Modulation of Membrane Fluidity Performed on Model Phospholipid

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Modulation of membrane fluidity performed on model phospholipid membrane and live cell membrane: Revealing through spatiotemporal approaches of FLIM, FAIM and TRFS Dipankar Mondal, Rupam Dutta, Pavel Banerjee, Devdeep Mukherjee, Tapas Kumar Maiti, and Nilmoni Sarkar Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04044 • Publication Date (Web): 01 Mar 2019 Downloaded from http://pubs.acs.org on March 1, 2019

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Analytical Chemistry

Modulation of Membrane Fluidity Performed on Model Phospholipid Membrane and Live Cell Membrane: Revealing through Spatiotemporal Approaches of FLIM, FAIM and TRFS Dipankar Mondal1, Rupam Dutta1, Pavel Banerjee1, Devdeep Mukherjee2, Tapas Kumar Maiti2 and Nilmoni Sarkar1,* 1Department

of Chemistry, Indian Institute of Technology, Kharagpur 721302, WB, India.

2Department

of Biotechnology, Indian Institute of Technology, Kharagpur 721302, WB, India.

E-mail: [email protected] and [email protected] Fax: 91-3222-255303 Abstract We have elucidated the role of unsaturated fatty acid in the in-vitro model phospholipid membrane and in-vivo live cell membrane. Fluorescence microscopy and time-resolved fluorescence spectroscopy have been employed to uncover how modulation of vesicle bilayer fluidity persuades structural transformation. This unsaturation induced structural transformation due to packing disorder in bilayer has been delineated through spatially resolved fluorescence lifetime imaging microscopy (FLIM) and fluorescence polarization or anisotropy imaging microscopy (FPIM/FAIM). Structure-function relationship of phospholipid vesicle is also investigated by monitoring inter-vesicular water dynamics behavior, which has been demonstrated

by

temporally

resolved

fluorescence

spectroscopy

(TRFS)

techniques.

Nevertheless, it has also been manifested from this study that loss of rigidity in bilayer breaks down the strong hydrogen bond (H-bond) network around the charged lipid head groups. The disruption of this H-bond network increases the bilayer elasticity, which helps to evolve various kinds of vesicular structure. Furthermore, the significant influence of unsaturated fatty acid on membrane bilayer has been ratified through in-vivo live cell imaging. Introduction: Diverse structures of model phospholipid membranes offer to mimic beautiful and complex shapes of cell and cell organelles. Cellular membranes are known to change their conformations in a number of remarkable ways during various processes1-3 like movement, division, the 1 ACS Paragon Plus Environment

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extension of neuronal arbors and vesicle trafficking. Also, vesicles with local saddle geometry are essential during excretion process4,5 by a Golgi body. Each biological shape essentially evolves for specific physiological purposes6,7. In the traditional view, structure always directs and dictates the functional activities from molecular to cellular level thus, it is a very fundamental question how structure control function activity of biological and chemical processes. So, the pursuit for structure-function relationship, one needs a powerful tool with high spatiotemporal resolution. Fluorescence lifetime imaging microscopy (FLIM) and Fluorescence anisotropy imaging microscopy (FAIM) hold great promise for probing biological structurefunction relationship with high spatial resolution. On the other hand, time-resolved fluorescence spectroscopy (TRFS) enables us to probe location dependent fast dynamical behavior of water molecules with high temporal resolution. Combining FLIM, FAIM and TRFS techniques, we have investigated the role of membrane bilayer fluidity in transforming the vesicular structure, which dictates their inter-vesicular activities. In early living organisms, the ﬁrst membrane systems were chemically much more primitive8 than the current complex membrane lipids9. But the main principle of membrane modeling still relies on molecular shape. In this study, we have enlightened an unresolved problem, depicting the exact role of unsaturation in membrane structure or organization to control their function. It is reported that in certain neural membranes, the inclusion of double bonds (ω-fatty acid) is very much crucial for their proper functioning10,11. Fatty acids with multiple double bonds (unsaturation) are also abundant in cerebral and olfactory bulb12. Again, the near-native levels of 50 mol % of docosahexaenoic acid (22:6ω3 fatty acid) are required in retinal rod outer segment disk membranes for full production of metarhodopsin II, an intermediate molecular form of rhodopsin13, which is an essential receptor protein for the absorption of a photon. Therefore, the elucidation of the biophysical properties of unsaturated lipids are significantly important14,15 to gain a clear understanding of their role in membrane function and organization. To answer this fundamental question, we have linked between the structure and dynamics of vesicles prepared with a mixture of saturated and unsaturated phospholipids. We have studied an unsaturated phospholipid molecule with two unsaturated fatty acid residues in their hydrophobic tail. Additionally, we have elucidated the individual effect of those fatty acids16,17 on the topology of saturated phospholipid vesicle at a low molar ratio. The shape of a fatty acid residue in the phospholipid molecule and its area at the lipid-water interface 2 ACS Paragon Plus Environment

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is influenced by the presence of unsaturation18,19. We have explored this by combining FLIM and FAIM techniques. The results delineate that unsaturation disrupts the lipid bilayer packing20. This packing disruption mitigates the rigidity of the phospholipid vesicle bilayer, which essentially controls the solvation dynamics of water. The solvation time of water refers to the dynamic process of water reorganization in response to an abrupt change in charge distribution of a dye via electronic excitation. Based on structural properties, fluorescence dye molecules can be intentionally located in the hydrophobic backbone or in the hydrophilic head group region of the phospholipid bilayer21,22 to acquire information from their surroundings23. From the experimental measurement, we have gleaned that the water dynamics become faster as membrane fluidity increases. The faster water dynamics inside phospholipid vesicle is further ratified with single molecule diffusion in the vesicle water pool through fluorescence correlation spectroscopy (FCS). This unprecedented understanding of unsaturated fatty acid-phospholipid membrane interaction through spatiotemporal approaches intensifies the interest to know how unsaturated fatty acid interacts with the live cell membrane. In order to gain detailed insights, we have performed FLIM and FCS measurement to unveil the profound role of unsaturation on THP-1 live cell membrane. The results are very much comparable with model phospholipid membrane; depicting the fact that the unsaturation decreases the membrane rigidity substantially. Experimental Section: Materials and Sample Preparation: DMPC (1,2-Dimyristoyl-rac-glycero-3-phosphocholine), LAPC (L-α-Phosphatidylcholine) and 4H-pyran (DCM), Coumarin-153 (C-153), Rhodamine 6G (Rh-6G), 4-(dicyanomethylene)-2-methyl-6(p-dimethylaminostyryl) (Scheme 1) were purchased from Sigma Aldrich and used as received. We used different fluorescent probe molecules to characterize a vesicle, a naturally occurring structure, consisting of biologically relevant fluid enclosed by the phospholipid bilayer. We used DCM to discern the bilayer rigidity through FLIM study as this hydrophobic dye resides mostly in the hydrophobic bilayer region. Here, C153 was preferred to decipher the interfacial solvation dynamics of vesicles, because C-153 dye molecule shows very high Stokes’ shift depending upon the surroundings viscosity or polarity. Whereas, solvation dynamics is experimentally determined by observing the time-dependent Stokes’ shift24,25 of the fluorescence spectrum of a polar solute after laser pulse excitation. Kiton red or Sulforhodamine B (SRh-B) was received from Lambda Physik. This charged dye used to

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elucidate the packing behavior of interfacial charged hydrophilic head group through fluorescence anisotropy imaging. Finally, to infer about inter vesicular rigidity, we monitored the Brownian motion of a hydrophilic dye Rh-6G, which preferentially stayed in water pool of the vesicle. All the solvents were of spectroscopy grade and used without further purification. Vesicles were prepared using FILM method (See S1). The vesicles sizes were kept ~120 nm (See S2) for time resolved measurement. Vesicle solutions were added to the methanol evaporated C153 so that probe to lipid ratio became 1:10. The steady-state emission spectra (See S3) were recorded in a Shimadzu RF-6000 spectrofluorometer. The stock solution of DCM and SRh-B were prepared in methanol both having concentration 1 mM. At first, a small aliquot of the dye solutions from the stock was taken in the volumetric flask, and methanol was evaporated in hot air flow. The dye concentration of DCM was maintained as ∼10 μM for the FLIM measurements. Further, a required amount of 12 mM vesicular solution was added to adjust the above mentioned concentration of the dye ∼10 μM. The same procedure was followed for anisotropy imaging using SRh-B dye.

Scheme 1. Chemical structures of (a) C-153, (b) DCM (c) Rh-6G (d) SRh-B (e) DMPC and (f) LAPC. Instrumentation: We performed time resolved fluorescence measurement using time correlated single photon counting (TCSPC) method and Fluorescence Correlation Spectroscopy (FCS) technique. The 4 ACS Paragon Plus Environment

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imaging techniques, we used: Fluorescence Lifetime Imaging Microscope (FLIM), fluorescence polarization or anisotropy imaging microscope (FPIM/FAIM), Transmission electron microscope (TEM). The detailed descriptions of the experimental techniques are discussed in the supporting information (See S2-S7). Results and Discussion: Modulation of Membrane Fluidity through Unsaturated Fatty Acid: We have modulated membrane fluidity by incorporating unsaturated fatty acids in phospholipid vesicles as well as live cell membrane. This effect of unsaturation on membrane permeability is investigated through the FLIM technique. Our experimental results have explained the role of membrane properties in controlling vesicular and cellular structure26, which is further ratified with other spatiotemporal approaches. For FLIM experiments, we have used DCM dye to study the property of vesicle bilayer region27,28. Experimental results indicate that hydrophobic DCM molecules have high propensity to accumulate at the hydrophobic bilayer region of the DMPC vesicle, which seems to be the most rigid spherical vesicle. Because DMPC vesicles are made of saturated phospholipid molecules (Figure 1a) thus lipid molecules settle in tightly packed arrangement resulting in very rigid hydrophobic bilayer. As a result, FLIM lifetime distribution peak for DMPC vesicle show higher value (2066±112 ps) (Figure 1b-1d) compared to other two phospholipid bilayer made of 9:1 molar ratio of DMPC:LAPC (Figure 1e-1h) and 3:2 molar ratio of DMPC:LAPC (Figure 1i-1l) phospholipids. The broad distribution of the lifetime in a DMPC vesicle signifies the dynamic heterogeneity in the distribution of DCM in lipid bilayer. Whereas, 90:10 volume mixture (9:1 molar ratio) of DMPC and LAPC phospholipids produces mostly inter lipid vesicles thus there is perfect symmetry loss of lipid packing in the bilayer of saturated DMPC lipid due to incorporation of unsaturated LAPC lipid. Here, lifetime distribution peak is centered at ~1645±40 ps (Figure 1g and 1h) for 90:10 DMPC:LAPC vesicles and lowest among all three lipid vesicles (Figure 1b, Figure 1f and Figure 1j) indicating the increase in the membrane fluidity29,30. As soon as, we increase the amount of unsaturated LAPC lipid in saturated DMPC lipid, we have observed that lifetime distribution histogram moves towards higher value due to regaining the symmetry in the bilayer region. At 60:40 volume mixture of DMPC and LAPC (molar ratio 3:2) vesicle formation is guided by intra lipids as well as inter lipids. We have observed an increase in the lifetime distribution at 3:2 molar ratio compared to

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9:1 molar ratio and fitted value is 1796±70 ps (Figure 1j-1l). Thus, it is predicted that there is a higher chance of intra lipid vesicle formation than inter lipid vesicle formation. During inter lipid bilayer formation between DMPC and LAPC, vesicle requires structural transformation from positive Gaussian curvature to negative and mostly zero Gaussian curvature31 at bilayer surface. We have proposed seven possible models of lipid bilayer arrangement (See S8) during vesicle formation between saturated and unsaturated phospholipids. We have expected that most random structure is given in S8 Figure S4g, which will provide most unsymmetrical packing of bilayer resulting in rod and pear-shaped vesicle in 9:1 molar ratio of DMPC:LAPC vesicle. This possible molecular arrangement induces the vesicle curvature from positive to zero or negative curvature, whereas at 3:2 molar ratio there is more chance of Figure S4a, S4b, S4c bilayer rather than other packing possibilities presented in S8 and thus vesicle regain packing order, which produces rigid bilayer. Few inter lipid vesicle formation have also been observed to form ring like shape possibly due to fatty acid chain length difference32. We have also confirmed structural deformation of the lipid vesicles through repeated fluorescence microscope images (See S9) as well as with TEM images (Figure 1a, 1e and 1i). The significant effect of unsaturation in controlling bilayer rigidity is further ratified by examining the vesicle made of 9:1 molar ratio DMPC:Oleic acid and 9:1 molar ratio of DMPC:Linoleic acid. In Figure 1, the lifetime distribution peak maxima centered at 1580±30 ps (Figure 1m-1o) and 1523±50 ps (Figure 1p-1r) for those vesicles respectively, which is substantially lower than the saturated DMPC vesicle. The mono-unsaturation in oleic acid limits the alignment of saturated lipid close packing thus the rigidity of the vesicle bilayer decreases. Furthermore, linoleic acid (ω-3 fatty acid) with polyunsaturation disrupts bilayer more extent. As a result, lifetime distribution peak shifts slightly towards lower value compared to the data obtained from DMPC-Oleic acid vesicle. In comparison with saturated DMPC vesicles, these results clearly indicate that the unsaturationinduced kink20 in hydrophobic tail breaks down the perfect packing order in bilayer as well as decreases their rigidity. The few dominant bilayer packing made of saturated and unsaturated lipid (or fatty acid) has presented in Figure 1s, 1t, 1u, 1v. We have further cross examined the membrane fluidity using fluorescence recovery after photobleaching (FRAP)33 technique using DCM dye. FRAP data (See S10 for Detail) proves that unsaturation is the predominant reason to increase membrane fluidity. We have compared half-life (t1/2) of fluorescence recovery between saturated DMPC and 90:10 DMPC:Oleic+Linoleic acid. The observed t1/2 of DMPC is 2.47±0.03 6 ACS Paragon Plus Environment

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s, whereas 9:1 molar ratio phospholipid and fatty acid mix vesicle recovers from photobleach at very faster rate which is 0.41±0.07 s (see figure S6a and S6h).

Figure 1: DMPC vesicle (a) TEM image (b) Fluorescence Confocal Image (FCI) (c) Fluorescence lifetime distribution histogram (FLDH) (d) FLDH fitted. 90:10 DMPC:LAPC vesicle (e) TEM image (f) FCI (g) FLDH (h) FLDH fitted. 60:40 DMPC:LAPC vesicle (i) TEM image (j) FCI (k) FLDH (l) FLDH fitted. 90:10 DMPC:Oleic acid vesicle (m) FCI (n) FLDH raw data (o) FLDH fitted. 90:10 DMPC:Linoleic acid vesicle (p) FCI (q) FLDH (r) FLDH fitted. (s) DMPC lipid bilayer symmetric packing. (t) unsaturated LAPC lipid bilayer symmetric packing. (u) Symmetric packing of DMPC and LAPC (v) most asymmetric bilayer formation in DMPCLAPC mixture. THP-1 cell (A) FCI (B) FLDH (C) FLDH fitted. THP-1 cell after 15 min addition of 12 mM (Oleic+Linoleic) acid mixture (D) FCI (E) FLDH (F) FLDH fitted. THP-1 cell after 25 min addition of 12 mM (Oleic+Linoleic) acid mixture (G) FCI (H) FLDH (I) FLDH fitted. THP-1 cell after 40 min addition of 12 mM (Oleic+Linoleic) acid mixture (J) FCI (K) FLDH (L) FLDH fitted. Further, we have investigated the effect of unsaturated fatty acid34 on THP-1 cell membrane. We have performed FLIM study on live cell35,36 membrane tagged with carbocyanine dye, 5 nM DiOC18 to delineate unsaturation induced membrane fluidity. From our experimental results; we have observed that DiOC18 in THP-1 cell (Figure 1A, 1B, 1C) show broad fluorescence lifetime distribution and it can be deconvoluted with two Gaussian distribution due to complete heterogeneity of cell environment. Remarkable change has been observed upon addition of 7 ACS Paragon Plus Environment


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unsaturated fatty acid mixture to the THP1- cell in PBS buffer. During experiments, we have maintained 12 mM concentration of oleic+linoleic (ω-3 fatty acid) acid in their equimolar mixture to perform the in vivo study on live cell membrane. After addition of oleic and ω-3 fatty acid, the time dependent FLIM data ensures the fact that unsaturation significantly increases the membrane permeability. After 15 minutes, addition of fatty acid mixture (Figure 1D, 1E, 1F), the lifetime histogram seems to become less heterogeneous because we have obtained Gaussian peak separations (de-convoluted with two Gaussian) is 182 ps which is less than 286 ps obtained from control experiment. Further delay of 10 minutes, membrane fluidity increases (Figure 1G, 1H, 1I), as we have observed that fluorescence lifetime distribution fits with single Gaussian at peak maxima at 885 ps and FWHM 230 ps. Final FLIM measurement of THP-1 cell after 40 minutes addition of fatty acid mixture (Figure 1J, 1K, 1L), the peak maxima shifts towards lower lifetime at 815 ps and provide substantial reduction in FWHM as 150 ps. This result solely due to unsaturation as we have not observed any time-dependent change in lifetime distribution of DiOC18 in a different batch of THP-1 cell (See S11) without fatty acids. Although data presented in Figure 1 and Figure S7 have same characteristic features (deconvoluted with two Gaussian) still fitting amplitude differs due to local microenvironment. Because individual cell have their independent size and structure and thus they have little extent of rigidity difference. Furthermore, we have also performed concentration-dependent study with 6 mM and 15 mM (See Figure S8 of S12) of an oleic+linoleic acid mixture to check the consistency of the fatty acid effect on the live cell membrane. In both cases, we have observed that unsaturated fatty acid mixture decreases the membrane fluidity but interestingly lower concentration takes a little longer time compared to higher concentration. Molecular Unsymmetrical Packing in Bilayer induces Vesicle Shape Transformation: To elucidate packing behavior of the lipid molecules, we have used a charged and highly fluorescent molecule SRh-B to study the fluorescence polarization or anisotropy image (FPI/ FAI), as used by Axelrod37. In Figure 2, the cross sections of scanned vesicles in the focal plane of the fluorescence microscope are presented. The intensity of FPI/FAI reveals the relative mobility of the fluorophores in the phospholipid vesicle environment38. Here, the higher fluorescence anisotropy values, coded with yellow color, signify that the transition dipoles of SRh-B molecules orient slowly due to higher local viscosity39. Whereas, the lower anisotropy value, coded with red color, implies that the rotational motion of SRh-B is faster due to lower 8 ACS Paragon Plus Environment

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local viscosity. As local viscosity solely depends on the H-bonds formed between charged head groups and interfacial water molecules thus the increase in inter spacing between two head groups decrease the local viscosity at the interface region of the vesicle. The spherical vesicle in Figure 2a reveals that most of the part has very high fluorescence anisotropy value, which indicates that the lipid packing is highly ordered and the interspacing between two head groups is the smallest. Because such an arrangement of head groups lead to very strong H-bond network, which has been further confirmed from TCSPC experiment. The reason behind this fact is that whenever the spacing between the head group increases the H-bond network is broken and water molecules become free to move and as a result of local viscosity decreases. This decrease in local viscosity helps to enhance the rotational diffusion rate of SRh-B at the interface.

Figure 2: Fluorescence anisotropy image of SRh-B in vesicle obtained at excitation wavelength 488 nm (a) DMPC vesicle, (b) 90:10 lipid mixture of DMPC:LAPC vesicle (c) 90:10 lipid mixture of DMPC:Oleic acid vesicle, (d) 90:10 lipid mixture of DMPC:Linoleic acid vesicle. In Figure 2b, FAI of the vesicle (9:1 molar ratio of DMPC and LAPC) shows very low fluorescence anisotropy value. So it is evident that in this vesicle, the spatial arrangement of the lipid molecules in lateral direction has an unusual pattern that increases the separation between two head groups and as a result, “bound” water dynamics become faster. Similar kind of pattern is also available in the vesicle prepared with DMPC and oleic acid mixture and DMPC and 9 ACS Paragon Plus Environment


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linoleic acid mixture. We have observed from Figure 2c and 2d that vesicles prepared with DMPC and linoleic acid are more elastic compared to the vesicle prepared with DMPC and Oleic acid. This study can be further used to monitor the interaction between membranes and other biomolecule40 like, protein, DNA etc. Effect of Bilayer Nature on Inter-Vesicular Water Dynamics: The structure, reactivity, and dynamics of the biomolecules inside cell are essentially regulated by the water molecules present in that confined system. The confinement has a major role in determining the fluid dynamics as it dictates the fluid distribution as well as diffusion41. Thus there is a paramount interest to study the water solvation dynamics inside confined media42-45. Here, we have accentuated on “bound” and “exchange” water dynamics of vesicle to unveil the salient role of the bilayer on inter-vesicular behaviour46. We have observed that “bound” water dynamics are 2050 ps for pure DMPC (Figure 3a, black line) vesicle whereas 1100 ps for 9:1 molar ratio DMPC:LAPC mix lipid vesicle (Figure 3a, red line) and 1860 ps for 3:2 molar ratio of DMPC:LAPC mix lipid vesicle (Figure 3a, blue line). The bound water relaxes very slowly as the relaxation procedure involves rupture of strong hydrogen bonds between water molecules and phospholipid charged head groups along with a coherent motion of the phospholipid hydrophobic long chains47. There are also water molecules those are continuously exchanging hydrogen bonds between bound water and unbound water known as “exchange” water. Our measured interior “exchange” water dynamics time scales are 850 ps, 630 ps and 700 ps in pure DMPC vesicle, 9:1 molar ratio DMPC:LAPC and 3:2 molar ratio DMPC-LAPC mix lipid vesicle, respectively. From Table 1 it is evident that dynamics of bound water is slow as a result the exchange rate also becomes slow, which eventually slows down average solvation time of water. We have not measured free water dynamics as these water molecules reside far form bilayer and depict least information about bilayer fluidity. Yet a considerable percentage of C153 molecules stay in pool water and least amount outside bulk water confirmed through FLIM image. Still, our solvation dynamics measurement is not affected by bulk water dynamics at all because bulk water dynamics varies from few femtosecond48 to less than few picoseconds. Thus, we have predominantly captured the interfacial water dynamics and our solvation timescale completely independent from the chain dynamics of surfactants as they relax on a very slow47,49 time scale at ~100 ns. The average solvation time of water reveals how the compact lipid head groups are packed. The most organized and compact packing occur between saturated DMPC 10 ACS Paragon Plus Environment

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lipids, whereas inter-lipid bilayer between two un-symmetric lipid molecules (DMPC and LAPC) produces more fluidic bilayer. In case of 9:1 molar ratio, lipid vesicle bilayer is loosely packed and head group separation is higher which results in weak H-bond formation and as a result, water dynamics become faster. On the other hand, in the competing lipid concentration of 3:2 molar ratio, mostly intra-lipid vesicle formations happen and even though there is a chance of proposed model bilayer formation as given in Figure S4g of S8. These symmetric structures again decrease the inter lipid separation and thus comparatively strong H-bond network reform and solvation dynamics of water molecules become slowed down.

(a)

(b)

Figure 3: The decay of solvent response function, C(t) of C-153 in vesicle obtained from TCSPC measurement at excitation wavelength 400 nm (a) in DMPC vesicle (black line), 90:10 lipid mixture of DMPC:LAPC vesicle (red line), 60:40 lipid mixture of DMPC:LAPC vesicle (blue line), (b) 90:10 lipid mixture of DMPC:Oleic acid vesicle (red line), 90:10 lipid mixture of DMPC:Oleic+Linoleic acid vesicle (green line). Moreover, the effects of unsaturation residues present in LAPC vesicle have also been studied individually (Figure 3b). We have mixed saturated DMPC lipid with single unsaturated Oleic acid and observed that water dynamics become faster. The bound water relaxation time is 1340 ps and exchange water dynamics is 580 ps (Figure 3b, red line). Nevertheless, we have also tried to mimic the structure obtained from 9:1 molar ratio mixture of DMPC and LAPC lipid. In this respect, we have experimentally investigated the solvation dynamics of vesicle made of 9:1 molar ratio of DMPC and two fatty acids equimolar mixture. Interestingly the bound water dynamics is 1110 ps (Figure 3b, green line) and that value matches well with the most deformed vesicle obtained from 9:1 molar ratio of DMPC and LAPC. To construct the decay of the solvent 11 ACS Paragon Plus Environment


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correlation function C(t), we have fitted TRES (See S13) with lognormal function and used peak frequency to obtain exponential decay curves. The acyl chain induces saturated alkyl chains to become "fatter" at the bilayer center than near the interfacial region, leading to packing disorder toward the lipid-water interface. As a consequence, the bilayer thickness changes slightly. Thus, the small change in thickness (vertical organization) may actually result a significant perturbation near the interface (lateral organization). This perturbation highly regulates the Hbonds strength between charged phospholipid head groups and interfacial water molecules. The ordering of H-bonded water molecules extends several layers beyond the first hydration shell of the polar head group50. The perfect order makes perfect H-bond network and results in very rigid water structure. Whenever first hydration cell transforms to a fragile structure due to breakdown in symmetric packing of head groups attached to unsaturated hydrophobic tails, H-bond network collapses and water molecules become free to move very fast. Table 1. Picosecond Decay parameter of C-153 in vesicle made of different proportions Lipid-Lipid and Lipid-Fatty acid mixture. Excitation Sample τ1 τ2 a1 a2 avg wavelength (ps) (ps) (ps) 2050±120 850±150 0.76 0.24 1760 402 nm DMPC vesicle 90:10 DMPC:LAPC lipid mix 1100±100 630±50 0.72 0.28 970 vesicle 60:40 DMPC:LAPC lipid mix 1860±50 800±50 0.72 0.28 1560 vesicle 90:10 DMPC:Oleic acid mix 1340±70 1150 580±60 0.75 0.25 vesicle 90:10 DMPC:(Linoleic +Oleic) 1110±50 1070 280±50 0.95 0.05 acid mix vesicle Bilayer Rigidity and Vesicle Pool Water Behavior observed by FCS: We have chosen a hydrophilic dye Rh-6G to probe the vesicle pool water behavior at single molecular level inside mixed DMPC-LAPC lipid vesicles (Figure 4a) and DMPC-fatty acid (oleic and linoleic acid) vesicles (Figure 4b). This single molecular level FCS study explains inter vesicular environmental changes due to frictional force arises by the solvent molecules within confined media51-54. Experimentally, we have observed that Rh-6G diffuses slowly inside DMPC vesicle because the nice symmetric packing of saturated DMPC lipid molecules forms a highly rigid H-bond network. Thus, Rh-6G experiences the highest frictional force with this rigid H-bond, which is the reason behind the slowest diffusion rate in DMPC vesicle. Consistent with 12 ACS Paragon Plus Environment

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our TCSPC results, our FCS data also indicates that Rh-6G diffuse faster inside vesicle made of 9:1 molar ratio between DMPC and LAPC lipid (Figure 4a, red line). As previously described, at this particular mixture the most structural deformation occurs as they form inter lipid bilayer. This bilayer has the most unsymmetrical packing ensuring the least rigid bilayer formation, which essentially prevents the formation of a strong H-bond network of water molecules at the first hydration shell of the polar head group. Interestingly, at 3:2 molar ratio between DMPC and LAPC in the lipid mixture, mostly intra lipid vesicle formation occurs. As a result, we have observed spherical and few ring-like shaped vesicular aggregates. Here, bilayer rigidity increases due to an increase in lipid packing order as evident from the spherical vesicular structure. Increase in packing order decrease the separation between two head groups thus again strong Hbond network forms between water molecules. This H-bond network extends several layers beyond the first hydration shell and as a result local viscosity increases. Thus, diffusing Rh-6G at the inner interface of vesicle (made of 3:2 molar ratio DMPC and LAPC) experiences more frictional force and has slower diffusion (Figure 4a, blue line) compared to the vesicle formed between DMPC and LAPC unsaturated lipids at 9:1 molar ratio. As described in FLIM and FAIM data analysis, unsaturation in membrane bilayer produces a twist in the alkane chain resulting in disruption of the lipid packing20. This restricts saturated hydrophobic tail to align closely. As a consequence, bilayer rigidity decreases and fluidity of bilayer55 increases.

(a)

(b)

(c)

Figure 4: The FCS traces of Rh-6G obtained when diffuses inside (a) DMPC vesicle (black line), 90:10 lipid mixture of DMPC:LAPC vesicle (red line), 60:40 lipid mixture of DMPC:LAPC vesicle (blue line), (b) 90:10 mixture of DMPC:Oleic acid vesicle (red line), 90:10 mixture of DMPC:Linoleic acid vesicle (blue line). (c) FCS traces of DiOC18 labeled only THP-1 cell in PBS buffer (black line) and time interval data the with addition of 12 mM (Oleic + Linoleic) acid mixture; after 25 min (red line) and after 40 min (dark yellow line). [See inset for individual residuals of FCS fitting] 13 ACS Paragon Plus Environment


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We have also studied DMPC and unsaturated fatty acids (with 9:1 molar ratio) mix vesicles to unveil the individual effect of fatty acids. From Table 2 it is clearly evident unsaturation substantially increases the lipid membrane fluidity and decreases H-bond network of water pool. As a result, the diffusion rate of Rh-6G increases (Figure 4b, red line) when oleic acid having single double bond has entered inside DMPC lipid bilayer. The same hydrophilic dye diffuses at a faster rate inside more fluidic vesicle at interfacial region, made of (Figure 4b, blue line) DMPC and linoleic acid, which has two double bonds in the acyl chain. The main reason is that these unsaturated fatty acids continue to diffuse around freely rather than forming a stationary alignment with the saturated hydrophobic chain. In their movement, these molecules can jostle and slide in between the tightly packed saturated fatty acid chains leading to complete disorder in lipid packing. This result clearly indicates that the increase in unsaturation deeply decreases the lipid packing in bilayer and thus membrane fluidity increases extensively. Table 2. Diffusion Coefficient (D) of Rh-6G inside vesicles Obtained Traces Excitation Sample τD (μs) Avesicle/Cell wavelength 488 nm DMPC vesicle 2558±131 0.60±0.02 90:10 DMPC:LAPC lipid mix 1224±86 0.38±0.01 vesicle 60:40 DMPC:LAPC lipid mix 1505±60 0.56±0.02 vesicle 90:10 DMPC:Oleic acid mix 1473±98 0.45±0.02 vesicle 90:10 DMPC:Linoleic acid mix 1289±182 0.20±0.01 vesicle THP-1 cell in PBS 902±123 0.65±0.04 Cell after addition of 12 mM 267±24 0.58±0.03 (Linoleic +Oleic) acid for 25 mins Cell after addition of 12 mM 186±20 0.50±0.03 (Linoleic +Oleic) acid for 40 mins

from the Analysis of FCS Awater

D (μm2.S-1)

0.40±0.01 13.02±0.66 0.62±0.02 27.21±1.90 0.44±0.02 22.13±0.97 0.55±0.02 22.61±0.85 0.80±0.01 25.83±1.60 0.35±0.02 36.92±5.03 0.42±0.03 124.74±11.21 0.50±0.04 179.07±19.25

In order to gain further practical insight, we have elucidated the effect of unsaturation on live cell membrane fluidity by quantifying a single molecular mobility56,57 of a membrane binding dye DiOC18. Using this dye, we have monitored the THP-1 cell membrane fluidity by fitting with a model58 that describes the simultaneous translational diffusion of a fast (3D) and a slow (2D) component (see S6) of DiOC18 at membrane and near to the membrane respectively. We have fixed the value of fast component τ3D=64 μs as reported earlier59 in order to obtain the accurate 14 ACS Paragon Plus Environment

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value of τ2D during fitting of the data shown in Figure 4c. The experimentally measured, D2D (mentioned as D in Table 2), of DiOC18 at live cell membrane is ~37 μm2.S-1 keeping D3D =520 μm2.S-1 as a constant parameter in our fitting equation. But when we have added 12 mM equimolar mixture of oleic and linoleic acid, the diffusion rate of membrane binding dye increases substantially reflecting the increase in membrane fluidity. The diffusion rate calculation after adding unsaturated fatty acid mixture at two different times has been selected from the FLIM image analysis. The experimentally observed diffusion rate after 25 minute and 40 minute result as 124.74±11.21 μm2.S-1 and 179.07±19.25 μm2.S-1 indicating ~3.5 and ~5 times faster compared to control respectively. In the fitting equation α parameter also relates to the degree of diffusion constraint or membrane rigidity. In the case of Brownian motion, α=1, else 0

Modulation of Membrane Fluidity Performed on Model Phospholipid

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