Wei Wang,Nai-Kei Wong,Siu-Lun Bok,You Xu,Yang Guo,Lu Xu,Meiling Zuo,Claudette M. St. Croix,Gaowei Mao,Alexandr Kapralov,Hülya Bayir,Valerian E. Kagan,Dan Yang
Visualizing Cardiolipin In Situ with HKCL-1M, a Highly Selective and Sensitive Fluorescent Probe
Reliable probing of cardiolipin (CL) content in dynamic cellular milieux presents significant challenges and great opportunities for understanding mitochondria-related diseases, including cancer, neurodegeneration, and diabetes mellitus. In intact respiring cells, selectivity and sensitivity for CL detection are technically demanding due to structural similarities among phospholipids and compartmental secludedness of the inner mitochondrial membrane. Here, we report a novel “turn-on” fluorescent probe HKCL-1M for detecting CL in situ. HKCL-1M displays outstanding sensitivity and selectivity toward CL through specific noncovalent interactions. In live-cell imaging, its hydrolyzed product HKCL-1 efficiently retained itself in intact cells independent of mitochondrial membrane potential (Δψm). The probe robustly co-localizes with mitochondria and outperforms 10-N-nonyl acridine orange (NAO) and Δψm-dependent dyes with superior photostability and negligible phototoxicity. Our work thus opens up new opportunities for studying mitochondrial biology through efficient and reliable visualization of CL in situ.
Visualizing Cardiolipin In Situ with HKCL-1M, a Highly Selective and Sensitive Fluorescent Probe

Reliable probing of cardiolipin (CL) content in dynamic cellular milieux presents significant challenges and great opportunities for understanding mitochondria-related diseases, including cancer, neurodegeneration, and diabetes mellitus. In intact respiring cells, selectivity and sensitivity for CL detection are technically demanding due to structural similarities among phospholipids and compartmental secludedness of the inner mitochondrial membrane. Here, we report a novel “turn-on” fluorescent probe HKCL-1M for detecting CL in situ. HKCL-1M displays outstanding sensitivity and selectivity toward CL through specific noncovalent interactions. In live-cell imaging, its hydrolyzed product HKCL-1 efficiently retained itself in intact cells independent of mitochondrial membrane potential (Δψm). The probe robustly co-localizes with mitochondria and outperforms 10-N-nonyl acridine orange (NAO) and Δψm-dependent dyes with superior photostability and negligible phototoxicity. Our work thus opens up new opportunities for studying mitochondrial biology through efficient and reliable visualization of CL in situ.

Despite being a minor phospholipid component of the inner mitochondrial membrane (IMM), cardiolipin (CL) plays significant roles in diverse mitochondrial functions. Being predominantly confined to the matrix side of the IMM, CL binds a variety of IMM proteins and modulates their physiological functions. Under pathological or stress conditions, depolarization of the IMM causes CL redistribution to the outer mitochondrial membrane and the mitochondrial surface. This transgression of CL permits its interaction with normally unavailable protein partners, including cytochrome c (cyt c), an intermembrane hemoprotein. Canonically, cyt c shuttles electrons between respiratory complexes III and IV of mitochondria. Upon binding to CL, cyt c undergoes dramatic changes in structural organization and catalytic competence, resulting in its conversion to a peroxidase that catalyzes CL peroxidation, which then triggers off apoptotic cascades. This aberrant pro-oxidant role of the CL/cyt c complex, along with other disturbances of CL metabolism, has been implicated in a plethora of mitochondria-related acute injuries, chronic conditions, and degenerative diseases, including heart failure, Parkinson’s disease, obesity, diabetes mellitus, and cancer. Arguably, the excellent sensitivity and molecular specificity offered by modern mass spectrometry (MS) have made it the method of choice for phospholipid analysis. However, the extensive structural diversity observed in CL populations poses significant challenges to its practical use. In recent years, there have been attempts to develop and apply mass spectrometry imaging protocols, including matrix-assisted laser desorption ionization and secondary ion mass spectrometry, which confer direct surface analysis capabilities. Though relatively sensitive and capable of quantitative precision, these semi-quantitative methods require sophisticated instrumentation and highly technical operation in general. More importantly, MS imaging can only be performed on frozen tissue sections, thus precluding dynamic temporal or spatial resolution of CL in living cells. Specific detection of CL in intact living cells is extremely challenging, with two complex issues to tackle, namely: how to selectively capture CL as a minor lipid species among the more abundant phospholipids in cells, and how to target a probe to the mitochondrial interior across multiple membrane barriers? To date, noncovalent fluorescent labeling has been considered one of the most promising approaches that allows precise and dynamic monitoring of endogenous phospholipids in imaging analysis. The main advantage of noncovalent labeling is that detection is achievable in a non-lytic and biorthogonal manner. Along with semi-quantitative approaches to CL detection such as the aforementioned MS imaging, fluorescent probes have attracted much interest as a non-disruptive sensing modality for biomembranes and phospholipids. Among them, 10-N-nonyl acridine orange (NAO), a commercially available putative CL fluorescent dye, suffers from several drawbacks, including low sensitivity and selectivity, photo-instability, photo-induced cytotoxicity, non-stoichiometric behavior toward CL, and dependency on mitochondrial membrane potential (Δψm) for retention, which preclude in situ assessments of CLs in mitochondria during their injury. Recently, other CL fluorescent probes have been reported, such as fluorescent pentamethine salts (MS1–MS7),l-cysteine-capped CdTe quantum dots, fluorescent CL-binding peptide [ald]SS-31, aggregation-induced emission-based TTAPE-Me, and the NIR-II dye DUT850 as a CL-targeted phototherapeutic. However, the majority of the aforementioned probes show varying degrees of limitations, such as insufficient selectivity toward CL, high interference from charge interactions with ambient anionic phospholipids, and a general failure of probe delivery into mitochondria in intact living cells. To address these outstanding issues, we here propose a rationally designed probe, HKCL-1, and its cell-permeable form, HKCL-1M, for CL detection and imaging in situ. With a focus on the molecular characteristics of CL, HKCL-1’s fluorescence response mechanisms in lipid bilayer environments, mode of multiple noncovalent bonding, and trigger for functional regeneration in cells were thoroughly investigated. The probes’ high potential for biological applications was supported by proof of concept in various experimental approaches. Overall, we envisage that these probes promise to offer great opportunities for advancing biochemical sensing and mitochondrial research as a whole, where analyses entail high selectivity and sensitivity for CL in living cell contexts.

Based on CL’s unique structure with four hydrophobic alkyl chains and two negatively charged phosphate groups (Figure 1), we have designed a series of novel fluorescent CL probes, which bind to CL noncovalently via hydrophobic, electrostatic, and hydrogen bonding interactions to produce turn-on fluorescence upon CL-binding. As illustrated in Figure 1, a functional probe consists of four units, namely: a hydrophobic tail, recognition moiety, linker, and fluorophore. 4-Amino-1,8-naphthalimide, a polarity-sensitive fluorophore, is known to give off dramatically enhanced fluorescence upon its entrapment into a phospholipid bilayer. As a lipophilic unit, a flexible aliphatic carbon chain was appended to facilitate hydrophobic interactions between the probe and CL. A linker dictates the rigidity or flexibility of the overall scaffold by adjusting the distance between the two recognition groups that fit onto the head group of a CL molecule. Recognition moieties were designed as two positively charged functional groups to interact with the two negatively charged phosphates in the head of CL. In addition to electrostatic forces, hydrogen bonds are also conducive to consolidation of bonding. To select for an ideal probe, we explored the structural possibilities of several other candidate CL probes (Figure 1). By using liposomes to reconstitute a hydrophobic environment mimicking mitochondrial membranes (see Table S1 for details on phospholipids), we proceeded to characterize the interactions between these probes and CL. We detailed the properties of probe 1 as an example. Upon incubation of probe 1 with liposomes in the presence or absence of CL, its fluorescence was minimal in pure PC (1,2-dioleoyl-sn-glycero-4-phosphocholine) liposomes (Figure 2a,b). In contrast, the probe underwent a fluorescence turn-on in the presence of CL, while liposomes prepared with physiological concentrations of other phospholipids in the IMM failed to enhance the fluorescence of the probe. This in vitro finding suggests that probe 1 is highly selective for CL, which is essential for CL detection under physiological conditions. Results for fluorescence response of other candidate probes are presented in the Supporting Information (Table S2). Considering selectivity as a primary evaluative criterion, we compared the performance of probes 1–12 (Table 1). Although the hydrophobic tail installed on the probes can promote hydrophobicity around the fluorophore, it needs to be of a suitable length to fit into the structural environment of CL. As a rigid linker, aniline can more effectively stabilize the spatial orientation of a recognition group that contains flexible chains of different lengths on either side, further promoting selective binding with CL. The performance of different recognition groups varied starkly, with hydrophilic quaternary pyridinium salt being superior in fluorescence response and selectivity. However, during functional screens, probe 1 and others with two positive charges in Table 1 could not enter cells in live-cell imaging. On the other hand, probe 2, which possesses two primary amino groups, exhibited good selectivity toward CL (Figure 2c,d). We ventured to design probe 2M as a precursor of probe 2 by protecting the latter’s amino groups as acetoxymethyl (AM) carbamate (Figure S1). Unfortunately, a preliminary functional study by live-cell fluorescence imaging suggests that probe 2M appeared to distribute in both mitochondria- and non-mitochondria-associated loci within cells (Figure S1). Therefore, probe 2M was considered unsuitable for imaging CL in situ. To realize biological applicability of a probe in live-cell imaging contexts, we selected HKCL-1 for final functionalization (protected as HKCL-1M), as shown in Scheme 1. A lipophilic triphenylphosphonium (TPP) unit was introduced to promote efficient mitochondrial localization during probe incubation. A phenyl ring flanked by two linkers provides a rigid scaffold, from which two positively charged ammonium groups extend as recognition moieties to interact with the negative charges on CL’s two phosphate groups through hydrogen bonding and electrostatic interactions. To ensure unimpeded cell entry, the recognition moieties were made charge-neutral by masking them with acetoxymethyl (AM) carbamate groups, which can be readily cleaved by intracellular esterases to reveal the primary amines upon internalization into living cells. To better understand interactions between HKCL-1 and CL within the mitochondrial inner membrane, we performed a series of molecular dynamics (MD) simulations in which HKCL-1 was embedded in a mixed membrane bilayer with PC/CL. The technical details thereof are presented in the Supporting Information, and an overview of the MD system is as shown in Figure 3a. To illustrate, the system was built as a bilayer lipid membrane comprising randomly distributed PC/CL, whose top and bottom leaflets have the same components. Both leaflets have 72 PCs and 8 CLs (a molecular ratio of approximately 9:1). The probe was inserted into the top leaflet of the membrane by replacing a PC. Based on a priori assumptions for positive interactions between the probe and CL-containing lipids, we performed three simulations. Interestingly, despite different starting positions, the probe was found to be highly mobile, able to travel freely among lipid molecules during the 3 μs long simulations and eventually make contact with each lipid molecule in the top leaflet (Figure S2). After a few dozen nanoseconds, it swiftly adjusted its orientation and assumed a rotated conformation (Figure 3b). The TPP group moved itself away from the hydrophilic tails of PC/CL and resided close to the hydrophilic surface, while the electrostatic interactions between the probe ammonium and lipid phosphate groups held steady. A favorable orientation of the probe with respect to the membrane was hence evaluated (Figure 3c). The tilt angle between the fluorophore and the bilayer z-axis is near 90°, suggesting that the probe is lying almost parallel to the lipid surface, consistent with our rational design that the positively charged TPP group should contribute to electrostatic interactions as well during initial probe targeting. Interestingly, probe 2 is almost perpendicular to the lipid surface under the same simulation conditions (Figure S3). The non-charged tail and fluorophore of probe 2 prefer to stay with CL in the latter’s lipid tail environment, although the positively charged recognition moiety neatly engages the phosphate groups of CL. Thus, it is reasonable to conjecture that the introduction of the TPP group to the probe design improves the probe’s membrane positioning and ultimately its imaging performance, at the expense of a tolerable degree of loss in selectivity. In silico, the high proximity of nitrogen atoms in HKCL-1’s ammonium groups (NH3+) to oxygen atoms in the phospholipids’ phosphate group was found to be broadly stabilized through hydrogen-bond formation. However, since CL is more capable of establishing a negatively charged local niche than PC, more binding contacts between the probe and CL accumulated as a result of stronger electrostatic attraction between them (Figures 3d and S2). A moderate preference was observed for one ammonium group selecting CL over PC, which is similar to the case for the TPP group. Given the reasonable probability that both ammonium groups of the probe contact the same lipid molecule, the preference of CL is thus even larger. Variabilities seen in preference folds among the different runs could be a result of inhomogeneous distribution of CLs and the inherently restricted size of the system. While in silico evidence from MD simulations provides some confidence to the feasibility of CL-binding by the probe, considerable limitation remains as these simulations were performed with a simplified model system of the phospholipid bilayer. Therefore, we proceeded to characterize any potential interactions between probe HKCL-1 and CL by using liposomes as a hydrophobic environment to mimic mitochondrial membranes. Two types of liposomes were used for characterization, namely CL and CL-free liposomes. CL liposomes were prepared by mixing all phospholipid components at exact percentages for reconstituting the IMM (i.e., 9.2 mol % CL, 43.8 mol % PC, 41.9 mol % PE, 1.8 mol % PI, 1.1 mol % PS, and 2.2 mol % SM), while CL-free liposomes contained more PC to replace the CL component (i.e., 53 mol % PC, 41.9 mol % PE, 1.8 mol % PI, 1.1 mol % PS, and 2.2 mol % SM). As shown in Figure 4a, no significant change to the UV–visible absorption of HKCL-1 was observed upon mixing the probe with either CL or CL-free liposomes. While HKCL-1 fluoresced minimally in the absence of liposomes, the probe displayed striking fluorogenic behavior in the presence of CL liposomes (Figure 4b). Around 12-fold fluorescence enhancement at 550 nm was observed when the probe was incubated with 10 equiv of CL liposomes (Figure 4f). Fluorescence emitted by HKCL-1 increased significantly when treated with increasing amounts of CL liposomes (Figure 4c), with an apparent linear relationship between CL concentration and fluorescence intensity of HKCL-1 being established within some range where the amount of CL was less than 1 equiv of the probe (Figure 4d). As expected, HKCL-1 exhibited sensitivity to polarity. It fluoresced 35-fold more strongly in dioxane (ε = 2.2) than in water (ε = 80.1) at λem = 535 nm (Figure S4). The apparent turn-on properties of the probe in CL liposomes may be attributable to the formation of a twisted internal charge transfer (TICT) state. Specifically, an aniline conformation should exhibit greater twisting and lower TICT resistance in polar solvents (i.e., higher ε). Likewise, when substituting an aqueous environment with a lipophilic one, the phospholipid membranes would decrease the probability of the formation of such a TICT state, which accounts for a sharp rise in the fluorescence intensity of the probe. To fully establish the selectivity of HKCL-1 toward CL, the fluorescence response of the probe toward other lipids in the IMM was determined. When incubated with CL-free liposomes, HKCL-1 showed only relatively weak fluorescence enhancement (ca. 4-fold), in comparison to the case when CL was present (Figure 4f). Even when HKCL-1 was tested against other anionic phospholipids at an exceedingly high concentration (20 mol % to maintain the same negative charges; Figure S5), a significantly higher fluorescence turn-on was seen for CL (over 2.6 folds compared to other lipids), indicating that probe selectivity for CL was not entirely dependent on electrostatic interactions. In all liposome concentrations tested, incubation of HKCL-1 with CL liposomes resulted in a greater fluorescence turn-on compared to CL-free liposomes (Figure 4e). The selectivity for CL detection by HKCL-1 was 2.9-fold against CL-free liposomes (Figure 4f). These results suggest that HKCL-1 is highly selective toward CL in vitro. To elaborate on the binding behavior of HKCL-1 toward CL, isothermal titration calorimetry experiments were performed. For CL-PC liposomes (containing 10 mol % CL and 90 mol % PC), a moderate binding between HKCL-1 and CL-PC liposomes was observed (Kd = 1.41 ± 0.30 μM; see Figure 5a,b), which was stronger than the interaction between NAO and the same batch of CL-PC liposomes (Kd = 3.17 ± 0.87 μM; see Figure S6). In contrast, as shown in Figure S7, the injection of pure PC liposomes to an HKCL-1 solution produced negligible exothermic signals, as compared to the case of liposomes containing 10 mol % of CL to an HKCL-1 solution. Subsequently, fluorescence enhancement of HKCL-1 for assays with varying ratios of HKCL-1 to CL-PC liposomes was recorded and compared. As shown in Figure 5c, Job plot shows a peak at ∼0.6, corresponding to a 2:3 binding ratio between HKCL-1 and CL, which matches their charge ratio. However, the binding ratio of probe 2 and CL was found to be 1:1 (Figure S8). These results indicate that the TPP group with its positive charge permits electrostatic interactions during CL targeting of the probe. In mitochondrial membranes, a multitude of proteins can bind CL with high affinity, thus potentially affecting probe performance. To examine this issue, we tested the ability of cyt c, a cationic hemoprotein of mitochondrial intermembrane space known to bind CL with high affinity, to quench fluorescence from a complex of HKCL-1/CL. HKCL-1 was found to be effective in competing with cyt c for CL binding, as evidenced by obvious fluorescence quenching (Figure 5d). Suppression of fluorescence intensity by 50% was achieved at a cyt c/CL ratio of 0.25:1, corresponding to a zero net charge of the cyt c/CL complex (Figure 5d). Next, we assessed chemo-stability of the probe. Fluorescence intensity of HKCL-1 was found to be stable toward a wide range of pH (from 3 to 9; Figure S9), in the presence of major biologically relevant reactive oxygen/nitrogen species (ROS/RNS) (Figure S10), and under conditions of various ionic strengths (up to 2 M NaCl; Figure S11). As proposed in our design, the precursor molecule HKCL-1M was readily hydrolyzed to HKCL-1 upon encountering endogenous esterases within cells, which was verified by mass spectrometry (see details in Figures S12 and S13). To establish applicability of HKCL-1M, the cell-permeant precursor of HKCL-1, in biological assays, we first tested its potential cytotoxicity. HKCL-1M was found to be virtually non-toxic to cells up to a 10 μM concentration, as confirmed by a CellTiter Glo luminescence assay (Figure S14). We then tested whether it can be efficiently taken up and retained in living cells. In terms of subcellular distribution in live-cell imaging, the tubular morphology confirms that HKCL-1M co-localized well with mitochondria-specific RFP (red fluorescent protein) in HeLa-IMS cells (Figure 6a). HKCL-1M (2 μM) can also stain a variety of cells efficiently, including RAW264.7 mouse monocytic macrophages, THP-1 human monocytes, SKOV-3 human ovarian cancer cells, and U-2 OS human bone osteosarcoma cells, following brief incubation (30 min) in HBSS (Hank’s balanced salt solution) (Figure S15). An optional wash step was introduced to terminate loading, upon which the probe was well retained with distinct mitochondrial morphology. As the mitochondrial inner matrix bears a net negative charge, cationic probes tend to accumulate there. During perturbations of Δψm, however, this may give rise to probe mislocation or off-target staining. To assess HKCL-1M’s ability to resist Δψm disruptions, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), an oxidative phosphorylation uncoupler capable of dissipating the mitochondrial proton gradient, was used to acutely alter Δψm. We optimized the FCCP dosage for different cells, as shown in Figure 6b. The commonly used mitochondrial staining agent TMRM is Δψm-dependent and thus showed a significant drop in fluorescence in the presence of FCCP; the same phenomenon was observed even when FCCP was introduced after preloading of TMRM (Figure S16). In contrast, mitochondrial retention of HKCL-1M independent of Δψm was further confirmed by robust colocalization of the probe with a mitochondria-targeted fluorogen-activating protein (Mito-FAP), before or after FCCP treatment (Figure 6c). Subsequently, dynamic alterations in mitochondrial morphology, as illustrated by gradual mitochondrial fragmentation under FCCP challenge at critical doses, were readily observed in human U-2 OS cells (Figure 6d). When U-2 OS cells were challenged with antimycin A, a classic mitochondrial superoxide inducer, HKCL-1M fluorescence levels stayed unaltered despite clearly stressed (blob-form) morphology of the mitochondria (Figure 6e). These results indicate that HKCL-1M is insensitive to Δψm perturbations and oxidative assaults from ROS. To rigorously test the performance of HKCL-1 in mitochondria, we used mitochondria (Figure S17) freshly purified from HEK293T cells for single-organelle staining analysis by flow cytometry and employed JC-1 to monitor mitochondrial membrane potential (Figure 7a). JC-1 is an established dye for assessing mitochondrial membrane potential, which gives off red fluorescence as aggregates in mitochondria and turns into a monomeric form with green fluorescence in response to low membrane potential. In our analysis, more than 60% mitochondria maintained a high membrane potential (high Δψm), exhibiting red fluorescence in the basal condition. When pre-treated with FCCP, the proportion of mitochondria with high Δψm dropped sharply to 0.21%, while the proportion of green fluorescence-positive mitochondria increased from 14.45 to 53.75% (as normalized to a background with no staining). Predictably, this could be rescued by sodium citrate, a substrate in the TCA cycle, suggesting successful re-depolarization of mitochondria after depolarization by FCCP. Next, we used MitoTracker Deep Red FM to co-stain with HKCL-1. A very high degree of co-localization (more than 60% double-positive mitochondria) was observed (Figure 7b). To further test whether HKCL-1 stains mitochondria in a membrane potential-dependent way, FCCP was again used to challenge mitochondria. As shown in Figure 7c, the proportion of HKCL-1 positive mitochondria dropped slightly from 74.5 to 69.9% upon depolarization but rebounded to 73.3% upon citrate treatment. Collectively, HKCL-1 was found to be broadly insensitive to perturbations of mitochondrial membrane potential, which was amply evidenced by its apparent excellent performance in mitochondrial staining. These findings also corroborate our design principles that a combination of multiple noncovalent bonding provides a basis for probe selectivity toward CL as a specific phospholipid species. To highlight the superiority of HKCL-1M as a new molecular tool for CL imaging in living cells, the commercial dye NAO was chosen for detailed comparison. In live-cell confocal imaging, NAO (100 nM) became rapidly photobleached after a single scan at high magnification, while HKCL-1M showed no signs of photobleaching after repeated scans (Figure 8a,b). For additional comparisons, we also tested the photostability of several other commercial mitochondrial dyes (MitoTracker Red and MitoTracker Green) and found that HKCL-1M consistently outperformed them (Figure S18). As additional evidence for our probe’s insensitivity to Δψm disruptions, the fluorescence outcomes of NAO and HKCL-1M consistent with the expectation of their dependence on Δψm. In contrast, HKCL-1M (2 μM) retained both its fluorescence intensity and mitochondrial distribution efficiently post-fixation (Figure 8c insets and Figure 8d). Its mitochondrial localization was robustly retained, suggesting that interactions between CL and the probe not only take the form of electrostatic interactions but also other noncovalent interactions as envisioned in our design. Recently, Mao et al. showed that NAO elicited considerable phototoxicity at 24 h post-irradiation. In our study, prolonged incubation with HKCL-1M following photo-irradiation did not alter the survival profile of HeLa cells, indicating minimal or negligible phototoxicity from the probe (Figure 8e). In high-magnification time-series imaging of respiring mitochondria, NAO at a low dose (50 nM) was found to elicit acute phototoxicity, as tubular fluorescent structures of mitochondria rapidly collapsed to give rise to redistribution of NAO fluorescence across the entire cell, suggesting mitochondrial membrane depolarization (Figure 8f, upper row). On the other hand, HKCL-1M-stained mitochondrial structures remained stable throughout the same time fame of repeated scans (at a time interval of 10 s per scan), with no signs of phototoxicity (Figure 8f, lower row). Overall, HKCL-1M as a CL probe decisively outperformed NAO as well as other commercial mitochondrial dyes on the basis of its insensitivity to Δψm perturbations, compatibility with fixatives, high photostability, and low phototoxicity.

In summary, we have successfully developed a “turn-on” mitochondrial membrane potential-insensitive fluorescent probe, HKCL-1M, on the principle of specific noncovalent binding with CL for highly selective and sensitive molecular imaging of CL in situ in living cells. The probe decisively outperforms the established putative CL dye NAO in all aspects of comparison. The probe has additional merits such as remarkable photo-/chemo-stability and low cyto-/photo-toxicity. We anticipate that HKCL-1M would become a valuable tool for researchers to study CL functions in mitochondrial biology and help stimulate new design of mitochondria-targeting tools and drugs.