Retinal absorbs blue light and induces translocation of PIP2 sensor to the cytosol

The objective was to examine whether retinal or blue light excited retinal, independent of photoreceptors, elicits PIP2 hydrolysis and inositol (1,4,5) triphosphate (IP3) generation in cells, because calcium and its regulatory pathways are suggested as key players of cytotoxicity in the retina5,23. We employed HeLa cells as the major cell line here to eliminate potential response contamination due to retinal and blue light activating endogenous photoreceptors in cells derived from retina. Upon retinal addition to HeLa cells expressing PIP2 sensor (mCherry-PH), cells did not show any change in sensor distribution (Fig. 1A, left). These cells were exposed to blue light at every one-second interval for 10 minutes. Starting blue light intensity was set to 0.22 µW, since this power is sufficient to activate retinal bound to photoreceptors. Cells were imaged for mCherry while gradually increasing blue light intensity. Intensities at 1 μW and above, cells exhibited mCherry-PH translocation to cytosol (Fig. 1A,B, Movie-S1). However, at these intensities, photobleaching of fluorescence proteins was not observed. Interestingly, the translocated mCherry-PH did not recover, even after termination of blue light exposure, suggesting the likelihood of irreversible photochemical perturbation of PIP2 by blue light excited retinal.

Figure 1 Comparison of photoreceptor dependent PIP2 hydrolysis vs photoreceptor independent PIP2 sensor translocation by photoexcited retinal. (A) Images of HeLa cells incubated with 50 µM ATR (retinal) expressing PIP2 sensor (mCherry-PH). Both images and the plot of F cy vs time show that cells exposed to 0.22 µW 445 nm blue light did not respond, while cells exposed to 4.86 µW and 9.70 µW blue light exhibited mCherry-PH translocation to cytosol (mean ± S.E.M.). (B) The plot of initial PIP2 sensor dislodging rate vs laser power of 445 nm blue light. (mean ± S.E.M., n = 6 cells in each experiments). (C) Images of HeLa cells expressing Gq-coupled Melanopsin and mCherry-PH. Cells were incubated with ATR (50 µM) for 5 minutes. A significant PIP2 hydrolysis was observed upon optical activation (OA = blue box) of melanopsin using short pulses of blue light (0.22 µW of 445 nm). Recovery of PIP2 sensor to the PM was observed even the blue light exposure is continued. The plot shows the dynamics of PIP2 sensor translocation in cytosol. Mean and S.E.M. are from 3 < independent experiments. (blue light (BL) = blue box). Scale = 5 µm. Full size image

Response signatures of mCherry-PH translocation induced by blue light (4.86 μW) excited retinal (BLE-retinal) were compared with PIP2 hydrolysis elicited by melanopsin-Retinal Schiff Base (RSB), to examine if both processes trigger similar chemical changes to PIP2. HeLa cells expressing melanopsin and mCherry-PH were incubated with ATR (50 µM) for 5 minutes in dark to allow RSB formation. Cells exhibited PIP2 hydrolysis upon exposure to low intensity blue light (0.22 µW), as indicated by the translocation of mCherry-PH from PM to cytosol (Fig. 1C, Movie-S2). The observed PIP2 hydrolysis was transient and recovered within 2–3 minutes. Gq-coupled GPCR activation induced-PIP2 hydrolysis recovers over time due to signaling adaptation24,25. Similar to melanopsin activation, addition of carbachol to HeLa cells expressing M3R and PIP2 sensor exhibited PIP2 hydrolysis. A similar recovery of PIP2 was also observed (Fig. S1, Movie-S3).

During PIP2 hydrolysis, IP3 moiety of PIP2 dissociates from PM anchored diacylglycerol (DAG) domain. The PH domain of PLCδ1 in the PH sensor moves to cytosol with IP3 upon PIP2 hydrolysis26. The irreversible translocation of mCherry-PH by BLE-retinal was further characterized to determine if this is due to PIP2 hydrolysis or a photochemical perturbation to PIP2, either by breaking the molecule or preventing PIP2 sensor binding. To examine whether BLE-retinal induces mCherry-PH translocation due to PIP2 hydrolysis, leaving DAG on the PM, DAG generation was imaged using cytosolic DAG sensor, YFP-DBD, that translocates to PM. Cells incubated with ATR failed to exhibit a detectable mCherry-PH translocation (Fig. 2A). When a selected cell in the field of vision (Fig. 2C, yellow arrow) was exposed BLE-retinal for 400 seconds, a robust mCherry-PH translocation only in blue light exposed cell was observed (Fig. 2B). However, the DAG sensor did not translocate to PM (Fig. 2B), indicating that, exposure to BLE-retinal does not leave DAG at PM. We also comparatively examined Gq-coupled M3-muscarinic receptor (M3R) activation in the same HeLa cells transiently expressing M3R. When cells were exposed to 10 µM carbachol, both PIP2 hydrolysis and DAG formation were observed (Fig. 2A). When this cell was exposed to YM254890 (1 µM), which inhibits Gαq heterotrimer activation, both PIP2 and DAG sensors reversed to their basal pre-activation status, suggesting PIP2 recovery (Fig. 2A). Interestingly, the cell exposed to BLE-retinal did not show such a recovery upon addition of YM254890 (Fig. 2B). This further confirms that BLE-retinal induced PIP2 distortion in cells is irreversible.

Figure 2 Comparison of M3-muscarnic receptor mediated PIP2 hydrolysis vs blue light excited retinal (BLE-retinal) induced PIP2 sensor translocation. Images of HeLa cells expressing M3-muscarinic receptor, mCherry-PH (PIP2 sensor), DBD-YFP (DAG sensor). (A) Without blue light exposure, retinal addition does not change PIP2 or DAG sensor distribution (left), while the addition of carbachol resulted in PIP2 hydrolysis and DAG formation (middle). The addition of Gq inhibitor, YM254890 (1 µM) to cells resulted in reverse translocation of PIP2 and DAG sensors. (mean ± S.E.M., n = 6 cells) (B) When cells were exposed to blue light (4.86 µW of 445 nm) in the presence of retinal, they only showed PIP2 sensor translocation while no change in DAG sensor was observed (left). The addition of carbachol to these cells exhibited an additional PIP2 sensor translocation to the cytosol with a mild DAG sensor translocation to the PM (left) (mean ± S.E.M., n = 4 cells). Interestingly, addition of YM254890 only reversed both PIP2 and DBD responses elicited by carbachol. (C) The field of vision of cells shown in A and B. Mean and S.E.M. are from 3 < independent experiments. (blue light (BL) = blue box). Scale = 5 µm. Full size image

Characteristics of retinal photoexcitation in cellular environment

Compared to the light power required for opsin activation (~0.2 μW), a higher power of blue light (>1 μW) was required for free retinal to induce PIP2 distortion. This can be partly due to the bathochromic shift in the absorption spectrum of melanopsin-retinal Schiff base (m-RSB) with a λ max = 478 nm, compared to the λ max = 383 nm of free retinal (Fig. 3A,B). This allows m-RSB to have a greater spectral overlap with blue light (445 nm) that drives a larger population of molecules to the excited S 1 (π* ← π) state, compared to free retinal (Fig. 3C). Since quantum yield of m-RSB isomerization is estimated to be at near unity27,28,29, all absorbed photons result in GPCR activation. Additionally, the lower power requirement for melanopsin activation induced PIP2 hydrolysis is due to the enhanced photosensitivity of m-RSB and PIP2 hydrolysis being an enzymatic process while BLE-excited retinal induced PIP2 distortion is likely to be a non-enzymatic and stoichiometric reaction.

Figure 3 Energy and wavelength requirement for PIP2 solubilization by photoexcited retinal. (A) UV-VIS absorption spectra for both 11-cis retinal (11CR) and all trans retinal (ATR). Note that 445 nm blue light spectrally overlaps with both absorption spectra. (B) The absorption spectra of melanopsin and retinal (left), (ε ATR = 44180.0 M−1cm−1). (C) The energy level diagram and the population (pop.) of energy levels of free retinal (blue) and melanopsin (red) according to their respective absorption maxima (right). Note that blue light (445 nm) can highly populate melanopsin compared to that of free retinal. (D–F) Images of HeLa cells expressing PIP2 sensor (mCherry-PH). (D) Cells were incubated with ATR (50 µM) for 5 minutes. A substantial PIP2 sensor translocation was observed upon exposing cells to short pulses of blue light (4.86 µW of 445 nm). The plot shows the dynamics of PIP2 sensor translocation to cytosol. (E) In the absence of retinal, cells did not show a detectable PIP2 sensor translocation when exposed to blue light or other wavelengths. (F) Both blue light excited ATR (50 µM) and 11CR (50 µM) exhibited a permanent accumulation of PIP2 sensor cytosol. Compared to exposed cell (yellow arrow), control cell without blue light (BL) exposure (white arrow) did not show any detectable PIP2 response. The plots show the dynamics of PIP2 sensor translocation in cells shown in F (mean ± S.E.M., n = 6 cells). (G) All trans retinal and blue light induce PIP2 sensor translocation in cells with distinct origins. Images of RAW264.7, NIH3t3, ARPE-19, MDA-MB-468, BT-20, HCT116 and HEK293 cells expressing mCherry-PH (PIP2 sensor). ATR (50 µM) was incubated in cells for 5 minutes followed by continuous exposure of blue light for 5 minutes. Blue light exposure induced PIP2 sensor translocation from PM in all the cell types tested while cells that were not exposed to blue light did not respond. Mean and S.E.M. are from 3 < independent experiments. (blue light (BL) = blue box). Scale = 5 µm. Full size image

To examine the wavelength dependency of BLE-retinal induced PIP2 distortion, HeLa cells expressing mCherry-PH were exposed to 445 nm, 488 nm, 515 nm and 594 nm wavelengths of light at 4.86 µW respectively, in the presence or absence of retinal. Cells exposed to 488, 515 and 594 nm light, in the presence of retinal did not show a detectable signal (Fig. 3D). Control cells also did not exhibit PIP2 distortion with blue light in the absence of retinal (Figs 3E and S3). Retinal addition alone (without blue light) also showed no PIP2 distortion (Fig. 3F). Only the selected cell in the field of vision exposed to blue light exhibited PIP2 distortion (Fig. 3F, yellow arrow, Movie-S4). Similar to ATR, 11CR also induces PIP2 distortion when exposed to blue light (Fig. 3F). Unless specified, all the experiments henceforward employed blue light with the power of 4.86 µW to excite retinal. At this light power, photobleaching of fluorescent biosensors was not observed. All the cell types examined including RAW264.7 (mouse macrophage), ARPE-19, MDA-MB-468 (triple negative breast cancer), BT20 (triple negative breast cancer), HCT116 (colon cancer), NIH3t3 (mouse embryonic fibroblast), and HEK293 (human embryonic kidney) exhibited mCherry-PH translocation upon exposure to BLE-Retinal (Fig. 3G) which indicates a universal mechanism operated by BLE-retinal.

To decipher molecular features of retinal for its blue light-induced cellular effects, compounds with analogous functional groups such as retinol, retinoic acid, β-ionone (β-ionone ring of retinal) and conjugated linoleic acid (CLA- mimicking the extended π conjugated system) were examined as follows. HeLa cells expressing mCherry-PH were incubated for 10 minutes with the retinal-like molecules at 50 μM concentrations (Fig. 4). Interestingly, none of these compounds were able to induce PIP2 distortion upon exposure to blue light (Fig. 4A–D). Examination of absorption spectra of these screened molecules show that they do not have sufficient spectral overlaps with the blue light (Fig. S3). However, both retinol and retinoic acid exhibited mCherry-PH translocation when exposed to 365 nm ultra violet light (Fig. S4).

Figure 4 Retinal-like molecules exhibited no effect on PIP2 upon blue light exposure. Images of HeLa cells expressing PIP2 sensor (mCherry-PH) on PM. In all of the experiments conducted, cells were exposed to blue light (4.86 µW of 445 nm) which is indicated by the white box. Cells were incubated with (A) β-ionone (50 µM), (B) 10E, 12Z linoleic acid (50 µM), (C) Retinol (50 µM) and (D) Retinoic acid (50 µM), for 10 minutes followed by continuous exposure of short pulses of blue light for 200 s. In all experiments, cells did not show a detectable PIP2 sensor translocation. Plots show the dynamics of PIP2 sensor translocation in cells shown in A–E. (mean ± S.E.M., n = 5–10 cells). Mean and S.E.M. are from 3 < independent experiments. (blue light (BL) = blue box). Scale = 5 µm. Full size image

BLE-retinal induced PIP2 distortion is independent of GPCR-G protein activation

Retinal and blue light mediated activation of Gq-coupled GPCRs are suggested to trigger signaling and cytotoxicity in ARPE-19 photoreceptor cells5. This study measured degeneration of cells in the mouse retina, however the underlying mechanism was unclear. Upon GPCR activation, both Gαq-GTP and Gβγ can induce PIP2 hydrolysis since they activate PLCβ30. To examine whether BLE-retinal activates GPCR-G proteins pathways, PIP2 distribution was studied in the presence of Gαq inhibitor-YM254890 (1 µM) and Gi-pathway inhibitor-pertussis toxin (PTX) (50 ng/mL) respectively (Fig. 5A–D). Previously, we showed that pertussis toxin inhibits Gβγ mediated PIP2 hydrolysis since inhibition of Gi-pathway activation prevents generation of free Gβγ31. After inhibitor treatment, cells incubated with retinal were exposed to blue light for 2–3 minutes while imaging the PIP2 sensor response. Both inhibitors failed to inhibit BLE-retinal induced PIP2 distortion (Fig. 5B,D). Additionally, HeLa cells were treated with both Gαq and Gβγ inhibitors simultaneously to rule out the possibility of activation of PLCβ by BLE-retinal. Regardless of the collective inhibition of Gβγ and Gαq, BLE-retinal induced PIP2 disruption in cells (Fig. S5). This indicates that BLE-retinal induced responses are not due to the GPCR-G protein pathway activation. Control experiments show YM254890 and PTX inhibit Gq-pathway induced PIP2 hydrolysis and Gi-pathway induced Gγ9 translocation, respectively (Fig. 5A,C)32. In addition, retina-derived ARPE-19 cells exhibited PIP2 disruption upon BLE-retinal in the presence of Gαq, Gβγ and Gi pathway inhibitors (Fig. S6). Contrary to the previous reports7, these experiments and data confirm that free retinal with or without light does not activate GPCR pathways.

Figure 5 Photoexcited retinal induced PIP2 translocation is not due to GPCR pathway activation. (A) HeLa cells expressing M3 receptor and mCherry-PH, carbachol (10 µM) was added to activate M3R in the presence (left) and absence (middle) of Gq inhibitor (YM254890, 1 µM, 5 min). Only control cells (no YM254890), showed PIP2 hydrolysis (left). (B) Even in the presence of Gq inhibitor, blue light excited retinal induced PIP2 sensor translocation (middle). The plots show the dynamics of PIP2 sensor translocation in the cells shown in A and B. (C) To HeLa cells expressing CXCR4-GFP, mCh-γ9, SDF1α (100 ng/mL) was added to activate CXCR4 in the presence (left) and absence (middle) of Gαi inhibitor (pertussis toxin = Ptx, 50 ng/mL, overnight incubation). Only control cells with no added Ptx exhibited mCh-γ9 translocation from PM to IMs (left). (D) Exposure to photoexcited retinal induced PIP2 sensor translocation in cells treated with Ptx. The plots show the dynamics of mCh-γ9 and PIP2 sensor translocation. In all the experiments conducted, cells were exposed to 4.86 µW of 445 nm blue light. (mean ± S.E.M., n = 5–10 cells). Mean and S.E.M. are from 3 < independent experiments. (blue light (BL) = blue box). Scale = 5 µm. Full size image

PIP2 distortion by BLE-retinal is associated with an increase in intracellular calcium, [Ca2+] i

According to the crystal structure of IP3 receptor33, IP3 molecule forms H-bonds with the putative interacting domain (Fig. 6A, PDB code:1N4K). To examine whether distorted PIP2, that may be entering cytosol could also increase cytosolic calcium, HeLa cells expressing mCherry-PH were incubated with cell permeable calcium sensor fluo4-AM and exposed to BLE-retinal. Cells exhibited a robust upsurge of calcium and a synchronized mCherry-PH translocation response was observed (Fig. 6B,C, Movie-S5). Unlike Gq-coupled GPCR induced calcium response, which recovers over time (Fig. S7A), BLE-retinal induced response did not exhibit signaling adaptation (Fig. 6C). To examine the source of calcium mobilized by BLE-retinal, cellular calcium modulators were deployed as follows and percent change in fluo-4 fluorescence in the cell upon exposure to BLE-retinal was calculated with respect to fluo-4 fluorescence observed prior to blue light exposure. Upon BLE-retinal exposure, cells without a calcium modulator was considered as the control and exhibited a 55 ± 8% increase in fluo4 fluorescence compared to basal fluorescence. Cells incubated in extracellular calcium free medium (using BAPTA) and with intracellular calcium chelator, BAPTA-AM, induced only 25 ± 5% and ~16 ± 1% increases respectively, (Fig. 6D). Treating cells with IP3 receptor inhibitor, 2-aminoethoxydiphenyl borate (2-APB) resulted in ~21 ± 4% increase (Fig. 6D). These data indicate possible interactions of the IP3 domain of distorted PIP2 with IP3 receptors33. Calcium modulators above were validated as follows (Fig. S7). Cells incubated with BAPTA-AM showed only PIP2 hydrolysis but no calcium response upon M3-muscarinic receptor activation (Fig. S7B), while 2-APB treated cells showed a transient PIP2 hydrolysis with no calcium response (Fig. S7C). Compared to BLE-retinal induced calcium responses, M3R activation induced responses were rapid and transient. These results also show the ability of BLE-retinal to induce downstream signaling, independent of GPCR activation. Collectively, these data suggest that BLE-retinal induced distortion of PIP2 not only induces mobilization of stored calcium, but also stimulate influx of extracellular calcium.

Figure 6 All trans retinal and blue light induced signaling in cells. (A) Crystal structure of the IP3 receptor bound with IP3 with H-bonding interactions (PDB code:1N4K), suggesting the PIP2 can have the majority of interactions exhibited by IP3. (B) HeLa cells expressing PIP2 sensor (mCherry-PH) and incubated with calcium sensor Fluo4. Fluo4 stained cells were incubated with ATR (50 µM) for 5 minutes, followed by exposure of blue light (4.86 µW of 445 nm) for 3 minutes. Here the whole cell was exposed to blue light. A substantial increase in cytosolic calcium is observed. (C) Dynamics of calcium responses and PIP2 translocation in the cells shown in B (mean ± S.E.M., n = 5 cells). (D) Calcium responses in control and calcium modulator-incubated (using BAPTA-AM and 2-APB) HeLa cells in regular and extracellular calcium free (using BAPTA) buffers. Here, cells were pre-incubated Fluo4 were incubated with 2-APB (5 µM for 15 min), BAPTA-AM (10 µM for 30 min), or BAPTA (5 µM for 5 min) in calcium free HBSS buffer. The cells were then incubated with ATR (50 µM) for 5 minutes, followed by continuous exposure of blue light for 5 minutes. The bar chart shows the changes in calcium sensor fluorescence in the cytosol before and after blue light exposure on cells for all the above mentioned experiments (mean ± S.E.M., n = 5–15 cells). Mean and S.E.M. are from 3 < independent experiments. (blue light (BL) = blue box). Scale = 5 µm. Full size image

Blue light-excited retinal induces cytotoxicity

The possibility of PIP2 distortion and calcium mobilization elicited by BLE-retinal to induce cytotoxicity was examined. Morphology of HeLa cells incubated in retinal containing medium was observed using time-lapse microscopy while only selected cells were exposed to blue light. PIP2 distortion and substantial morphological changes, including extensive bleb formation, were observed only in blue light exposed cells (Fig. 7A, Movie-S6). Unexposed control cells in the same field remained morphologically intact. To identify whether exposure of BLE-retinal induces cell death, HeLa cells were incubated with cell-death marker, propidium iodide (PI) and examined its inclusion, since apoptotic cells fail to exclude PI34,35. Cells were supplemented with 100 µL of PI (50 µg/mL) and 50 µM ATR, while selected cells were exposed to blue light. Control cells were either exposed to blue light without retinal or incubated with retinal without blue light exposure. Cells exposed to blue light in the presence of retinal showed gradual incorporation of PI, indicated by the increase in red fluorescence (Fig. 7B). Observed time curves of PI incorporation into cells were heterogenous (Fig. 7B, plot). Cells in both the control experiments did not incorporate PI (Fig. 7B). These results indicate that prolonged exposure of cells to BLE-retinal leads to cell death.

Figure 7 Photoexcited retinal induced cytotoxicity. (A) Fluorescence and DIC images of HeLa cells treated with ATR (50 µM) followed by blue light exposure (4.86 µW of 445 nm). Only the middle cell (yellow arrow) expresses PIP2 sensor. Blue light exposed (blue box) cell showed substantial change in cell shape and morphology. The PIP2 sensor also accumulates in cytosol upon blue light exposure. (B) HeLa cells were incubated with propidium iodide (PI) with ATR (50 µM) and exposed to blue light (4.86 µW) for 45 minutes. Incorporation of PI in to cells were observed upon light exposure. The control experiments performed with cells exposed to only to blue light or only to ATR, did not show PI incorporation into cells over time. Plot shows the different rates of PI incorporation into cells compared to control experiments. (C,D) Solvent dependent degradation and isomerization of ATR. (C) ATR (20 µL of 50 mM in ethanol) was exposed to blue LED light for 30 minutes. The blue light exposed ATR (injection sample: 1 µL of exposed ATR was diluted in 1 mL of ethanol) was analyzed by HPLC where degradation of ATR is observed by reduction of corresponding ATR peak in chromatogram. right: The degraded ATR (dATR) (1 µL) was added to HeLa cells (final volume of imaging buffer = 1 mL) expressing PIP2 sensor and continuously exposed to 445 nm light (4.86 µW). Cells did not show detectable PIP2 translocation upon blue light. Exposure of cells to fresh ATR (50 µM) and blue light (BL) induced PIP2 distortion (mean ± S.E.M., n = 12). (D) HPLC analysis of retinal in different solvents after exposing to white light for varying durations. Note that retinal in water degrades in seconds while in ethanol and hexane show over 100 times enhanced stability. Improved isomerizations were seen as well. Mean and S.E.M. are from 3 < independent experiments. (blue light (BL) = blue box). Scale = 5 µm. Full size image

mCherry-PH sensor translocation by BLE-retinal indicates distortion of the PIP2 molecule

To determine whether translocation of mCherry-PH is a common response exhibited by fluorescence proteins on exposure to BLE-retinal, a membrane anchor with a transmembrane domain (from mouse ICAM5) and a C-terminal mCherry, DenMark-mCherry was used36. In addition to DenMark-mCherry, HeLa cells were also expressed with YFP-PH. Upon exposure to BLE-retinal, PM localization of DenMark did not change, while YFP-PH translocated to cytosol (Fig. S8A,C). A saturated lipid anchor bearing glycoprotein, glycosylphosphatidylinositol (GPI) which anchors extracellularly to cells also did not respond to BLE-retinal (Fig. S8B,C). HeLa cells expressing GPI-GFP and NIH3t3 cells expressing GPI-mCherry were used for the experiment. BLE-retinal did not remove GPI anchored proteins from PM. This suggests that specifically PIP2, that contains an unsaturated arachidonoyl chain is susceptible to photochemical reactions elicited by BLE-retinal. Since PIP2 also contains one stearyl anchor, a saturated lipid, it is not clear how oxidation of only arachidonoyl anchor induces PIP2 solubilization. One possibility is that only a small fraction of distorted PIP2 undergoes additional cleavage from the stearyl anchor. This could result in a fraction of PIP2 molecules solubilizing to cytosol and induce calcium mobilization.

Cell membranes are likely to facilitate retinal photoexcitation and cellular damage

As the location of retinal in the cellular environment could be crucial, the next objective was to examine the subcellular environment that can support the photochemical reactions of retinal. When a 20 µL solution of ATR in ethanol (50 mM) was exposed to blue LED light (5 W, 460–470 nm) for 30 minutes, a significant retinal degradation was observed (Fig. 7C). The retinal content in the solution was analyzed using high performance liquid chromatography (HPLC), before and after blue light exposure. HeLa cells expressing mCherry-PH sensor were incubated with 1 µL of this pre-blue light exposed retinal for 10 minutes. However, the addition of pre-blue light exposed retinal did not exhibit PIP2 distortion in cells. Exposure of these cells to blue light at 4.86 μW intensity also failed to exhibit a detectable mCherry-PH translocation, suggesting that (i) the mixture does not have sufficient retinal left and (ii) extracellularly generated retinal photoproducts are not able to disrupt cellular PIP2. Nevertheless, addition of fresh retinal (50 µM) followed by blue light exposure resulted in mCherry-PH translocation (Fig. 7C). Next, we examined if household white fluorescent light also induces retinal degradation, since both fluorescent and LED light contain a peak ~450 nm. Retinal degradation observed under white light was lower in ethanol (Fig. 7D). When retinal in hexane was exposed to white light, it showed even lower degradation. Interestingly, retinal in water exhibited over a 100 times faster degradation (within seconds) than in ethanol or hexane (within minutes) (Fig. 7D). These experiments collectively suggest that in cellular context, retinal must retain in a relatively non-aqueous and hydrophobic environment, not in the cytosol. These data also show that retinal undergoes efficient isomerization, in non-aqueous media upon photon absorption. PM lipid bilayer of cells is likely to provide an ideal environment for retinal to undergo continuous photoisomerization over degradation.

Ability of retinal to harvest light and induce PIP2 distortion was examined to understand whether this process is governed by photosensitization of retinal. A known photosensitizer rose bengal (RB) absorbs green light (Fig. S9) and exhibits singlet oxygen generation37 and cell death38. HeLa cells expressing PIP2 sensor were incubated with RB (50 µM) and exposed to blue and green light respectively. Cells were imaged to observe whether RB too induces a similar PIP2 distortion. Both blue and green light exposure induced a significant PIP2 sensor translocation (Fig. 8A). To assess whether observed PIP2 distortion requires dissolved oxygen in cell culture media to produce singlet oxygen, HeLa cells were treated with CoCl 2 for 12 hours to expose cells to hypoxia39. Upon exposure to BLE-retinal, compared to control normoxic cells, hypoxic cells exhibited an attenuated PIP2 sensor translocation (Fig. 8B). To validate that BLE-retinal induced PIP2 distortion was governed through free radical/ROS mechanisms, prior to blue light exposure, cells were incubated with antioxidants, glutathione ethyl ester and alpha-tocopherol respectively (Fig. 8C,D). Alpha-tocopherol is lipid soluble while glutathione is water soluble and cytosolic. Only cells incubated with alpha-tocopherol exhibited an attenuated PIP2 sensor translocation (Fig. 8C,D). This indicates that reactions of oxidative damage induced by BLE-retinal are likely taking place in lipid membranes (Fig. 8E).

Figure 8 Comparison of PIP2 sensor dissociation from PM by retinal vs a known photosensitizer, rose bengal. (A) Images of HeLa cells expressing PIP2 sensor (mCherry-PH). (A) PIP2 sensor translocation was induced by rose bengal (50 µM), incubated with cells for 5 minutes, in the presence of blue (4.86 µW of 445 nm) and green (light (0.22 µW of 515 nm) respectively. Plot shows the cytosolic fluorescence of PIP2 sensor in HeLa cells upon exposing to light. (mean ± S.E.M., n = 6). (B) HeLa cells were incubated with CoCl 2 (100 µM) for 24 h to expose cells to hypoxia. The control cells were kept in same conditions without CoCl 2 treatment. Cells were incubated with ATR (50 µM) and 445 nm imaging for 10 minutes was performed. Cell in hypoxic condition did not exhibit detectable PIP2 sensor accumulation in cytosol while control cells showed a gradual PIP2 sensor accumulation from PM to cytosol. (C,D) Antioxidants were tested to examine if they prevent PIP2 sensor translocation induced by retinal and blue light. HeLa cells expressing PIP2 sensor were incubated with antioxidants, alpha-tocopherol (1 mM) and reduced-glutathione ethyl ester (500 µM) overnight. Prior to imaging experiments ATR (50 µM) was added and incubated for 5 minutes followed by exposure of blue light (4.86 µW of 445 nm) for 5 minutes. (C) Cells treated with reduced-glutathione ethyl ester exhibited PIP2 sensor translocation from PM to cytosol upon blue light exposure. Plot shows the dynamics of PIP2 sensor (mean ± S.E.M., n = 14 cells). Overview of the antioxidant mechanism exert by reduced glutathione in vivo (right). (D) Cells treated with alpha-tocopherol showed a reduced rate and extent of PIP2 sensor translocation from PM to cytosol upon blue light exposure. Plot shows the dynamics of PIP2 sensor translocation (mean ± S.E.M., n = 6 cells). Note the reduction of PIP2 sensor accumulation in IMs of cells. Right: Overview of the antioxidant mechanism exert by alpha-tocopherol in vivo. (E) Proposed mechanism for blue light excited retinal induced PIP2 distortion process. (F) TD-DFT calculations (CAM-B3LYP/6–31++G**) of retinal’s energy states and the Jablonsky diagram shows strong absorption band due to the π → π* transition where triplet excited states are energetically and symmetrically matched to allow for efficient intersystem crossing and energy transfer to O 2 which allows for singlet oxygen and ROS generation. Mean and S.E.M. are from 3 < independent experiments. (blue light (BL) = blue box and green light (GL) = green box). Scale = 5 µm. Full size image