Solvation Dynamics and Intermittent Oscillation of Cell Membrane

Article pubs.acs.org/JPCB

Solvation Dynamics and Intermittent Oscillation of Cell Membrane: Live Chinese Hamster Ovary Cell Shirsendu Ghosh, Shyamtanu Chattoraj, and Kankan Bhattacharyya* Department of Physical Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India ABSTRACT: Dynamics of the exofacial thiols (i.e., cell surface thiol containing membrane proteins) of a live Chinese hamster ovary (CHO) cell is probed by timeresolved confocal microscopy. For this purpose, a fluorescent probe, 7-(diethylamino)-3(4-maleimidophenyl)-4-methylcoumarin (CPM) is covalently attached to the exofacial thiols. The emission maximum of CPM bound exofacial thiols indicates a highly exposed and polar environment. Using CPM, we studied solvation dynamics, for the first time, at the membrane of a live cell. The thiol containing membrane proteins shows ultraslow response with average solvation time, ⟨τs⟩ = 475 ps. CPM labeled exofacial thiols also show spontaneous, intermittent oscillation in fluorescence intensity with a period of 0.5−1.0 s. This is ascribed to reversible, intermittent changes in the structure and conformation of the membrane proteins.

nonfluorescent in pure water. It exhibits strong fluorescence only when it is covalently tagged with thiol containing proteins, and thus one need not worry about the fluorescence of the free dye. The structure of a cell membrane is dynamic in nature.23−30 Various factors cause oscillations of the structure and function of cell membranes. Such oscillations trigger many biological processes (e.g., transport of ions through an ion channel),23−27 biological rhythms (e.g., beating motion of heart cells28), and other nanomechanical motions.29,30 The time period of such oscillation is a few seconds for the natural beating motion of cardiomyocyes of a heart cell28 and for mechanical motion (0.05−0.4 Hz, 2−20 s) of human airway smooth muscle cells.29 The oscillations of membrane potential occur in 0.1−0.5 s (2− 10 Hz) in a pancreatic-β cell,27 hippocampal interneurons,24 glycolytic oscillations,26,27 and a grid cell.23 In contrast, the time scale of nanomechanical motion of cell walls of Saccharomyces cerevisiae is much faster (0.8−1.6 kHz, i.e., ∼1 ms).30 Such oscillations are accompanied by periodic changes in the structure of the membrane. Recently, several groups have reported intermittent, coherent oscillations in 0.1−0.5 s time scale for aqueous solutions of various proteins/enzymes using single-molecule fluorescence spectroscopy.31−34 We now search for similar oscillations at the cell membranes of the CHO cell using the time dependence of fluorescence intensity. We have specifically tried to understand the dynamics of exofacial thiols (i.e., cell surface thiol containing membrane proteins). Exofacial thiols (i.e., cell surface thiol containing membrane proteins) play a crucial role in intracellular redox signaling pathways, in delivery of ions or therapeutic agents

1. INTRODUCTION Water is an indispensable ingredient of life and is often considered as an “active matrix for life”, not as a mere solvent.1,2 The hydration layer of proteins and nucleic acids controls biological function, molecular recognition, structural integrity, and flexibility.3−13 Thus, there is long-standing interest to understand “biological water” (i.e., water inside a biological system) at the molecular level.1−22 In the vicinity of biomolecules, the solvation dynamics is found to be ultraslow (10−100 times slower than that in bulk water). This is ascribed to binding of the water molecules to the protein and other biomolecules by hydrogen bond.1−22 Although a large number of in vitro studies3−22 have been carried out on the dynamics of biological water, there are very few reports on the dynamics of water in in vivo condition, e.g., inside a live cell. Very recently, we have reported on solvation dynamics in the nucleus, cytoplasm, and lipid droplet regions of a live Chinese hamster ovary (CHO) cell using time-resolved confocal microscopy.14,15 Solvation dynamics in the cytoplasm, nucleus, and lipid droplets of a live biological cell are found to be significantly slower than that in bulk water.14,15 However, virtually nothing is known about the solvation dynamics at the membrane region of a live cell. In this work, we report, for the first time, on the solvation dynamics at the membrane of a live CHO cell. In our previous works,14,15 we used a noncovalent probe and, hence, location was not fully certain. In this work, we study solvation dynamics at the membrane of a live cell using a covalent probe. To study the dynamics of cell surface protein thiols (i.e., exofacial thiols), we have covalently labeled the exofacial thiols of a live CHO cell using a fluorescent probe CPM (7(diethylamino)-3-(4-maleimidophenyl)-4-methylcoumarin; Scheme 1). CPM covalently tagged to a free thiol (i.e., cysteine residue) containing protein is a very good probe for solvation dynamics.10,21 A unique property of the CPM dye is that it is © 2014 American Chemical Society

Received: December 26, 2013 Revised: February 23, 2014 Published: February 26, 2014 2949

dx.doi.org/10.1021/jp412631d | J. Phys. Chem. B 2014, 118, 2949−2956

The Journal of Physical Chemistry B

Article

Scheme 1. (A) Structure of CPM. (B) Protein Labeling Reaction Scheme: Michael Addition

Exciton Inc.), dimethyl sulfoxide (DMSO, Sigma, biological grade for cell culture), and human serum albumin (HSA, Sigma) were used as received. 2.2. Methods. 2.2.1. Covalent Labeling of HSA with CPM. For the covalent labeling of HSA at the free Cys-34 residue by CPM, we followed the procedure reported by Wang et al.21 (Scheme 1B) with minor modifications.22 Sufficient quantity of CPM (dissolved in a minimum amount of DMSO) was added to 5 mL of 50 μM HSA solution (in 0.1 M phosphate buffer (pH ∼ 7.0)) to yield a molar ratio of HSA to CPM of 1:1. The mixture was stirred gently in the dark and maintained at room temperature for 10 h. The resultant solution was dialyzed for 4 days at 4 °C against 500 mL of phosphate buffer (0.1 M) with a change of dialysis buffer after every 12 h. The solution containing the labeled protein was passed through a Sephadex G-50 gel column for further removal of any unreacted and free CPM and HSA. The concentration of the labeled protein was determined spectrophotometrically following Lowry et al.,56 and the labeling efficiency was found to be ∼70%. 2.2.2. Cell Preparation. As discussed in our previous publications,14,15 CHO cells were grown in phenol red free DMEM medium with 10% fetal bovine serum (FBS) and 1% pen strep glutamine (from Gibco) in an atmosphere of 5% (v/ v) CO2 enriched air at 37 °C. For microscopy, a 35 mm glass bottom Petri dish was used for the cell culture. For the MCS measurement, after rinsing the cells three times with phosphate buffered saline (PBS) buffer solution, 10 μL of 2 μM CPM dye solution in DMSO was added to the Petri dish containing 2 mL of the phenol red and FBS free DMEM medium. Then it was incubated for about 30 min. For solvation dynamics studies, ∼200 nM dye was used and it was also incubated for 30 min. For the in vivo MCS measurement of CPM labeled HSA, 10 μL of 2 μM CPM labeled HSA solution in PBS buffer was added to the Petri dish containing 2 mL of the phenol red and FBS free DMEM medium, and it was incubated for 6 h. The stained cells were rinsed with PBS solution for 5−6 times. After staining, cells were used for microscopic study within 10 min. All experiments are carried out in ∼25 °C. All experiments are repeated at least three times. 2.2.3. Experimental Setup for One-Photon Microscopy. Experimental setup for one-photon microscopy has been described in our previous publication.14,15 Briefly, a combination of confocal microscope (Olympus IX-71) and timecorrelated single-photon-counting (TCSPC) setup (PicoQuant, MicroTime 200) have been used in this study. A water immersion objective (magnification, 60×; numerical aperture (NA) ≈ 1.2) and a pulsed picosecond diode laser (PDL 828-S “SEPIA II,” PicoQuantat, 405 nm) were used. Thus the diffraction limited spot size is 0.6λ/1.2 ∼ λ/2. For live cell studies, we kept laser power at or below ∼0.27 μW. Fluorescence from the dye in the cells was separated by a dichroic mirror (Z405RDC, Chroma). To block the exciting

across the membrane (transfection or internalization), and in controlling protein function.35−41 A thiol−disulfide redox system controls the stability and function of proteins containing cysteine residues (i.e., free thiol groups, -SH). Under oxidative stress (in the extracellular region) the thiol groups are oxidized to form disulfide bonds.35 This has been implicated in transcription factors, molecular adapters, chaperones, protein tyrosine phosphatases, and proteases.35−41 It is suggested that the activation and proliferation of T-cells are regulated by the redox status of the exofacial thiols on T-cells.36 The level of cell surface protein thiols on normal donor lymphocytes is reported to be different from that of patients with HIV.37 Various membrane receptors, ion channels,38 and extracellular thiol− disulfide oxidoreductases are also redox sensitive39 in many cells including CHO cell.41 Targeting of intracellular redox signaling pathways has been suggested as a therapeutic approach for cancer.40 The function and dynamics of different proteins,42−52 lipid,53 and DNA54,55 have been extensively studied using singlemolecule spectroscopy (SMS). From real-time single-molecule measurements, Lu and co-workers studied the hinge-bending motion of the T4 lysozyme protein during the course of sequential enzymatic hydrolysis reactions of the polysaccharide walls of Escherichia coli (E. coli) B cells.43 They also studied nanoscale domains of redox protein on bacterial cell surfaces.44 They identified a redox heme protein as one of the major components of the cell surface domains.44 Schutz and coworkers reported on the feasibility of simultaneous fluorescence imaging and electrical recording to study single ion channels in planar bilayer membranes.46 Demuro and Parker used total internal reflection fluorescence microscopy to optically monitor the activity and localization of multiple Ca2+-permeable channels in the plasma membrane.47 Majima and co-workers reported two kinds of motions in the folding dynamics of a unfolded redox protein, cytochrome c.51 Using two-color z-scan fluorescence correlation spectroscopy, Hof and co-workers observed cross-linker triggered formation of nanodomains in the model membranes at lipid compositions closely approaching the optically resolvable phase separation boundary.53 Ishii and Tahara developed two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS)54 and used this to unravel spontaneous structural dynamics of DNA hairpin.55 In this work, we will show that free thiol containing membrane proteins (i.e., exofacial thiol) of the CHO cell not only show ultraslow solvation dynamics but there is also a spontaneous, intermittent oscillation in the fluorescence intensity time trajectory of these membrane proteins.

2. EXPERIMENTAL SECTION 2.1. Materials. Laser-grade dye, 7-(diethylamino)-3-(4maleimidophenyl)-4-methylcoumarin (CPM, Scheme 1A, 2950



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Figure 1. Confocal image of CHO cells stained by (A) 10 nM CPM, (B) 200 nM CPM, (C) 800 nM CPM, and (D) 10 nM CPM labeled HSA.

laser light, a suitable filter (430LP, Chroma) was used before the detectors. The fluorescence was focused through a pinhole (30 μm). The emitted light with different polarizations were separated using a polarizer cube (Chroma) and were detected by two single-photon-counting avalanche photodiodes (SPAD1 and SPAD2). Appropriate narrow band-pass filters are used (e.g., XBPA510, 540, and so on; Asahi Spectra) to collect TCSPC decay at specific emission wavelengths. The signal was subsequently processed by the PicoHarp-300 time-correlated single-photon-counting module (PicoQuant) to generate a TCSPC histogram. Since the CPM dye covalently bonded to the immobilized cell does not diffuse, we could not record the autocorrelation trace using fluorescence correlation spectroscopy (FCS). As a result, we do not know the value of G(0) which gives the exact number of dye molecules (N) in the focal volume. To estimate N, we first obtained the focal volume as follows. The structure parameter (ω) of the focal volume is given by ω = ωz/ωxy, in which ωz and ωxy are the longitudinal and transverse radii of the focal volume, respectively.57 From the reported diffusion coefficient of rhodamine 6G in water (426 μm2 s−1)57 we obtained ωxy ∼ 319 nm, and ω ∼ 5. This corresponds to a focal volume 0.9 fL. From this value of focal volume, we estimate that the number of dye molecules (N) in the focal volume for a 10 nM dye solution is ∼5. Emission Spectra. The emission spectra of CPM in CHO cell was recorded using an electron multiplying charge-coupled device (EMCCD, ANDOR Technology) attached to a spectrograph (ANDOR Technology, Shamrock series). 2.2.4. Picosecond Time-Resolved Fluorescence. The parallel (I∥, parallel to the polarization of the exciting light), and perpendicular (I⊥) components of the fluorescence were recorded by using the two detectors (SPADs). I∥ and I⊥ were combined to generate the fluorescence lifetime decays at magic angle conditions as follows:

Fluorescence Lifetime Measurement. For recording Instrument Response Function (IRF), we used a bare slide and collected the scattered laser light. The full width at halfmaximum (fwhm) of the IRF for excitation at 470 nm is ∼100 ps. The fluorescence decay is deconvoluted using the IRF and DAS6 v6.3 software. 2.2.5. Solvation Dynamics under a Microscope. The timeresolved emission spectra (TRES) were constructed using the parameters of best fit to the fluorescence decays and the steadystate emission spectrum following the procedure described by Maroncelli et al.60,61 The solvation dynamics is described by the decrease in emission energy (frequency, ν) with an increase in time. If ν(0), ν(t), and ν(∞) are the emission frequencies at time 0, t, and ∞, respectively, the decrease in emission energy with time are described by v(t ) = v(∞) + [v(0) − v(∞)] ∑ ai e−t/ τi i

The time constants for solvation (τi) are obtained from the fitting of ν(t) verses t curves using eq 3. The other parameters, ν(0) and ν(∞), are also obtained from this fitting. The solvent correlation function, C(t), can be defined as C(t ) =

(1)

Fluorescence Anisotropy Measurement. The anisotropy function, r(t), was obtained using the formula r (t ) =

I∥ − GI⊥ I∥ + 2GI⊥

v (t ) − v (∞ ) v(0) − v(∞)

(4)

3. RESULTS 3.1. Confocal Image. Figure 1 shows the confocal image of a single Chinese hamster ovary (CHO) cell stained by CPM and CPM labeled HSA. In order to minimize autofluorescence, we used a phenol red free culture (DMEM) medium. From the image (Figure 1A,B), it is clearly seen that CPM dye (up to 200 nM) localizes in the membrane region of the CHO cell. It is previously mentioned that CPM dye shows fluorescence only when it is covalently attached to a free thiol group of a protein.10,21 Thus, it is obvious that CPM dye covalently labeled the free thiol containing membrane proteins. At a high concentration of the dye (at or above 0.8 μM ≈ 800 nM) the entire cell gets labeled by CPM (Figure 1C). Therefore, all experiments were carried out at a low dye concentration (∼10−200 nM, Figure 1A,B) so that the dye is localized in the membrane region. As a control, we labeled HSA (containing a single cysteine group) by CPM. The CPM labeled HSA protein is found to be distributed over the entire membrane and cytoplasm region of the CHO cell (Figure 1D). Thus CPM labeled HSA does not provide any information specific to the membrane region. 3.2. Emission Spectra Recorded under the Microscope. Figure 2 shows the emission spectra of CPM labeled cell membrane inside the CHO cell. The CPM labeled cell

Imagic(t ) = I∥(t ) cos2(54.75°) + I⊥(t )G sin 2(54.75°) = (1/3)I∥(t ) + (2/3)GI⊥(t )

(3)

(2)

The G factor for this microscope setup was measured by tail fitting of fluorescein58 and was found to be ∼1.2. Fisz59 carried out a detailed analysis for one-photon excitation fluorescence polarization microscopy, under high-aperture excitation and/or detection. We have used eq 2 as an approximation to analyze the fluorescence anisotropy decays. 2951



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nonpolar medium. Since the absorption spectrum of CPM under a few molecules condition is impossible to record, we could not apply this method. The emission maximum of CPM at the CHO membrane (476 nm) is very close to that (472 nm) of CPM labeled HSA denatured by 6 M guanidinium hydrochloride (GdnHCl)10 for which using the Fee− −1 10 Maroncelli procedure υtheo Assuming the em (0) is 21555 cm . theo same υem (0) for CPM bound to CHO cells, the amount of solvation missed in our setup is ∼27%. Figure 3B shows the decay of emission energy (ν(t)) at CPM labeled membrane proteins of the CHO cell. The decay parameters of ν(t) are given in Table 1. The decay of ν(t) of CPM labeled membrane proteins of the CHO cell displays two components150 ps (44%) and 1400 ps (29%) with an average solvation time (⟨τs⟩) ∼ 475 ps (Table 1). 3.4. Fluorescence Anisotropy Decay under a Confocal Microscope. The fluorescence anisotropy decay of CPM bound to the membrane of a CHO cell was fitted to a biexponential decay,

Figure 2. Emission spectra of a CPM labeled membrane and CPM labeled HSA proteins inside the CHO cell.

membrane exhibits an emission maximum at 476 nm. Inside the CHO cell, the CPM labeled HSA also exhibits an emission maximum at the same position (476 nm). However, the width of the emission spectrum (fwhm) of CPM labeled HSA proteins inside the CHO cell are much larger than that of CPM labeled cell membrane. The larger fwhm for CPM labeled HSA may be ascribed to multiple locations and resulting heterogeneity inside the CHO cell. 3.3. Solvation Dynamics. Since CPM is a solvation probe, we studied solvation dynamics at the membrane of the CHO cell. For CPM labeled membrane protein in the CHO cell, the fluorescent transients display decay at blue end (at short emission wavelength) and rise preceding the decay at the red end (at long emission wavelength). This clearly indicates that solvation dynamics is occurring at the membrane of the cell. Figure 3A shows the wavelength dependent fluorescence decays of CPM labeled membrane proteins of a single live cell. We did not observe any growth at long wavelength regions (red end) in the case of CPM labeled HSA inside the CHO cell, presumably because of its multiple locations. Since the IRF of our setup is about 100 ps, we have certainly missed the ultrafast component of solvation occurring in a time scale shorter than 100 ps. Fee and Maroncelli62 proposed a simple method of calculation of true emission frequency at time zero, υtheo em (0) as follows

r(t ) = r0[β exp(−t /τslow ) + (1 − β) exp(−t /τfast)]

(5)

Figure 4 shows the anisotropy decay of CPM labeled membrane, and the decay parameters are listed in Table 2. The high value of initial anisotropy suggests most of the rotational relaxation dynamics is captured in our setup. From Figure 4 and Table 2 it is readily observed that the anisotropy decays exhibit a major (80%) fast component of 200 ps (=0.2 ns) and a long component (12.8 ns) with an average rotation time (⟨τrot⟩ = ar1τ1 + ar2τ2) of 2.7 ns. The lifetime of the covalent probe CPM is too short (∼3.5 ns) to faithfully report the long component (>10 ns) of anisotropy decay. The segmental motion of the protein or the local motion of the probe (CPM) molecule may be responsible for the relatively faster component (0.2 ns). 3.5. Oscillation in Fluorescence Intensity. Figure 5 shows intensity versus time trajectories for CPM labeled membrane proteins in CHO cell. It is readily seen in Figure 5C,D that the CPM labeled membrane proteins display an intermittent oscillation in the fluorescence intensity. As shown in Figure 5A−D in the 90 s period (5−95 s, Figure 5A) such oscillations were observed for the following periods: 5−10 s, 60−70 s (Figure 5C), and 85−90 s (Figure 5D). No such oscillations are observed at other time periods (Figure 5B). To ascertain whether this oscillation is common for any CPM labeled protein inside living cells, we performed a control experiment with CPM labeled HSA inside the CHO cell. No such oscillations in fluorescence intensity were detected in the case of CPM labeled HSA (Figure. 3E,F). Thus, the observed intermittent oscillation is associated exclusively with the CPM

p np theo np υem (0) = υabs − (υabs − υem ) p np where υabs and υabs denote the steady-state absorption frequency in the polar medium (system) and in a nonpolar medium, respectively. υnp em is the emission frequency in the

Figure 3. CPM labeled membrane in the CHO cell: (A) Fluorescence transients at λex = 405 nm; (B) decay of emission energy (ν(t)). (Inset shows time-resolved emission spectra, TRES.) 2952



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Table 1. Parameters of Decay of Emission Energy, υ(t) of CPM Attached to the Membrane of a CHO Cell

a

region

emission maxima (nm)

τ1 (ps) (a1)

τ2 (ps) (a2)

⟨τs⟩a (ps)

% missed

CPM labeled membrane

476

150 (0.44)

1400 (0.29)

475

27

±100 ps.

4. DISCUSSION At the outset, it may be noted that the confocal image of CPM labeled membrane indicates that the probe (CPM) molecule is largely immobilized at the membrane region of the CHO cell. Thus CPM reports on the polarity and dynamics of the microenvironment at the cell membrane. The steady-state emission maximum of CPM bound to the cell membrane is a good indicator of the local polarity of the microenvironment. The emission maximum of CPM bound to cell membrane (λem ∼ 476 nm) is red-shifted by 16 nm compared to CPM labeled HSA protein in pH ∼ 7 buffer.10 This suggests that the microenvironment of CPM at the membrane of the CHO cell is more polar and exposed than that inside HSA. The emission maximum of CPM at the CHO membrane is very close to that (472 nm) of CPM labeled HSA denatured by 6 M GdnHCl.10 Since CPM bound to HSA denatured by GdnHCl corresponds to a highly exposed site, at the cell membrane, CPM experiences an environment highly exposed to the extracellular region. As a result, the polarity sensed by CPM bound to exofacial thiols is quite high. Solvation time (⟨τs⟩) in the CPM labeled membrane is 475 ps (Figure 3). Using a noncovalent coumarin 153 probe, we have previously observed that the solvation time in a CHO cell is 750 ps for nucleus, 1100 ps for cytoplasm, and 3600 ps for lipid droplets.15 Figure 6 shows a comparison of solvation dynamics in four different regions of the CHO cell. Note, the observed solvation time in the membrane region (475 ps) is ∼3 times faster than that observed in the membrane of a giant lipid vesicle (⟨τs⟩ = 1100 ± 200 ps).18 The solvation time in the CPM labeled membrane is almost the same as that of CPM labeled HSA under in vitro condition in bulk solution.9−11,16,17 The solvation time of CPM labeled HSA under in vitro

Figure 4. Fluorescence anisotropy decay of CPM labeled membrane of a single live CHO cell, at λem = 450 nm.

Table 2. Parameters of Fluorescence Anisotropy Decay of CPM Attached to the Membrane of a CHO Cell region

r0

τr1 (ns) (ar1)

τr2 (ns) (ar2)

⟨τrot⟩a (ns)

CPM labeled membrane

0.37

0.2 (0.80)

12.8 (0.20)

2.72

a

±100 ps.

covalently attached to the exofacial thiols at the membrane of the CHO cell and is not caused by any other CPM labeled extracellular protein (such as HSA). The confocal image of the cell labeled with CPM recorded at 5 min intervals did not display any change in position. This shows that the fluctuation in fluorescence intensity is not due to motion of the living cell. Further, CPM labeled HSA protein inside the CHO cell does not show any fluctuation in fluorescence intensity. Thus intensity fluctuation is not peculiar to CPM bound to a thiol group of a protein.

Figure 5. Fluorescence intensity time trajectories. CPM labeled membrane in CHO cell: (A) 5−95 s, (B) 51−56 s (no oscillation), (C) 60−70 s, and (D) 85−90 s. CPM labeled HSA proteins inside the CHO cell: (E) 0−90 s and (F) 40−45 s. 2953



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Figure 6. Comparison of C(t) of different regions of a single live CHO cell under a confocal microscope.

Figure 7. Intensity time trajectories. CPM labeled membrane in the CHO cell: (A) 5−10 s and (B) 5−10 s smoothed by FFT (showing halfperiods).

Scheme 2

condition (in phosphate buffer with pH = 7) is 650 ps.10 The average solvation time (475 ps) in the cell membrane of the CHO cell is substantially slower compared to that (1 ps) in the bulk water. Thus the dynamics of water molecules bound to the membrane proteins, though significantly slower than bulk water, is faster than that of CPM bound to HSA in bulk water. We have noted that the emission maximum of CPM bound to the membrane of the CHO cell (476 nm) is close to that of CPM bound to HSA (472 nm) under strongly denatured condition (6 M GdnHCl). The solvation dynamics of CPM− HSA denatured by 6 M GdnHCl is however nearly 8 times faster (⟨τs⟩ ∼ 60 ps).10 The observed slow dynamics of the membrane bound water may play a crucial role in controlling the efflux of ions and other biomolecules through the membrane. Perhaps, the most important finding of this work is the intermittent oscillation in fluorescence intensity for CPM attached to the membrane proteins of the CHO cell. The time period of oscillation was obtained by carefully determining the peak position by smoothing using a fast Fourier transform (FFT) filter (Figure 7A,B). It is observed that the period of

oscillation is not constant and the half-period varies between 0.5 and 1.0 s (for various gaps in one bunch of oscillation and for different bunches in different sets). In summary, the time trace suggests the fluorescence intensity oscillates intermittently with a varying period separated by long silent (“quiescent”) periods in which there is no oscillation. It may be recalled that such intermittent oscillations are previously reported by Lu and co-workers31 for sm-FRET in an enzyme (HPPK). They attributed this to the transition between different conformationally active states. The time scale of the intermittent oscillation in the present case (0.5−1.0 s) is close to the time period (0.5 s) observed by Lu and co-workers31 for HPPK. The observed intermittent oscillations may be attributed to the redox cycle of the cell surface thiols. Xie and co-workers32,33 also observed cycles of duration 0.5 s (500 ms) for the on (oxidized) and off (reduced) state of an enzyme. We have already noted that, for many different kinds of cell, the membrane potential oscillates in the time scale ranging from a few seconds to milliseconds.23,24,26,27 The oscillation of fluorescence intensity of CPM attached to the membrane 2954



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provides a direct proof of the dynamic nature of the membrane and its intermittent and reversible structural changes. Finally, we discuss the origin of the observed oscillation of the fluorescent intensity of the CPM probe bound with cell surface thiols. As shown in Scheme 2, CPM is covalently attached to one or a few of the large number of thiols of the proteins, present at the surface of the cell. The extracellular region contains a large number of oxidants. As discussed by Torres and Gait,35 during oxidative stress, free thiol groups (not bonded to CPM) at the cell surface are oxidized by extracellular oxidants to form disulfide bonds. This leads to stabilization and folding of cell surface thiol containing proteins. The reducing environment inside the cytoplasm causes reduction of the disulfides back to thiols. The repeated cycles of oxidation of cell surface thiols by extracellular oxidants and the reduction of disulfides inside the cell gives rise to reversible, intermittent changes in the structure of the membrane (Scheme 2).35 This plays a key role in cellular delivery and internalization. The fluorescent intensity of coumarin dyes with a flexible dialkylamino substituent is (e.g., CPM) extremely sensitive to polarity and decreases with an increase in solvent polarity.63 The change in local polarity during structural changes gives rise to the fluctuation in florescence intensity. So far the intermittent change in membrane has been studied by oscillations in membrane potential.23−30 This is the first report of direct observation of structural fluctuation of cell membrane using fluorescence.

5. CONCLUSION This work demonstrates that the selective and covalent labeling of membrane proteins of a CHO cell (i.e., exofacial thiols) may be achieved by using a low concentration of CPM dye (10−200 nm). The emission maximum of CPM bound to exofacial thiols indicates a highly polar and exposed microenvironment. The average solvation time at the membrane of the CHO cell is faster than that in other regions (cytoplasm, nucleus, and lipid droplets) of the CHO cell. The intermittent oscillation in fluorescence intensity in 0.5−1.0 s time scale provides direct evidence of the dynamic nature of the membrane of the CHO cell. The new information obtained regarding the intermittent change in structure of the membrane and its solvation environment may have implications in redox signaling pathways.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: (91)-33-2473-2805. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Thanks are due to the Department of Science and Technology, India for the IRHPA Project entitled “Center for Ultrafast Spectroscopy and Microscopy” (Project No. IR/S1/CU 02/ 2009), Council for Scientific and Industrial Research (CSIR), and the J. C. Bose Fellowship for generous research support. S.G. and S.C. thank CSIR for awarding fellowships.

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REFERENCES

(1) Bagchi, B. Water in Biological and Chemical Processes from Structure and Dynamics to Function; Cambridge University Press: Cambridge, U.K., 2013. 2955



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Solvation Dynamics and Intermittent Oscillation of Cell Membrane

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