Potential of Engineered Bacteriorhodopsins as Photoactivated

Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

pubs.acs.org/journal/abseba

Potential of Engineered Bacteriorhodopsins as Photoactivated Biomaterials in Modulating Neural Stem Cell Behavior Hao-Wei Han,† Ling-Ning Ko,‡ Chii-Shen Yang,*,‡ and Shan-hui Hsu*,†,§,⊥ †

Institute of Polymer Science and Engineering, ‡Department of Biochemical Science and Technology, and §Center of Tissue Engineering and 3D Printing, National Taiwan University, No. 1, Sec. 4 Roosevelt Road, Taipei, Taiwan 10617, R.O.C. ⊥ Institute of Cellular and System Medicine, National Health Research Institutes, No. 35 Keyan Road, Zhunan, Miaoli County, Taiwan 35053, R.O.C.

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S Supporting Information *

ABSTRACT: Bacteriorhodopsin (BR), a light-sensitive bacterial proton pump, has been demonstrated the capacity for regulating the neural activity in mammalian cells. Because of the difficulty in production and purification in large quantities, the BR proteins have neither been directly employed to biomedical applications nor verified the functionality by protein administration. Previously, we have invented a highly expressible bacteriorhodopsin (HEBR) and established the massive production protocol. In the current study, we massproduced the two types of HEBR proteins that have normal or abnormal activity on the proton pumping, and then we treated murine neural stem cells (NSCs) with these HEBR proteins. We discovered that the cell behaviors including growth, metabolism, mitochondrial inner membrane potential, and differentiation were obviously affected in NSCs after the treatment of HEBR proteins. Particularly, these effects induced by HEBR proteins were correlated to their proton pump activity and could be altered by cell culture substrate materials. Current findings suggest that the engineered light-sensitive HEBR protein can serve as a biological material to directly influence the multiple behaviors of mammalian cells, which is further modified by the cell culture substrate material, revealing the versatile potential of HEBR protein in biomaterial applications. KEYWORDS: bacteriorhodopsin, HEBR, neural stem cells, cell behavior

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behavior, that is, “optogenetics”, especially in the regulation of neural activity.11−13 To develop microbial rhodopsins as biomedical materials, the reasonable amount of protein and reliable production process are prerequisites. BR-containing purple membrane of Halobacterium salinarum is the most prevalent source for microbial rhodopsin,14 but the process of sample preparation prevents it from being a plausible source to meet the need of large quantity. In addition, the organic solvent is involved in the removal of biological lipid to reach the desired protein form. Although these light-sensitive proteins can create photoelectric effects on biological systems, their influences on the behavior of neural-related cells are neither tested directly nor verified by dysfunctional mutant because of the difficulty in production and purification of these proteins in large quantities. Previously, we have developed a highly expressible bacteriorhodopsin (HEBR) using E. coli system. HEBR is functionally intact with a slightly retarded photocycle kinetics.15 It is thus suitable for the functional studies of

INTRODUCTION Microorganism has been used to produce protein-based biopolymers, foods, and biofuels because of low-cost, effective, and scalable production as well as strong tolerance for the extreme environment.1−3 The bioactive compounds produced by these tiny organisms have even been considered as the potential biomaterials or therapeutic agents for cancer treatment.4,5 Rhodopsins are a group of transmembrane proteins that respond to the wavelength-specific lighting to exert various biological functions.6 Microbial rhodopsins, a subgroup of rhodopsins, are naturally found in microorganisms from archaea, bacteria, fungi, and algae, and several light-driven anion or cation ion pumping or light-gated channel types were identified.7 For instance, bacteriorhodopsin (BR) is a lightdriven outward proton pump, halorhodopsin (HR) is a lightdriven inward chloride pump, while channelrhodopsin II (ChR2) is determined as an nonselective cation channel. The structures and operating mechanisms are all well studied.8,9 Functionally, the microbial rhodopsins sustain the survival of these tiny organisms by regulating energy production, osmotic homeostasis, migration behavior, and so on.10 Since these microbial rhodopsins independently respond to the light stimulation and execute ion transportation, they have been combined with light illumination to modulate mammalian cell © XXXX American Chemical Society

Received: March 15, 2019 Accepted: May 9, 2019

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DOI: 10.1021/acsbiomaterials.9b00367 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

∼2 cm. Except for the period of green light illumination, cell samples were prevented from lighting as far as possible during the other operation procedures. Analysis of Cell Growth. The protocols of isolation and culture of NSCs were performed as previously described,16 and the detail is provided in the Supporting Information and Methods. The growth of NSCs was evaluated by direct cell counting and WST-8 cell viability assay (Sigma-Aldrich). After NSCs were seeded and attached on TCPS overnight, they were incubated in the presence of HEBR or HEBR-D83N proteins at the final concentration of 3.75−15 μg/mL. After incubation for 3 d, the resultant cell numbers of NSCs were counted by a hemocytometer using the standard protocol. The viability of NSCs was measured by WST-8 assay after seeding and culturing overnight, which was considered as the initial cell viability (100% relative viability) before the HEBR treatment. After the HEBR treatment for 1, 2, and 3 d, the viability of NSCs was determined again by WST-8 assay. Cell Cycle and Vitality Analysis. NSCs were seeded and cultured under the serum starvation condition overnight for the synchronization of cell cycle. The medium was then changed to the complete culture medium containing 15 μg/mL normal or mutant HEBR proteins. The profiles of cell cycle of NSCs were determined by DNA contents measured on the NucleoCounter NC-3000 system using the kit of two-step Cell Cycle Assay (Chemometec) after the treatment of HEBR proteins for 1 and 3 d. When NSCs were maintained in the medium containing 15 μg/mL normal or mutant HEBR proteins with green light illumination for 3 d, the vitality of HEBR-treated NSCs was evaluated by the VB-48 Vitality Assay on the NC-3000 system according to the instructions provided by manufacturer (Chemometec). Mitochondrial Metabolism. The mitochondrial respiratory capacity (oxygen consumption) was monitored by a Seahorse Bioscience Extracellular Flux Analyzer (XFp, Seahorse Bioscience). After treatment with HEBR proteins (7.5 μg/mL) with or without green light illumination for 1 d, NSCs were subjected for analysis of the metabolic profiles by using the Seahorse Cell Mito Stress test. The procedure of the test was performed according to the manufacturer’s instructions, and the final concentrations of the compounds/ inhibitors used in the test were 1 μM, 1 μM, and 0.5 μM for oligomycin, FCCP, and rotenone/antimycin A, respectively. Mitochondrial Membrane Potential Assay. The mitochondrial membrane potential of neural cells was determined by JC-1 staining as described previously.17 NSCs were treated with 7.5 μg/mL HEBR proteins and illuminated with green light for 1 d, and then the JC-1 aggregates (red color) as well as monomers (green color) were stained with the dual-fluorescent dye (Chemometec). After the staining, the intensity levels of red and green fluorescence in NSCs (20 000 cells for each group) were analyzed by the Guava Easy-Cyte flow cytometer (Millipore). Immunostaining. Immunostaining was performed by the general procedures. The antibodies used: 6X His tag antibody (GTX628914, 1:1000 dilution, GeneTex), nestin (GTX26142, 1:200 dilution, GeneTex), GFAP (840001, 1:500 dilution, BioLegend), CNPase (836404, 1:500 dilution, BioLegend), Alexa Fluor 594 anti-Rabbit (ab150080), and anti-Mouse (ab150116) IgG. On the other hand, the protein levels of nestin, GFAP, and CNPase in NSCs were also semiquantified after the induction of HEBR proteins. After the HEBR treatment and immunostaining, the NSCs (20 000 cells for each group) were subjected to flow cytometric analyses to determine the relative fluorescence levels. The mouse and rabbit IgG isotype antibodies (GTX35009 and GTX35035, GeneTex) were used as negative control in flow cytometric analyses. Statistical Analysis. All experiments were conducted at least for three times, and the sample size of each group was 2−4 in each independent experiment. The quantitative results were presented as the mean ± standard deviation, and statistical analysis was performed by one-way ANOVA using GraphPad Prism 6.0 software. The p value of less than 0.05 was considered statistically significant.

rhodopsins for mammalian cells. In the current study, we produced and characterized two types of HEBR proteins that have normal or abnormal function on the outward proton pumping and then directly treated neural stem cells (NSCs) with these proteins combined with green light illumination. After the treatment, we examined whether the HEBR proteins could enter NSCs, and we investigated the cell behavior and physiology including growth, metabolism, mitochondrial membrane potential, and differentiation to evaluate the functionality of the engineered BR proteins as biological materials in mammalian cells. A comparison of the information derived from normal and mutant HEBR proteins would provide the first evidence if BR proteins may alter cell fate by their proton pumping activity. Besides, we also investigated if the alteration in cell differentiation and metabolism may be modulated by the cell culture substrate materials.

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MATERIALS AND METHODS

Protein Expression and Purification. E. coli C43(DE3) cells were adopted for all protein expression. Protein expression and purification were executed as described in our previous study.15 The detailed procedure is provided in the Supporting Information and Methods. The purified HEBR and HEBR-D83N samples were prepared in pure water for UV−vis spectra measurements with a U1900 spectrophotometer (Hitachi) at 25 °C to confirm the stability of purified protein. Preparation of Hyaluronan-Grafted Chitosan Substratum. Chitosan powder (molecular weight of 510 kDa and deacetylation of 77%) was purchased from Sigma-Aldrich (USA), and hyaluronan powder (molecular weight of 1880 kDa) was obtained from SciVision Biotech Inc. (Kaohsiung, Taiwan). To prepare hyaluronan-grafted chitosan (CS-HA) substrates on culture plates, 1% chitosan solution (1.5 mL) was prepared in 1% acetic acid and then poured onto each well of the six-well tissue culture polystyrene (TCPS) plates. After airdried in a laminar flow cabinet, 1.5 mL of hyaluronan solution (3 mg/ mL in pure water) was then coated on the surface of chitosan membrane. After the evaporation of solvent, the chitosan and hyaluronan were cross-linked by ethyl (dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS, weight ratio 1.84:0.23) solution for 48 h to form the final CS-HA substrates. The culture plates coated with CS-HA substrates were stored at 4 °C before use. Flash-Laser-Induced Photocycle Measurements. A Nd:YAG laser (532 nm, 6 ns pulse, 40 mJ) was connected with a laboratoryconstructed laser cross-beam flash spectrometer for flash photolysis as described.15 The purified proteins with absorbance of 0.3 at their corresponding maximum wavelengths (λmax, or Abs-max) and the transient absorbance changes were recorded at their ground state. The curves represent the loss and recovery of absorbance at the target wavelengths upon induced with the green laser (λex = 532 nm). Light-Driven Photocurrent Measurements. Two indium tin oxide (ITO) coated glass slides were assembled and served as a proton-detection material as described in our previous study.15 A sample chamber was assembled from top to bottom in an order of ITO-coated slide, sample, dialysis membrane, blank solution, and enclosure with another ITO-coated slide. After those two ITO-coated slides were connected by wire, the whole device was further linked to a signal amplifier (SR570, Stanford Research Systems, Sunnyvale, CA) for photocurrent recording. A modulated 532 nm continuous 0.5 W laser controlled by a data acquisition system was set to stimulate samples for a duration of 850 ms. For each sample, a total of 64 trials were averaged. Procedures of Green Light Stimulation. A green light-emitting diode (LED, 530 nm, 5 W) was used to induce the transient activation of HEBR proteins in NSCs. After the treatment of HEBR proteins for 3 h, NSCs were illuminated with the green light three times (8 h interval) per day. Each illumination lasted for ∼10 s, and the illumination distance from the cell samples to the light source was B


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Figure 1. Protien expression and characterization. (A) Protein visual colors of HEBR (left) and HEBR-D83N (right). (B) Absorption spectrum of HEBR analyzed by UV−vis spectroscopy. Flash-laser-induced photolysis recording of (C) HEBR and (D) HEBR-D83N. Light-driven proton pumping activity of (E) HEBR and (F) HEBR-D83N probed with the photocurrent signals recorded with an ITO-based device. Samples in all measurements were prepared in pure water without buffer capacity.

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RESULTS Characterization of HEBR and HEBR-D83N Proteins. HEBR and HEBR-D83N were expressed and purified as described in the Methods. The purified proteins (Figure 1A) were first checked for its photochemical properties. UV−vis spectroscopy showed that the maximum absorbance was 551 and 583 nm for HEBR and HEBR-D83N, respectively (Figure 1B). Both samples were stable as there was no other absorbance peaks shown in the absorption spectrum. The photocycle measurement of HEBR (Figure 1C) and HEBRD83N (Figure 1D) clearly showed both proteins responded to the light. However, light-driven photocurrent recording of HEBR (Figure 1E) demonstrated the light-driven proton pumping capability was intact, while no photocurrent was detected in HEBR-D83N (Figure 1F). Cellular Distribution of HEBR Proteins in NSCs. Bacteriorhodopsin executes the proton pump activity when integrated on the bacterial membrane. Here, we investigated if the recombinant HEBR proteins could enter NSCs, and, if they

entered NSCs, they were located in the cells. For the purpose, NSCs were incubated with HEBR proteins supplied in the culture medium for 24 h, and then the cellular distribution of HEBR was observed by immunostaining. As shown in Figure 2, HEBR proteins spread in the whole NSCs with a punctate pattern, and in particular, they displayed a greater density in

Figure 2. Intracellular distribution of HEBR proteins in NSCs. After incubation with the HEBR proteins for 24 h, the distribution of HEBR proteins in NSCs was revealed by immunostaining. The aggregation of HEBR proteins in nuclei was indicated by white arrows in panel A. The magnified image of selected region in panel B was indicated by white box a. Scale bar, 10 μm. C


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cell number when cultured in the medium containing 3.75 and 7.5 μg/mL of HEBR proteins, and further had 59% reduction at the higher HEBR concentration of 15 μg/mL. Meanwhile, we also determined the growth curves of HEBR protein-treated NSCs, and results are shown in Figure S1. By comparing Figure S1 and Figure 3, the tendency of NSC growth in the presence of HEBR was consistent with the reduction observed in cell number. NSCs were further exposed to the green light to determine whether the growth inhibition effect was associated with proton pump activity of intracellular HEBR proteins. Upon green light exposure, the number of NSCs in the presence of 15 μg/mL of HEBR proteins further went down to 24%. For NSCs treated with the mutant HEBR proteins (HEBR-D83N, 15 μg/mL), the growth inhibition was alleviated as compared to the HEBR groups either with or without green light. This finding confirmed that the growth inhibition effect of HEBR was related to the proton pump activity. HEBR-Induced Cell Cycle Arrest in NSCs. To investigate if abnormal cell death occurred for NSCs treated with HEBR proteins (15 μg/mL) and green light, the health status of cells at 3 d was determined by the levels of intracellular oxidative stress. Meanwhile, the dead NSCs were revealed by PI staining. As shown in Figure S2, the respective proportion of each cells, which were healthy, unhealthy, and dead, was not significantly different among three groups (untreated, HEBR, or HEBRD83N treated). This observation indicated that HEBR proteins could not result in the extra death of NSCs. On the other hand, the cell cycle of HEBR-treated NSCs was analyzed by quantifying the DNA contents at 24 and 72 h, and results are respectively shown in Figures 4 and S3. At 24 h, the

nuclei (indicated by white arrows, Figure 2A). Meanwhile, HEBR proteins were also observed on or proximately to the cell membrane of NSCs (indicated by the white box a in Figure 2B). Growth Inhibition of HEBR Proteins on NSCs. After entering cells, the effects of HEBR proteins on the growth of NSCs were first evaluated by direct cell counting. After the treatment with HEBR proteins of different concentrations for 3 d, the cell numbers of NSCs were compared to the untreated control group (Figure 3). NSCs had ∼40−50% reduction in

Figure 3. Relative number of NSCs after incubation with HEBR proteins for 3 d. NSCs were treated with normal or mutant (D83N) HEBR proteins at different concentration as indicated for 3 d, and then the cell number was determined by cell counting. The cell number of each group was represented by the relative ratio (%) to the control group (without HEBR treatment). ∗p < 0.05, among the indicated groups.

Figure 4. Cell cycle arrest in NSCs in vitro by HEBR proteins. The cell cycle was analyzed using the two-step cell cycle assay following 24 h of treatment with 15 μg/mL normal or mutant HEBR proteins as indicated. All groups were subjected to green light illumination. Cells in different cell cycle stages were demarcated by the markers in panel A and quantified in panel B. ∗p < 0.05, ∗∗p < 0.01, among the indicated groups. D


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of HEBR on mitochondrial respiration was alleviated when NSCs were not illuminated with the green light (right panel in Figure 5B). Mitochondrial Hyperpolarization in HEBR-Treated NSCs. Inner membrane potential of mitochondria is closely related to the aerobic respiration and ATP production, and depolarization of the mitochondrial inner membrane is also a critical phenomenon for cell apoptosis. In addition to the blockage of proton/electron transport in mitochondria, we investigated the changes of membrane potential of mitochondria by JC-1 staining in HEBR-treated NSCs. The mitochondrial membrane potential of NSCs revealed by the intensity levels of red (JC-1 aggregates) and green (JC-1 monomers) fluorescence is shown in Figure 6A. By the indication of crisscross reference lines, the apparently upward shift of entire cell population was observed for the HEBR-treated NSCs as compared to the mock and HEBR-D83N-treated groups. This finding confirmed the enhancement of JC-1 aggregates in mitochondria of NSCs by HEBR proteins. Meanwhile, the untreated and mutant HEBR-treated NSCs were quite similar in the intensity plots of JC-1 red/green fluorescence (Figure 6A). The mean intensities of JC-1 red and green fluorescence were respectively quantified and are shown in Figure 6B. With the treatment of HEBR proteins, the mean fluorescence intensity (MFI) was increased for JC-1 red fluorescent signals and decreased for JC-1 green fluorescent signals. Nevertheless, these effects upon JC-1 intensities were not as obvious when HEBR was replaced by mutant HEBR. The above data demonstrated that the uptake of HEBR proteins by NSCs induced the mitochondrial hyperpolarization in the cells. Effects of HEBR Proteins on the Cell Fate Determination of NSCs. The functionality of NSCs relies on their differentiation tendencies and capacities. After incubation with HEBR proteins for 3 d, the mRNA expression levels of several neural-related genes in NSCs were analyzed by qRT-PCR as described in the Supporting Information to evaluate the effect on cell differentiation. Results are shown in Figure 7A (with green light illumination) and Figure S4 (without green light illumination). As shown in Figure 7A, the expression level of nestin was maintained after the HEBR treatment, which indicated that the stemness of NSCs was not affected by HEBR. The differentiation potential of NSCs toward neural and glial cells was determined by the expression of βIII-tubulin (Tubb3)/microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP)/2′,3′-cyclic nucleotide 3′ phosphodiesterase (CNPase), respectively. With the treatment of HEBR, the expression levels of GFAP and CNPase in NSCs were both elevated, while those of neural markers were either down-regulated (Tubb3) or not significantly altered (MAP2) as compared with the mock group. In addition, the mRNA expression levels of these five neural-related markers in the HEBR-D83N group were all comparable and displayed similar tendencies to those in the HEBR-treated group. Regarding the effect of green light, we also observed that no matter with or without green light illumination, NSCs tended to differentiate into glial cells rather than neurons in the presence of either HEBR protein (Figure S4). The expression of nestin, GFAP, and CNPase was further compared among each group in the protein levels determined by flow cytometry at 3 d. As shown in Figure 7B, the protein levels of nestin, GFAP, and CNPase were all increased in NSCs by the addition of HEBR proteins. Meanwhile, the treatment of HEBR-D83N proteins did not increase the protein

percentage of NSCs situated in the G0/G1 stage increased to 70% for NSCs treated with HEBR, while the entire cell cycle of NSCs was not obviously affected by the treatment of HEBRD83N proteins versus mock (Figure 4). After 72 h, the G0/G1 arrest (from the mock ∼60% increased to ∼75%) was observed for both HEBR and mutant groups (Figure S3). These data suggested the immediate effect of HEBR with green light on the induction of cell cycle arrest in NSCs. Influence of HEBR Proteins on Mitochondrial Respiration of NSCs. The oxygen consumption rate (OCR) was analyzed to investigate whether the mitochondrial bioenergetics of NSCs was altered in the presence of HEBR. In Figure 5A, the mitochondrial respiratory capacity of NSCs was

Figure 5. Analysis of the mitochondrial respiratory activity in HEBRtreated NSCs. (A) NSCs were treated with HEBR proteins for 24 h, and exposed to the green light during the treatment. The profiles of mitochondrial respiration in NSCs were represented by the oxygen consumption rate (OCR). During the measurement, oligomycin, FCCP, and antimycin-A/rotenone were sequentially added as indicated. (B) Relative spare respiratory capacity of mitochondria in the NSCs treated with normal or mutant HEBR proteins as compared to the corresponding control groups. ∗p < 0.05, ∗∗p < 0.01, among the indicated groups.

presented by Seahorse Cell Mito Stress test. After the treatment of both types of HEBR proteins with green light exposure, the basal respiration (the OCR value before the addition of oligomycin) of NSCs was reduced by more than two-fold compared with the mock group. Another important indicator provided by the test is the spare respiratory capacity, which is the difference of OCR values between the maximal (induced by FCCP addition) and basal respiration. As shown in Figure 5A, the extent of OCR enhancement following the FCCP addition was attenuated by both HEBR and mutant proteins. The relative spare capacities are summarized in Figure 5B. Apparently, the obstruction of mitochondrial respiration was more severe in the HEBR-treated NSCs than HEBR-D83N-treated ones. Meanwhile, this interfering effect E


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Figure 6. Analysis of the mitochondrial membrane potential in HEBR-treated NSCs by JC-1 staining. After the treatment of HEBR proteins for 24 h with green light illumination, NSCs were subjected to JC-1 staining. In panel A, the dot plots show the intensity of JC-1 red versus green fluorescence in every single NSC. The crisscross reference lines were used to clearly indicate the movement of cell population in the dot plots. The mean intensities of JC-1 red and green fluorescence in each group of NSCs are shown in panel B. ∗∗p < 0.01, among the indicated groups.

GFAP and CNPase were both lower for the spheroid-forming NSCs compared to those cultured on TCPS. In addition to cell differentiation, we further investigated the alteration of mitochondrial respiration capacities of spheroidforming NSCs upon HEBR treatment and green light illumination (Figure 8B). We discovered that the basal respiration and relative spare capacity was both reduced in the HEBR-treated NSC spheroids, while the reduction was not alleviated when HEBR proteins were replaced by HEBR-D83N proteins, which indicated that the influence of HEBR proteins on the respiratory activity in spheroid-forming NSCs did not result from the alteration of proton gradient.

expression for any of the three markers. The most significantly altered protein expression was observed in CNPase, which increased from 50% (mock) to ∼67% in average for NSCs treated with normal HEBR proteins and green light exposure. Results were further confirmed by the analysis of relative fluorescent intensity quantified by ImageJ software. The images of representative cells after immunostaining as well as semiquantified results are shown in Figure S5. Although the difference was not quite obvious, the expression tendencies for protein levels of nestin, GFAP, and CNPase revealed by immunostaining (Figure S5) were generally consistent with those determined by flow cytometry (Figure 7B). Differentiation and Mitochondrial Respiration of Spheroid-Forming NSCs after HEBR Treatment. We investigated if the functions of HEBR proteins in NSC differentiation and metabolism would be affected by culture substrates. For the purpose, we cultured NSCs on the CS-HA substratum and treated the cells with HEBR (or HEBR-D83N) proteins. On CS-HA, NSCs were assembled into 3D spheroids rather than attached on the plastic plate as monolayer. The expression of neural-related markers was analyzed at 3 d. As shown in Figure 8A, the differentiation tendencies of spheroidforming NSCs were similar after the respective treatment of HEBR and HEBR-D83N proteins. The transcription levels of neuronal markers, Tubb3 and MAP2, were up-regulated after the HEBR/HEBR-D83N treatment combined with green light illumination. Meanwhile, the enhanced expression levels of

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DISCUSSION Repression of mammalian neural cells can be temporally and spatially manipulated in vitro and in vivo through expressing bacteriorhodopsin, the light-triggered proton pump.18−20 In addition, the controllable neural activity was observed for the neural cells expressing NpHR (a type of HR) and ChR2 (a type of ChR) in the central nervous system (CNS) of transgenic mice21,22 and monkey.23 These findings implicate the therapeutic potential of microbial rhodopsins in the treatment of neural disorders such as epilepsy. Although the activities of rhodopsin-expressing neurons are rapidly manipulated by light excitation, the inappropriate expression levels and cellular localization of transgenic rhodopsin may result in the side effects on these neural cells,21 indicating the expression of transgenic rhodopsins must be tightly controlled F


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Figure 7. Stemness and differentiation potential of HEBR-treated NSCs with green light illumination. NSCs were treated with normal or mutant HEBR proteins for 3 d and with the green light-illuminated condition. (A) Expression levels of markers for stemness, neural, and glial cells, including nestin, Tubb3, MAP2, GFAP, and CNPase, were analyzed by qRT-PCR. The expression of each marker was normalized to that of GAPDH in each group and then presented as the relative expression level to the mock group. (B) Protein levels of nestin, GFAP, and CNPase in NSCs were determined using flow cytometry. The percentages of NSCs demarcated by the markers of M1 (nestin), M2 (GFAP), and M3 (CNPase) were shown for each group as indicated. n.s., not significant, ∗p < 0.05, ∗∗p < 0.01, among the indicated groups.

for use in precision medicine in the future.24 In the current study, we mass-produced the normal and abnormal HEBR proteins. We demonstrated that the direct administration of specific concentration of HEBR proteins followed by green light illumination could dramatically interfere with the behavior of NSCs, and these effects were generally correlated with the function of BR in outward proton pumping. The current work provides the first evidence that engineered rhodopsins may directly enter mammalian cells and exert an influence. The finding suggests the potential of artificially produced rhodopsins as biological materials in modulating cell behavior. During the purification of HEBR proteins, a trace of detergent (DDM) may still exist after the dialysis. To avoid the effects of HEBR proteins that resulted from the residual detergent, the residual detergent was further deprived by lipidabsorbing beads. The HEBR proteins with further lipid absorption were subjected to analyses of photocycle recording and light-driven photocurrent. As shown in Figure S6A, no significant difference was observed. Meanwhile, we also analyzed the inhibitory function of HEBR proteins in the growth of NSCs after the deprivation of DDM by lipidabsorbing beads. We found that the effects were not

significantly different for the HEBR proteins processed with or without further deprivation of DDM (Figure S6B). Therefore, we suggest that the alteration of cell behavior was not caused by the residual detergent. In bacteria, BR is distributed on the cell membrane and transports the protons outward to establish a transmembrane proton gradient.25 On the basis of the current data, we have confirmed that the HEBR proteins provided in the culture medium would enter NSCs, while the cellular localization of HEBR proteins could not be exactly defined. HEBR and HEBR-D83N proteins could be integrated on the cell membrane or the membrane of some organelles in NSCs, but only the former would result in the alteration of proton concentration and pH value across the cell membrane. Meanwhile, both HEBR and HEBR-D83N proteins could randomly and transiently change the local distribution of protons if they were spread in the cytoplasm rather than anchored on the membrane. According to the results of cell growth and metabolism, both HEBR and HEBR-D83N proteins were functional in NSCs, and the effects can be further enhanced by green light illumination. Meanwhile, the effects of HEBR were greater than those of HEBR-D83N. Therefore, we suggest that a part of HEBR proteins may be G


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Figure 8. Differentiation and aerobic metabolism of spheroid-forming NSCs after HEBR/HEBR-D83N treatment. NSCs cultured on CS-HA substrates were treated with HEBR or HEBR-D83N proteins (7.5 μg/mL) as well as green light illumination, and then the differentiation and metabolism of NSCs were analyzed, respectively. (A) Expression levels of several neural-associated markers were, respectively, determined by qRTPCR. The expression of each marker was normalized to that of GAPDH in each group and then presented as the relative expression level to the mock group. (B) Respiratory capacity was evaluated by the Seahorse Cell Mito Stress test. The profiles of mitochondrial respiration were represented by the OCR (left panel). The compounds of oligomycin, FCCP, and antimycin-A/rotenone were sequentially added as indicated. Relative spare respiratory capacity of mitochondria in the NSC spheroids treated with HEBR proteins as compared to the mock group (right panel). n.s., not significant, ∗p < 0.05, ∗∗p < 0.01, among the indicated groups.

integrated onto certain membrane and execute proton pumping, leading to the more obvious effects of HEBR on the modulation of cell behavior. The origin of mitochondria is still a mystery. The most prevalent hypothesis is that the mitochondria originated from the bacteria engulfed by eukaryotic cells. After the engulfment, the bacteria were reserved and then evolved into the mitochondria in eukaryotic cells.26 On the other hand, the electrochemical proton gradient established by BR is used to produce ATP in microorganisms.25 An identical mechanism is also adopted by the mitochondria for aerobic energy generation. Thereby, the mitochondria would be a possible location for delivered HEBR proteins in NSCs due to the biologically similar microenvironment. Using the Cell Mito Stress test, we demonstrated that the rates of basal and

maximal respiration were both reduced in mitochondria after the HEBR treatment. The decreased oxygen consumption indicated that the electron transport chain was interrupted and the proton cycle between the intermembrane space and matrix in mitochondria may be abnormal. The exogenous HEBR proteins may be located in the mitochondria and interfere with the proton gradient by their proton pump activity, resulting in the reduction of mitochondrial respiratory capacity. In addition to the aerobic respiration, the mitochondrial membrane potential was also affected by HEBR proteins. The mitochondrial depolarization (reduction of JC-1 aggregation) is an important indicator for cell apoptosis.27 In the current study, the mitochondrial depolarization did not occur in NSCs after the HEBR induction, suggesting that the apoptosis could not be triggered by HEBR in NSCs. These data correspond H


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ACS Biomaterials Science & Engineering with the findings in the vitality assay that the abnormal cell death was not observed in HEBR-treated NSCs (Figure S2). In general, the depolarization of mitochondria is accompanied by the lower energy production in cells.28,29 After the HEBR treatment, we discovered that the aerobic respiration of mitochondria was decreased, whereas the mitochondrial membrane potential was hyperpolarized instead. The normal value of mitochondrial membrane potential is maintained between 100 and 140 mV in living cells, and the maximal efficiency of ATP production occurs at ∼120 mV.30 Although it has been reported that the ATP levels elevated in the mitochondria of neurons when the mitochondrial inner membrane potential increased,31,32 the low ATP production was also observed in the hyperpolarized mitochondria in neural cells from the patients of frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17).33 According to these reports, the energy generation and membrane potential are not absolutely correlated, depending on the cell origin/type, hyperpolarized extent, or other experimental parameters. Thus, the hyperpolarization of mitochondrial membrane potential induced by HEBR proteins may be a cause of the reduced respiratory activity in NSCs. Nevertheless, the relationship and order of consequence among the membrane hyperpolarization, low energy production, and interfered proton gradient induced by HEBR proteins still need to be further investigated. The high mitochondrial membrane potential (over 140 mV) would dramatically increase the level of reactive oxygen species (ROS) in cells.34,35 Although HEBR proteins elevated the mitochondrial membrane potential, ROS were not further generated in NSCs (proven by the unchanged VB-48 intensity in Figure S2), showing the moderate effects of HEBR proteins on mitochondrial membrane potential. The dysfunction of mitochondria including membrane depolarization was related to several neural associated diseases.36,37 Therefore, the HEBR proteins could be tested in the future to transiently recover the reduced membrane potential by the appropriate light illumination, as a potential therapy for these diseases. The growth of NSCs was obviously inhibited by HEBR proteins in a dose-dependent manner (Figure 3). On the basis of the data of growth curves of HEBR-treated NSCs (Figure S1), NSCs still proliferated after the HEBR treatment. However, the proliferation rate was apparently declined. By taking the information derived from cell cycle analysis, the G0/ G1 arrest of cell cycle was induced by HEBR proteins, leading to the consequences of cell number reduction. In Figure 2, we demonstrated that HEBR proteins seemed to aggregate in the nuclear region. It has been reported that the progression of cell cycle was slowed down by a decrease of pH value.38,39 Therefore, the possibility could not be ruled out that the HEBR-induced cell cycle arrest actually accounted for the variation of pH value around the nuclei. On the other hand, the cancer cells situated in the different stages of cell cycle display the diverse responses to the drugs. For example, certain cancer cells were further sensitive to the TNF-related apoptosis-inducing ligand (TRAIL), a potential anticancer protein, when their cell cycle was blocked.40 We speculated that if the inhibitory effect of cell cycle triggered by HEBR could be acted on cancer cells, the simultaneous or sequential codelivery of antitumor drugs and HEBR may enhance the therapeutic efficiency through an inducible cell cycle arrest controlled by light illumination.

We also investigated the effects of HEBR proteins on the differentiation of NSCs to evaluate if HEBR proteins could be applied to neural induction. According to the analyses of expression levels of several neural-associated genes and proteins, we suggested that the exogenous HEBR proteins slightly promoted the differentiation of NSCs toward glial cells on TCPS plates. In our previous study, we demonstrated that NSCs would differentiate into neurons after the HEBR transfection using the chitosan (CS) hydrogel-based in situ three-dimensional (3D) approach.24 After the transfection by this approach, the NSCs were encapsulated in the soft CS hydrogel and gradually grew into the cellular spheroids. The stiffness of 3D growth environment provided by CS hydrogel is much lower than that of NSCs (plated on 2D plastic dish) in the current study. We thus cultured the NSCs on the CS-HA substrates and treated the cells with HEBR proteins to clarify the effects of culture condition on NSC differentiation. On CSHA substrates, NSCs were self-assembled into 3D spheroids that resembled neurospheres. Besides, the stiffness of TCPS may not exert full effect on individual cells. In the spheroidforming condition, the expression of neuronal-related markers was significantly promoted after HEBR treatment (Figure 8A), suggesting the neuronal differentiation in contrast to glial differentiation on TCPS. Differentiation of NSCs is associated with the status of mitochondrial metabolism,41,42 and we thus further verified the alteration of mitochondrial respiration capacities of spheroid-forming NSCs upon HEBR treatment and green light illumination (Figure 8B). We found that the reduction in both basal respiration and relative spare respiratory capacity was greater in monolayer- versus spheroid-cultured NSCs (Figures 5 and 8B). The higher aerobic respiration in spheroid-forming NSCs versus those cultured on TCPS after HEBR treatment is consistent with the reported relationship between NSC differentiation and metabolism during in vivo neurogenesis.41,42 Taken together, we suggest that the regulatory effects of HEBR proteins on the differentiation of NSCs may be partially derived from the modulation of mitochondrial respiration in NSCs, which may be further manipulated by the cell culture substrates in the future. On the other hand, our previous study has demonstrated that the photocycle of HEBR proteins can be triggered by green light illumination after encapsulation within the biocompatible hydrogels.24 Therefore, HEBR proteins may be embedded within certain injectable and biocompatible hydrogels and then delivered to injured neural tissues for functional recovery by promoting the neural or glial differentiation. A further combination of HEBR proteins and hydrogels will be worthy of investigations for in vivo delivery of HEBR proteins. Regarding the in vivo delivery of HEBR proteins, the delivery efficiency is also a critical issue. In the current experiments of NSC spheroids, NSCs were treated with HEBR proteins before the formation of spheroids. Therefore, we suggest that the amount of HEBR proteins is uniform in NSCs located on the surface and in the interior of the spheroids. To further address the concern about in vivo delivery efficiency of HEBR proteins, here, we also compared the amount of HEBR proteins entering NSCs before and after the spheroid formation. By the analysis of flow cytometry, the relative amount of HEBR proteins was quantified in NSCs. As shown in Figure S7, the percentages of NSCs detected with HEBR signal were ∼55% and ∼20%, respectively, when the HEBR proteins were provided before and after the spheroid formation. The finding may indicate that I


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HEBR proteins in the culture medium could not diffuse into the central part of the spheroid after the spheroid had formed. On the basis of the current findings, an efficient approach to deliver the HEBR proteins into cells still needs more investigation before the in vivo applications.

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CONCLUSIONS The current engineered HEBR proteins provided direct evidence that multiple physiological behaviors of mammalian NSCs were affected by BR, bacterial proton pump. The proton concentration and cellular acidity may be adjusted by HEBR proteins, resulting in the alteration of cell behavior. Therefore, the mass-produced HEBR proteins could be a potential biomedical material or drug due to the multiple regulatory effects on mammalian cells. Meanwhile, as a proton carrier, HEBR protein could also serve as an intelligent light-sensitive pH modulator and may have diversified application potentials through further development, especially in combination with the other biocompatible materials to form composite biomaterials used in vivo.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.9b00367. Materials and methods; growth curves; vitality analysis; cell cycle analysis; stemness and differentiation potential; protein expressions; functional assays; flow cytometry measurements; primer sequences (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +886-2-3366-5313. Fax: +886-2-3366-5237. *E-mail: [email protected]. Phone: +886-2-3366-2275. ORCID

Shan-hui Hsu: 0000-0003-3399-055X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the research plan of National Taiwan University Core Consortium (NTU-CC-108L891101) and the funding of National Taiwan University for the postdoctoral fellowship (NTU-107L4000).

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REFERENCES

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Potential of Engineered Bacteriorhodopsins as Photoactivated

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