Intermittent Fluorescence Oscillations in Lipid Droplets in a Live

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Intermittent Fluorescence Oscillations in Lipid Droplets in a Live Normal and Lung Cancer Cell: Time Resolved Confocal Microscopy Rajdeep Chowdhury, Md. Asif Amin, and Kankan Bhattacharyya J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp5120042 • Publication Date (Web): 12 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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The Journal of Physical Chemistry

Intermittent Fluorescence Oscillations in Lipid Droplets in a Live Normal and Lung Cancer Cell: Time Resolved Confocal Microscopy Rajdeep Chowdhury, Md. Asif Amin and Kankan Bhattacharyya* Department of Physical Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India

Abstract Intermittent structural oscillation in the lipid droplets of live lung cells is monitored using time resolved confocal microscopy. Significant differences are observed between lung cancer cell (A549) and normal (non-malignant) lung cell (WI38). For this study, the lipid droplets are covalently labeled with a fluorescent dye, coumarin maleimide (7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin, CPM). The number of lipid droplets in the cancer cell is found to be ~20-fold higher than that in the normal (non-malignant) cell. The fluctuation in the fluorescence intensity of the dye (CPM) is attributed to the red-ox processes and periodic formation/rupture of S-CPM bond. The amount of reactive oxygen species (ROS) is much higher in a cancer cell. This is manifested in faster oscillations (0.9 ± 0.3 sec) in cancer cell compared to that in the normal cell (2.8 ± 0.7 sec). Solvation dynamics in the lipid droplets of cancer cell is slower compared to that in the normal cell.

Keywords: Lipid droplet, live cancer cell, intermittent oscillation, ROS, red-ox. *

E-mail: [email protected]

Fax: (91)-33-2473-2805 1

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1.

Introduction A cancer cell differs from a normal cell in various ways. For instance, the amount of

reactive oxygen species (ROS) is much higher inside a cancer cell compared to its normal counterpart.1-2 As an intra-cellular defense mechanism, level of anti-oxidants is increased in malignant (cancer) cells.1-2 Glutathione and thioredoxin are the two major cytosolic thiol containing anti-oxidant molecules responsible for redox signalling.3-4 Glutathione content inside a lung cancer cell is about 7-fold higher compared to that in the normal lung cell.4-5 The ROS may oxidize DNA bases (e. g. from guanine to 8-oxogunaine) and thus may impair the biological functions, significantly.6-8 The ROS induced DNA damage is repaired by a DNA repair enzyme (e.g. oxoguanine DNA glycosylase 1 converts 8-oxogunaine back to guanine).9 The ROS oxidizes thiol containing proteins to di-sulphides.10-12 A number of reducing enzymes in a cell repairs this by converting di-sulphides to thiols.10-12 Such reduction of S-S bonds plays an important role in the internalization of certain species in a cell i.e. transfection from outside the cell to the interior.10 Apart from elevated level of cytosolic glutathione, a cancer cell is characterized by increased number of lipid droplets.13-17 Many recent studies indicate that lipid droplets not only store lipid molecules (“fat depot”) but also contain many crucial signaling proteins.18-22 The large excess of lipid droplet in a cancer cell is a result of enhanced rate of glycolysis producing pyruvate which are precursor of fatty acid and lipogenic molecules.13 This is known as the Warburg effect.13 Inhibition of synthesis of lipid droplets (e.g., by use of the fatty acid synthase inhibitor C75) has been suggested as a therapeutic cure of cancer.23 In this paper, we investigate the structural fluctuations in the lipid droplets and cytosol of a live cell arising from the red-ox processes. For this study, we have labeled thiol-proteins in the lipid droplets of a normal lung cell (WI38) and lung cancer cell (A549) by CPM (Scheme 1). CPM is highly fluorescent when bound to a protein and is almost non-fluorescent when the SCPM bond is ruptured.24-25 The repeated rupture and re-formation of S-CPM bond may give rise to oscillations in fluorescence intensity. The structural oscillations in a cell give rise to fluctuations of membrane potential (0.1–0.5 seconds in pancreatic-β cell,26 nano-mechanical motion of cell walls of Saccharomyces cerevisiae27 and other oscillations.28-30 Recently, using single molecule fluorescence spectroscopy, Lu and co-workers reported intermittent, coherent oscillations in 0.1-0.5 sec time scale for various proteins (enzyme).31-33 Most recently, we have 2 ACS Paragon Plus Environment

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detected such oscillations for CPM bound to cell-surface thiols24 and thiol proteins in the mitochondria.25 In this work, we compare fluorescence oscillations of CPM bound to thiols in lipid droplets and cytosol of a live normal lung cell (WI38) with a lung cancer cell (A549). We show that nature and period of fluorescence oscillations in the lung cancer cell are significantly different from that in the normal lung cell.

Scheme 1. Structure of CPM dye. Since CPM is well known solvation probe, we have also studied solvation dynamics of water molecules surrounding the thiol containing molecules (antioxidant molecules such as glutathione, thioredoxin etc.) in the cytosol as well as in the lipid droplets. Previously, we along with many other groups, have investigated solvation dynamics of water molecules near a biomolecule under in vitro condition in an aqueous solution.34-53 In this work, we report on the solvation dynamics of real biological water17 in the lipid droplets of both normal and cancer cell using a covalent probe (CPM). 2.

Experimental Section

2.1

Materials

CPM dye was purchased from Exciton and used without further purification. 2.2

Methods

A.

Cell Preparation The cells (A549 and WI38) were grown in a phenol red free DMEM (Dulbecco’s

Modified Eagle Medium) containing 10% fetal bovine serum, 1% Pen Strep Glutamine (Gibco) in an atmosphere of 5% (v/v) CO2 enriched air at 37°C. For selective staining of the most reactive thiols inside cells, dilute (200 µL of 100 nM) solution of the dye (CPM in phenol red 3 ACS Paragon Plus Environment


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free DMEM) were added to the cells in a confocal dish and allowed to incubate for a short time (1 hour). It is observed under these conditions, only the lipid droplets and cytosol are stained by CPM. Use of higher concentration (~800 nM) of dye and longer incubation periods result in extensive labeling of the entire cell.24 After incubation, the cells were washed 2-3 times with phosphate buffered saline (PBS) to eliminate trace amount of dye outside the cell surface and 200 µL fresh phenol red free DMEM was added to the confocal dish. Each experiment was repeated 3 times and all sets of experiments were carried out side by side under exactly identical condition (20°C) so that the results can be compared without any ambiguity. In each set, at least 20 lipid droplets in 4-5 different cells are studied. Thus, the average over three sets corresponds to an average of at least 15 cells and 60 lipid droplets (or different regions of cytosol). In order to avoid the photo-damage of live cell by high power of laser (405 nm), we have recorded the confocal images at very low laser power ~ 100 nW. Phenol red free DMEM is used as a media to minimize the possibility of auto-fluorescence. B.

Experimental Set Up Confocal images of the live cells (both normal and cancer) have been captured using a

confocal setup (PicoQuant, MicroTime 200) with an inverted optical microscope (Olympus IX71). The excitation light (405 nm) from a pulsed diode laser (PDL 828-S “SEPIA II”, PicoQuant) was focused onto the live cells (attached to the glass surface of petri dish) by a water immersion objective (60X, 1.2 NA). Fluorescence was separated from the excitation light (405 nm) using a dichroic mirror (Z405RDC, Chroma) and a filter (HQ430lp, Chroma). The fluorescence was then focused onto a MPD detector (Micro Photon Device) through a pinhole (75 µm). For co-localization study, fluorescence of CPM and nile red are captured separately using a dichroic mirror (540DCLP, Chroma) and two MPD (Micro Photon Device) detectors. For recording the emission spectra under the confocal microscope, we used an electron multiplying charge couple device (EMCCD) attached to a spectrograph (ANDOR Technology).

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C.

Picosecond Time-Resolved Fluorescence Decays under a Microscope The parallel (I‖, parallel to the polarization of the exciting light) and perpendicular (I⊥,

perpendicular to the polarization of the exciting light) components of fluorescence decay were recorded by using the two detectors (Micro Photon Device, MPD). Subsequently, they were combined in the following way to achieve the fluorescence decays at magic angle condition,17 Imagic(t) = I║(t) cos2(54.750) + I┴(t) G sin2(54.750) = (1/3) I║(t) + (2/3) G I┴(t)

(1)

The G value was obtained by tail matching of I║ and I┴ components of fluorescence intensity of fluorescein25 and was found to be ~ 1.2. It should however, be emphasized that equation (1) is not strictly valid for a confocal set up because of depolarization at high numerical aperture.54-55 Fisz et al.54 and Wei et al.55 have discussed a detailed procedure for taking in account of this. However, for the sake of simplicity, we have used equation (1) as an approximation. The fluorescence transients were deconvoluted using the temporal response (IRF) of the detectors at 405 nm. For this purpose, we used back-scattered light from a bare slide using a laser diode at 405 nm. FWHM of IRF is found to be ~100 ps. Fluorescence decays have been analyzed using DAS6 v6.3 software. D.

Analysis of Solvation Dynamics The time resolved emission spectra (TRES) of CPM inside the different regions of live

cell were constructed from the steady state emission spectra and the fluorescence transients following by Maroncelli and Fleming.56-57 The solvation dynamics is described by the decay of the solvent correlation function C(t), defined as, C(t) =

ν(t) − ν(∞) ν(0) − ν(∞)

(2)

where, ν(0), ν(t) and ν(∞) are the emission maxima (frequencies) at time 0, t and ∞, respectively. The denominator in the right hand side of equation 2 indicates total dynamic Stokes shift (Δν). The solvent correlation functions C(t) were fitted to a single or double-exponential decay as follows.

− C(t) = ∑ a i e

t τi

(3)



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3.

Results

3.1

Bright Field and Confocal Images of the Cells: Co-localization with Nile Red

(A)

A549

WI38

(B)

5 µm

5 µm

(C)

CPM (A549)

CPM (WI38)

(D)

5 µm

5 µm

Figure 1. Bright field image of (A) lung cancer cell (A549) and (B) lung cell (WI38). Corresponding confocal images: (C) A549 and (D) WI38 stained by CPM.

Figure 1A-B shows the bright field images of cancer (A549) and normal (WI38) lung

cells, respectively. The corresponding confocal images of the same cell in which the lipid droplets are labeled with CPM are shown in the same figure (1C-D). The bright field images (and hence, morphology) of both the cells remains unchanged during the course of the experiment (i.e. before and after the experiment). This suggests that the cells remain healthy and 6 ACS Paragon Plus Environment

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alive during the experiments. It is also seen that there is a preponderance of lipid droplets stained by covalent probe CPM in the cancer cell (A549). In contrast, there is little or no CPM labeled lipid droplets in the normal lung cell (WI38).

(A)

(B)

CPM (WI38)

10 µm

10 µm (C)

CPM (A549)

Nile Red (A549)

(D)

10 µm

Merged (A549)

10 µm

Figure 2. Confocal images: (A) lung cell (WI38) stained by CPM and lung cancer cell (A549) stained by (B) CPM, (C) nile red and (D) merged image.

Figure 2A-D show the confocal images with co-localization of CPM and nile red in lipid

droplets (the corresponding bright field images are not available now). CPM localizes inside the cytosol of both the cells (normal and cancer) giving rise to a blue fluorescence (confocal images are shown in green for a better contrast). 7 ACS Paragon Plus Environment


It is clearly seen that there are a large number of bright fluorescent dots (~70) inside the lung cancer cell (Figure 2B). Such bright dots are very few (1-4) in the normal lung cell (Figure 2A). Similar bright dots were detected earlier using a non-covalent probe (coumarin 153) and have been assigned to the lipid droplets.17 In order to confirm the origin of the bright dots, we carried out a co-localization experiment with a lipid marker, nile red.58 Figure 2B-D shows the confocal images A549 cell stained by CPM (Figure 2B), nile red (Figure 2C) and by both (merged image, Figure 2D). The merged image (Figure 2D) clearly reveals that CPM colocalizes with nile red in the bright dots. Co-localization of the CPM labeled bright dots with nile red conclusively proves that the bright dots are lipid droplets labeled by CPM. From figure 2A-B, it is also apparent that the number of lipid droplets in the lung cancer cell (~70 in A549) is significantly higher than that in the normal lung cell (1-4 in WI38). It may be recalled that in a previous study, using a non-covalent probe (coumarin 153), we have shown that number of lipid droplets in cancer cell is about 20 times higher compared to the normal cell.17 (A)

A549 Nile Red

600k 400k

CPM

200k 0k

Intensity

800k Lipid Droplet

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Intensity (a.u.)

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450

500

550

600

650

Wavelength (nm)

0.0

450

500

550

600

650

Wavelength (nm)

Figure 3. Emission spectra: (A) lung cancer cell (A549) stained by CPM and nile red; cancer cell (A549, B) and normal cell (WI38, C) stained by only CPM. Figure 3A shows steady state emission spectra of A549 cell stained by CPM and nile red.

The emission spectra recorded by focusing on the lipid droplets inside the cells exhibit two peaks at ~470 nm (due to CPM) and ~600 nm (nile red, Figure 3A). This further confirms that CPM and nile red co-localizes in the lipid droplet.


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In summary, co-localization with nile red and the resulting confocal images and emission spectra suggest that the bright dots inside both normal and cancer cell arise from lipid droplets stained by CPM. 3.2

Steady Sate Emission Spectra under a Confocal Microscope The size of the lipid droplets in the lung cells is ~ 500 nm.17 Since the size of the focal spot

of our confocal microscope setup is ~λ/2, i.e., ~200 nm for excitation at 405 nm, we could explore the spectroscopy and dynamics in a region of size ~200 nm inside the lipid droplets. Figure 3B and 3C describe steady state emission spectra of CPM, recorded under a confocal microscope, inside the cytosol and lipid droplets of A549 and WI38 cells, respectively. In the lung cancer cell (A549), emission maxima of CPM are at ~ 476 nm inside the cytosol and at ~ 468 nm in the lipid droplet (Figure 3B). This indicates that lipid droplet are less polar compared to cytosol.17 The emission maxima of CPM are observed to be ~474 nm and ~472 nm inside the cytosol and lipid droplet respectively inside normal lung cell (Figure 3C). The emission maximum of CPM in the lipid droplet of cancer cell is blue shifted by ~4 nm compared to that of normal cell. This indicates that lipid droplet of a cancer cell is less polar compared to that in the normal lung cell. 3.3

Intermittent Oscillation of Fluorescence Intensity in the Cytosol 200

(A)

Cytosol (A549)

Intensity

100 50 0

Cytosol (WI38)

500

150 Intensity

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(B)

400 300 200 100

0

20

40 60 Time (Sec)

0

80

0

20

40 60 Time (Sec)

80

Figure 4. Fluorescence intensity versus time: CPM-labeled thiol protein in the cytosol of (A) lung cancer cell and (B) normal lung cell. 9 ACS Paragon Plus Environment


Figure 4 shows fluorescence intensity versus time trajectories of CPM bound to thiol protein inside the cytosol of lung cancer cell (Figure 4A) and normal lung cell (Figure 4B). It is clearly seen that the fluorescence intensity of CPM fluctuates intermittently inside the cytosol of the cancer cell. Such oscillations may arise from red-ox signaling pathways and repeated formation/rupture of S-CPM bond inside the cytosol of lung cancer cell. CPM bound to thiol protein inside the cytosol of normal lung cell (Figure 4B) does not exhibit any detectable oscillation in fluorescence intensity. This is in contrast to the marked oscillations obtained in the cytosol of lung cancer cell. 3.4

Intermittent Oscillation of Fluorescence Intensity in the Lipid Droplet We now search for fluorescence oscillation inside the lipid droplet of both normal and lung

cancer cell. Figure 5A and B show time dependence of fluorescence intensity in the lipid droplets of cancer and normal cells, respectively. In the case of the lipid droplets, such oscillations were detected both for the normal lung and the lung cancer cell. The observed oscillation in fluorescence intensity of CPM bound to the thiol-containing proteins in the surface of lipid droplets may be attributed to red-ox processes and intermittent formation/rupture of S-CPM bonds. To the best of our knowledge, this is the first direct evidence of structural oscillations occurring inside the lipid droplet in a live cell. Lipid Droplet (A549)

(A)

300

Lipid Droplet (WI38)

(B)

Intensity

400

Intensity

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200

0

0

40

80

200 100 0

120

Time (Sec)

0

40 80 Time (Sec)

120

Figure 5. Fluorescence intensity versus time trajectories of CPM-labeled thiol protein in the lipid droplet of (A) cancer cell, A549 and (B) normal cell, WI38.

10

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3.5

Solvation Dynamics in Cytosol: Normal and Cancer Cell In order to study dynamics of confined biological water in the live cells, we carried out a

solvation dynamics experiment. Solvation dynamics is characterized by time-dependent red shift of the emission maximum of a polar solute to lower energy (Stokes shift) with increase in time. As a result of solvation dynamics, the fluorescence transient at the blue end (short wavelength, unsolvated species) displays a decay. At long emission wavelength (red end), the transient for the solvated species exhibits a distinct rise preceding the decay. Figure 6A-B show the wavelength dependent fluorescence decays of CPM in cytosol of cancer and normal cell. In both the cases, we have detected distinct rise (~ 150 ps) component at the red end of emission spectra. Hence, it is evident that CPM is undergoing solvation inside cytosol of live lung cells.

0.8

1.0

(B) Cytosol (WI38)1.0 540 nm

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t

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Normalized Intensity


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(A) Cytosol (A549) 540 nm 450 nm IRF

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t 0 ps 200 ps 1600 ps

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24000

0.4 0.2

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2

3 4 Time (ns)

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Figure 6. Fluorescence decays of CPM inside cytosol of (A) cancer and (B) normal cell. Corresponding TRES are shown in insets.

The time resolved emission spectra of CPM in the cytosol (TRES, inset of Figure 6) were

constructed from the steady state emission spectra and the fluorescence transients. The total dynamic Stokes shift (DSS) and solvation time (=a1τ1+a2τ2) of CPM inside cytosol of normal and cancer cells are listed in table 1. Figure 7A-B show decay of solvent response 11 ACS Paragon Plus Environment


function, C(t) of CPM in the live cells. The decay of C(t) in the cytosol of both normal and cancer cell is found to be single exponential (Figure 7A). Solvation dynamics of CPM in the cytosol of cancer cell (~ 230 ps, table 1) is slightly faster than that (~370 ps) in the normal cell. 1.0

Cytosol

(A)

(B)

Lipid Droplet

0.8 A549

WI38

0.6

C(t)

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WI38

A549 0.4 0.2 0.0 0

500

1000 Time (ps)

1500 0

1000

2000 Time (ps)

3000

4000

Figure 7. Decay of C(t) inside (A) cytosol and (B) lipid droplet of lung cancer cell (A549) and normal lung Cell (WI38). Data points denote values of C(t) and solid lines indicate best fit. Table 1. Decay parameters of solvent response function, C(t) of CPM in different regions of cancer and normal cells.

Cell

Δν [ν(0)][a]

τ1 (a1)

τ1 (a1)

[b]

cm-1

(ps)

(ps)

(ps)

Cytosol

290 [21340]

230

Lipid droplet

620 [21760]

400 (0.25)

Cytosol

500 [21490]

370

370

Lipid droplet

500 [21550]

800

800

Region

230

A549 2000 (0.75)

1600

WI38

[a]

± 50 cm-1, [b] ± 10 %, =a1τ1+ a2τ2


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3.6

Solvation Dynamics in Lipid Droplets: Normal and Cancer Cell Inside the lipid droplet of a lung cancer cell, CPM exhibits a rise time ~ 400 ps at the red

end of the emission spectra while the rise time is ~ 200 ps inside the lipid droplet of a normal lung cell (Figure 8A-B). This suggests that the solvation dynamics of CPM is slower inside the lipid droplets of cancer cell. TRES of CPM in the lipid droplet of cancer and normal cell are shown in the inset of figure 8A and B, respectively. Total dynamic Stokes shift are calculated from the TRES and summarized in table 1.

540 nm 450 nm

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0.2

2

Time (ns)

4

6

1

2

3 4 Time (ns)

5

6

Figure 8. Fluorescence decays of CPM inside lipid droplet of (A) lung cancer and (B) normal lung cell. Corresponding TRES are shown in the insets.

Figure 7B shows the decay of C(t) inside the lipid droplet of normal and lung cancer cell. In the lipid droplets of cancer cell, C(t) of CPM exhibits two components of solvation dynamics400 ps (25%) and 2000 ps (75%) with an average solvation time () of 1600 ps. In contrast to the cancer cell, lipid droplets of normal lung cell display only one component of ~ 800 ps (table 1). Thus solvation dynamics in the lipid droplets of the lung cancer cell is ~ 2 times slower compared to that in the lipid droplet of the normal lung cell.



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4.

Discussion The most important finding of this work is the detection of intermittent fluorescence

oscillations in the lipid droplets of normal lung cell and lung cancer cell. Before discussing that we wish to emphasize that selective binding of CPM to the lipid droplets confirms existence of highly reactive thiol-containing proteins inside the lipid droplets of a live cell. It may be mentioned that several groups recently reported the presence of thiol proteins in the lipid droplets. Using proteomics, Goodman et al. detected the presence of several thiol-containing enzymes (e.g. including protein-disulphide isomerase)21-22 in mammalian lipid droplets. Other thiol proteins associated with lipid droplets include lipid coat protein (perilipin 3 or TIP47 contains lone cys-345 residue), lipid anchored protein59 (contains side chain of cys residues) and stomatin (contains cys-120 residue).20 These cysteine residues may be labeled by CPM according to Scheme 2 as follows.

Fluorescent

Non-Fluorescent

Scheme 2. Michael addition reaction to label a cysteine residue by CPM.

It is readily seen that the number of lipid droplets in a cancer cell is far (~20 times) higher than that in a normal cell. We have discussed earlier that this is due to the so called Warburg Effect and enhanced glycolysis in cancer cell.13 The large number of lipid droplets in a cancer cell may be used as a diagnostic tool for detection of lung cancer. We now compare the time periods of oscillation in the cytosol of lung cancer cell with those in the normal lung cell. In the cytosol, the main candidates for labeling by CPM are the thiol-containing antioxidants (e.g. glutathione, thioredoxin etc.) whose levels are elevated by several folds in the lung cancer cell compared to its normal counterpart.4-5 In order to determine the time period of oscillation, we have used fast Fourier transform (FFT) method. The mean value (and standard deviations) for the half period of oscillation are obtained for ~15 cells and ~60 lipid droplets (or ~60 regions in the cytosol). 14 ACS Paragon Plus Environment

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For the cytosol of cancer cell, we have enlarged a portion (Figure 9A, 70-78 sec) of the total trajectory in figure 4A. As shown in FFT trace (Figure 9B), the half periods of oscillation is not a constant and is in the range ~0.5-1.5 sec with a mean value of 0.9 ± 0.3 sec. In contrast to the marked oscillation taking place inside the cytosol of cancer cell, normal cell does not exhibit any detectable oscillation in the cytosol. The oscillations in the cancer cell and its absence in the normal lung cell may be attributed to the increased level of ROS inside the cancer cell.

Cytosol (A549)

FFT Trace

90

80 60 70

100

(A)

Intensity

100 Intensity

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72 74 76 Time (Sec)

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1.6 1.5 0.6 1.1 0.9 1

80 70 60 70

72

74 76 Time (sec)

78

Figure 9. (A) Enlarged portion of Figure 4A and (B) its corresponding FFT trace.

For the lipid droplets of lung cancer cell, a portion of the trace in figure 5A is enlarged

and shown in figure 10A. The corresponding FFT trace (Figure 10B) indicates that the half periods of oscillation inside the lipid droplets of the lung cancer cell range from 0.5 to 1.5 sec (Figure 10B) with a mean value of 0.9 ± 0.3 sec i.e. same as that in the cytosol of cancer cell. The lipid droplets of the normal lung cell exhibit relatively longer half periods of oscillation (~2-4 sec, Figure 10C-D) with a mean value 2.8 ± 0.7 sec. Note, the cytosol of the normal cell do not exhibit such oscillations. In order to eliminate the possibility of any artifact due to motion of cells within the time of observation, we had taken time lapse images at 5 minutes of interval for a total period of 60 minutes. It is observed that the position of the cells and lipid droplets do not change in these time period. Thus the large lipid droplets are stationary in the highly viscous cytoplasm within the cell during the course of measurement. Since the fluctuations of the intensities are at a much shorter



time scale (0.9 and 2.8 seconds) the fluctuations are not due to change in position of the lipid droplets. 17

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240 Lipid Droplet (WI38) (C)

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160 120

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Figure 10. (A) Enlarged window of Figure 5A and (B) its corresponding FFT trace. (C) Enlarged portion of Figure 5B and (D) its corresponding FFT trace. We now discuss the possible sources of the fluorescence oscillations or fluctuations. The main sources of the oscillation seem to be the red-ox processes and intermittent rupture of the SCPM bond. The red-ox processes periodically convert –SH (thiol) to S-S (disulphide bonds) and vice versa. This causes a fluctuation of local environment. In a cell, the rupture of S-ligand (SCPM in our case) bond is essential to internalize a foreign ligand.10 Similar process may be occurring at the periphery of the lipid droplets inside the cell. The CPM is non-fluorescent in free state and becomes fluorescent only when bound to S (as S-CPM). Thus binding and unbinding of CPM causes oscillation in fluorescence intensity. The longer periods of oscillations observed in normal lung cell compared to those in the cancer cell may indicate slower red-ox processes


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(presumably because of the lower ROS level in normal cells) and also slow internalization process. We now compare the relative polarity of the lung cancer and normal lung cell from the position of emission maxima of CPM. CPM bound to thiol proteins inside the cytosol and lipid droplet of a normal lung cell display emission maxima at 474 nm and 472 nm, respectively. These emission maxima are very close to the emission maximum of CPM bound to another protein HSA denatured by 6M guanidium hydrochloride (GdnHCl).60 Thus the local polarity experienced by CPM in the cytosol and lipid droplets of the normal lung cell (WI38) is similar to that of HSA unfolded by 6M GdnHCl. For lung cancer cell (A549), emission maximum of CPM bound to cytosolic thiol protein (476 nm) is similar to that of CPM labeled HSA in a mixture of 6M GdnHCl and 1.5 M of a room temperature ionic liquid (477 nm).60 This clearly reveals more polarity of the cytosol in a cancer cell, probably due to the greater antioxidant content associated with a cancer cell. In the case of lipid droplets, the emission maximum of CPM in the lipid droplet (~468 nm) of the cancer cell is blue shifted by 4 nm compared to the lipid droplet in the normal cell (472 nm). Thus lipid droplet of a lung cancer cell is less polar than that in the normal lung cell. Because of higher number and lower polarity of the lipid droplets, the hydrophobic molecule may accumulate in the lipid droplet of a cancer cell. Finally, we compare solvation dynamics in the lung cancer cell with the normal lung cell. Solvation dynamics inside the cytosol of cancer cell (~ 230 ps) is slightly faster compared to that (~370 ps) of normal cell. It may be mentioned that faster solvation dynamics favors polar reaction. Hence, slightly faster solvation inside the cytosol of cancer cell indicates polar reactions may occur more easily inside the cytosol of cancer cell compared to normal cell. Solvation dynamics is in general found to be slower inside the lipid droplets. It is already noted that lipid droplet of cancer cell exhibits ~ 2 time slower solvation dynamics compared to that in normal cell. The slower solvation dynamics and lower polarity of the lipid droplets in cancer cell coupled with ~20 folds higher number of them may give rise to accumulation of hydrophobic signaling molecules in the lipid droplets and thus favor non-polar reactions. It will be highly interesting to find out its consequences in the biological processes in the cancer cell.



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5.

Conclusion This work demonstrates intermittent structural oscillations of CPM bound to thiol proteins in

the lipid droplet and cytosol of a lung cancer cell. The oscillations are attributed to fluctuation of local environment because of red-ox processes and repeated rupture/formation of S-CPM bond. In the case of normal lung cell no such oscillation is observed in the cytosol. Also the fluorescence oscillation in the lipid droplets (2.8 ± 0.7 s) of the normal cell is slower than those in the cancer cell (0.9 ± 0.3 s). This may be attributed to slower red-ox process in the cancer cell because of lower ROS level and slower internalization (by rupture of S-CPM bond). Compared to normal cell, the lipid droplet in a cancer cell is found to be less polar and display slower solvation dynamics. In contrast, the cytosol of the cancer cell is more polar and exhibit faster solvation dynamics relative to the normal cell. These results may provide new insight on the biochemistry of the cancer cells. Acknowledgement Thanks are due to Department of Science and Technology, India (KB) and J. C. Bose Fellowship (KB). RC and MAA thank CSIR for awarding fellowships. We also thank National Centre for Cell Science (NCCS), Pune for providing A549 cell line and Professor Sanghamitra Raha (SINP) for donating the WI38 cell line. We appreciate Dr. Surajit Ghosh for stimulating discussion and ungrudging help.


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Intermittent Fluorescence Oscillations in Lipid Droplets in a Live

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