entire process of lysosome-dependent apoptosis in real-time in living cells without transfection with cathepsin B-mLumin. Figure 7 shows that the CpFABP-G−protein complexes were released significantly into the cytosol with a portion remaining in lysosomes after the activation of apoptosis, consistent with previous reports.35,38,43 The stronger fluorescence from some of the lysosomes after the H2O2 treatment might be because these lysosomes slightly moved along the z axis during the successive imaging process in living cells. Quantitative analysis reveals that, after a significant increase, the 1314

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Figure 7. Process of the release of lysosomal cathepsins into the cytosol after H2O2-mediated induction of apoptosis. (a) Time-lapse confocal microscopy images and (b) increase of mean fluorescence intensities from the areas of the circles [chosen at random in (a) or Figure S8 of the Supporting Information] over time. The fluorescence intensity values (means ± standard error of the mean, n = 5) are expressed in fold increase in comparison with the intracellular signal at the beginning (RFI: relative fluorescence intensity). The images were obtained every 5 min. HeLa cells were labeled with CpFABP-G (green, 10 μM) for 30 min. After 4 h postincubation, the cells were then either left untreated (control) or treated with 1 mM H2O2 for 1.5 h. Scale bar: 10 μm.

fluorescence in the cytosol reached a plateau (Figure 7b). In the control group (without adding H2O2), no obvious changes of the fluorescence intensity were observed in the cytosol (Figures 7b and S8 of the Supporting Information). In contrast, LysoTracker Red was unable to track the induction of apoptosis and showed extensive punctate labeling before and after induction of apoptosis (Figure S9 of the Supporting Information).

suitable for imaging intracellular proteins directly in live cells.25 ABPs equipped with a mannose cluster could enter live cells via receptor-mediated endocytosis but are limited to several specialized cell types, such as dendritic cells and macrophage cells.24 To the best of our knowledge, no cathepsin-targeted ABPs as well as other ABPs have been evaluated as markers for labeling and tracking intracellular organelles in living cells. By exploiting the properties that cysteine cathepsins are typically localized to lysosomal compartments, we have developed a new approach to label lysosomes via novel cathepsintargeted CpFABPs, which contain a cell-penetrating peptide (CPP) to enhance their cellular permeability and an epoxysuccinyl scaffold to selectively form covalent bonds with cysteine cathepsins. The fluorophores attached to our probes are rhodamine B and 5(6)carboxyfluorescein, which are cheap and commercially available, thus facilitating their large-scale production and potential applications. Although CPPs have been widely used to deliver various membrane-impermeable molecules into living cells, very few ABPs containing CPPs have been reported.2,45,46 Our CpFABPs entered live cells not only efficiently but also quickly (Figure 2), possibly due to the ability of CPPs to efficiently carry molecules into cells.26 The extremely good colocalization of CpFABPs with both LysoTracker probes (Figures 2 and 3) and cathepsin B-mLumin (Figure 6) in living cells as well as the competitive studies with E-64

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DISCUSSION Although several cell-permeable ABPs containing the epoxysuccinyl scaffold have been developed for in vivo application, most of them are employed to profile and identify cysteine cathepsins through SDS-PAGE and/or MS analysis.2,23−25 DCG-04 labeled with membrane-permeable boron-dipyrromethene (BODIPY) was capable of penetrating cell membranes and visualizing proteases in live cells. However, a long incubation time (∼12 h) was needed for its efficient cellular uptake.31 Moreover, the requirement of using cell-permeable dyes, such as BODIPY and cyanine, would prevent large-scale production of the ABP because the commercially available activated ester forms of these cell-permeable fluorophores are extremely expensive. ABPs relying on the “click” chemistry are able to label proteins in intact cells. However, the visualization of target proteins with the reporter tag needs to be carried out in cell lysates or fixed cells; thus, these ABPs are not 1315

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(2) Reich, M., van Swieten, P. F., Sommandas, V., Kraus, M., Fischer, R., Weber, E., Kalbacher, H., Overkleeft, H. S., and Driessen, C. (2007) Endocytosis targets exogenous material selectively to cathepsin S in live human dendritic cells, while cell-penetrating peptides mediate nonselective transport to cysteine cathepsins. J. Leukocyte Biol. 81, 990−1001. (3) Guicciardi, M. E., Leist, M., and Gores, G. J. (2004) Lysosomes in cell death. Oncogene 23, 2881−2890. (4) Ghosh, M., Carlsson, F., Laskar, A., Yuan, X. M., and Li, W. (2011) Lysosomal membrane permeabilization causes oxidative stress and ferritin induction in macrophages. FEBS Lett. 585, 623−629. (5) Mizukami, H., Mi, Y., Wada, R., Kono, M., Yamashita, T., Liu, Y., Werth, N., Sandhoff, R., Sandhoff, K., and Proia, R. L. (2002) Systemic inflammation in glucocerebrosidase-deficient mice with minimal glucosylceramide storage. J. Clin. Invest. 109, 1215−1221. (6) Mohamed, M. M., and Sloane, B. F. (2006) Cysteine cathepsins: multifunctional enzymes in cancer. Nat. Rev. Cancer 6, 764−775. (7) Soreghan, B., Thomas, S. N., and Yang, A. J. (2003) Aberrant sphingomyelin/ceramide metabolic-induced neuronal endosomal/ lysosomal dysfunction: potential pathological consequences in agerelated neurodegeneration. Adv. Drug Delivery Rev. 55, 1515−1524. (8) Berthiaume, E. P., Medina, C., and Swanson, J. A. (1995) Molecular size-fractionation during endocytosis in macrophages. J. Cell Biol. 129, 989−998. (9) Teter, K., Chandy, G., Quinones, B., Pereyra, K., Machen, T., and Moore, H. P. (1998) Cellubrevin-targeted fluorescence uncovers heterogeneity in the recycling endosomes. J. Biol. Chem. 273, 19625− 19633. (10) Hu, Q., Bally, M. B., and Madden, T. D. (2002) Subcellular trafficking of antisense oligonucleotides and down-regulation of bcl-2 gene expression in human melanoma cells using a fusogenic liposome delivery system. Nucleic Acids Res. 30, 3632−3641. (11) Hanaki, K., Momo, A., Oku, T., Komoto, A., Maenosono, S., Yamaguchi, Y., and Yamamoto, K. (2003) Semiconductor quantum dot/albumin complex is a long-life and highly photostable endosome marker. Biochem. Biophys. Res. Commun. 302, 496−501. (12) Anderson, R. G., Falck, J. R., Goldstein, J. L., and Brown, M. S. (1984) Visualization of acidic organelles in intact cells by electron microscopy. Proc. Natl. Acad. Sci. U. S. A. 81, 4838−4842. (13) Svendsen, C., Spurgeon, D. J., Hankard, P. K., and Weeks, J. M. (2004) A review of lysosomal membrane stability measured by neutral red retention: is it a workable earthworm biomarker? Ecotoxicol. Environ. Saf. 57, 20−29. (14) Anderson, R. G., and Orci, L. (1988) A view of acidic intracellular compartments. J. Cell Biol. 106, 539−543. (15) Freundt, E. C., Czapiga, M., and Lenardo, M. J. (2007) Photoconversion of Lysotracker Red to a green fluorescent molecule. Cell Res. 17, 956−958. (16) Ohkuma, S., and Poole, B. (1978) Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc. Natl. Acad. Sci. U. S. A. 75, 3327−3331. (17) Bidere, N., Lorenzo, H. K., Carmona, S., Laforge, M., Harper, F., Dumont, C., and Senik, A. (2003) Cathepsin D triggers Bax activation, resulting in selective apoptosis-inducing factor (AIF) relocation in T lymphocytes entering the early commitment phase to apoptosis. J. Biol. Chem. 278, 31401−31411. (18) Xiao, Y., Forry, S. P., Gao, X., Holbrook, R. D., Telford, W. G., and Tona, A. (2010) Dynamics and mechanisms of quantum dot nanoparticle cellular uptake. J. Nanobiotechnol. 8, 13−22. (19) Shi, H., He, X., Yuan, Y., Wang, K., and Liu, D. (2010) Nanoparticle-based biocompatible and long-life marker for lysosome labeling and tracking. Anal. Chem. 82, 2213−2220. (20) Rejman, J., Oberle, V., Zuhorn, I. S., and Hoekstra, D. (2004) Size-dependent internalization of particles via the pathways of clathrinand caveolae-mediated endocytosis. Biochem. J. 377, 159−169. (21) Pryor, P. R., and Luzio, J. P. (2009) Delivery of endocytosed membrane proteins to the lysosome. Biochim. Biophys. Acta 1793, 615−624.

in vitro (Figure S5 of the Supporting Information) suggests that CpFABPs are effective in specifically labeling lysosomes. Compared with previous small-molecule markers, such as LysoTracker probes (30 min), the incubation time of CpFABPs was longer (4 h). However, our CpFABPs were capable of labeling lysosomes in fixed cells, permeabilized cells, and NH4Cl-treated cells, unlike these small-molecule markers, which show ineffective labeling under the same conditions. Although a relatively high incubation concentration was required (10 μM), CpFABP-G showed low cytotoxicity even at a concentration of 20 μM. When applied to monitor the process of lysosome-dependent apoptosis, CpFABP-G (Figure 7) not only displayed the expected release of lysosomal cathepsins from lysosomes into the cytosol, but also revealed additional information about the location of the cathepsins during apoptosis, which is undetectable by other chemical lysosome markers.

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CONCLUSIONS In summary, we have developed a new strategy for labeling and tracking lysosomes via a cell-permeable fluorescent activitybased probe containing a reactive warhead that covalently binds to select lysosomal proteins. We have demonstrated that CpFABP-G enters living cells efficiently and specifically labels lysosomes in different kinds of live cells. Moreover, we showed that the probe maintains its labeling ability in fixed cells, permeabilized cells, and NH4Cl-treated cells. In addition, CpFABP-G possesses low cytotoxicity. Perhaps most remarkably, CpFABP-G can track lysosome-dependent apoptosis in real-time in living cells. These results provide useful guidelines for the use of CpFABP-G as a lysosomal marker in lysosomerelated studies and expand the application ranges of activitybased probes.

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ASSOCIATED CONTENT

S Supporting Information *

Characterization data of probes CpFABP-G, CpFABP-R, 1, and 2, supplemental confocal microscopy images, and cell viability data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel. (+86) 27 87792033; fax (+86) 27 87792034. E-mail address: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Analytical and Testing Center (Huazhong University of Science and Technology) for spectral measurements. This study was supported by the National Major Scientific Research Program of China (Grant No. 2011CB910401), Science Fund for Creative Research Group of China (Grant No.61121004), the National High-Tech Research and Development Program of China (863 Program: 2008AA02Z107), and the National Natural Science Foundation of China (Grant No. 30800183).

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