Langmuir 1991, 7, 623-626
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Sulfate-Ion-Induced Slow Fusion of Dioctadecyldimethylammonium Bromide Vesicles David Yogev, Bruno C. R. Guillaume, and Janos H. Fendler' Department of Chemistry, Syracuse University, Syracuse, New York 13244-4100 Received October 15, 1990. In Final Form: December 7, 1990 Addition of equimolar sodium sulfate to vesicles, prepared from 4.0 x 10-4 M dioctadecyldimethylammonium bromide (DODAB), in their gel state at room temperature slowly increased the mean hydrodynamic diameters, DH, of the aggregates from 80 to 740 nm over severaldays. Fluorescence resonance M DODAB, 0.8 mol '36 of N47energy transfer efficiency in DODAB vesicles prepared from 4.0 X nitrobenz-2-oxa-1,3-diazol-4-oyl)phosphatidylethanolamine (donor), and 0.8 mol '36 of N-(lissamine Rhodamine B sulfony1)phosphatidylethanolamine(acceptor)was also found to decrease slowly upon dilution by equal volumes and equal concentrations of DODAB vesicles and NaZS04. Thus, the slow increase of DHfollowing Na2S04 addition corresponds to fusion rather than to nonproductive adhesion of the DODAB vesicles. Monitoring the fluorescence relaxation times, lifetimes, steady-state anisotropies, and average angles of distribution of diphenylhexatriene (fluorescence probe sensitive to its microenvironment) in DODAB vesicles undergoing fusion revealed systematic and parallel fluctuations of different and independently obtained parameters which were attributed to the subtle changes (flattening, adhesion, destabilization, inverse micelle or lipidic intramembranous particle formation, and merging of adjacent bilayers through ellipsoid shape to the final spherical structure) accompanying the fusion of the vesicle ensemble. Introduction Fusion is vitally important in physiological processes such as endocytosis, exocytosis, and fertilization.' It converts two cells, or their surfactant vesicle analogues, into one and thereby removes the barrier betwen the contents of the two merging entities. Fusion of membranes and their models (liposomes and surfactant vesicles, for example) have been extensively investigated by several different approaches.lP2 Protocols have been developed for distinguishing between vesicle-vesicle adhesion and fusion and for establishing the extent of internali~ation.~ Surprisingly, very few experiments have been designed to obtain a mechanistic insight into fusion at the molecular level, and none has captured subtle step-by-stepchanges in real time. Observation of intimate details in the sulfateion-triggered fusion of dioctadecyldimethylammonium bromide (DODAB) vesicles is the subject of the present report. Capturing the fine changes accompanying DODAB vesicle fusion was made feasible by judicious selection of experimental conditions, which extended the observable time frame of fusion to hours instead of the customary seconds to minutes time scale.4 This was accomplished by using a fairly low concentration of salt and by monitoring the process in the gel state of the vesicle a t room temperature (T,of DODAB vesicles = 37 "C) by diphenylhexatriene (DPH) as a fluorescence probe sensitively reporting microviscosity changes in the hydrophobic part of the bilayer.
purified by a Millipore Milli-Q system containing a 0.4-bm Millistack filter at the outlet. DODAB vesicles were prepared by the ultrasonic dispersal of the solid in water (microtipof a Braunsonic 1510sonicatorat70W and70OCfor 10min)andsubsequent centrifugation at 3000rpm to separate trace amounts of titanium released by the tip. DPH (3.0 ML in THF) was introduced into a stirred vesicle solution (3.0 mL in HzO) to give [DODAB]: [DPH] = 5001. DPH-labeled vesicles were incubated in the dark for 2 days at room temperature prior to their use. Hydrodynamicdiametersof vesicles were determined by dynamic light scattering using the exponential sampling method on a Brookhaven BI 2030 AT system. Fluorescence lifetimes, 7F, relaxation times, 7 R , residual anisotropies,r-, and steady-state anisotropies,?, of DPH were calculated from fluorescence timeresolved decays using a single-photon-counting system6 (see legend to Figure 4). Emission intensities for energy transfer measurementswere taken automatically (overa 60-h time period) on a SPEX 1681 Fluorolog equipped with a Tracor Northern TN-6500 rapid scan spectrometer detection system.
Results and Discussion In the absence of additives, vesicles prepared from 4.0 X M DODAB remained stable as indicated by the absence of variation ( < l o % )of the mean hydrodynamic diameter, DH (DH= 80 nm, variance = 1.067, skewness = 1.67, xu2 = 4.21 X 104),6 monitored for weeks. The DH value of DODAB vesicles determined here agrees well with that reported previously (80 nm).7 Addition of Na2S04 (to give 4.0 X M) steadily increased the DH value of samples kept a t 20.0 "C over several days (5 h, D H = 157 nm, variance = 1.075, skewness = 2.124, xv2 = 9.0 X Experimental Section 15 h, DH = 209 nm, variance = 0.049, skewness = 0.553, xv2 = 7.9 X 48 h, DH = 410 nm, variance = 1.654, DODAB (Aldrich), diphenylhexatriene (DPH, Molecular Probes, Inc.),NazSOd (Baker),N-(7-nitrobenz-2-oxa-1,3-diazol- skewness = 3.465, xu2 = 3.2 X 10-4)6to a plateau value of 4-oy1)phosphatidylethanolamine(D,Molecular Probes, Inc.),and 740 nm (variance = 2.08, skewness = 3.362, xs2 = 7.9 X N - (lissamine Rhodamine B sulfony1)phosphatidylethanolamine 10-3)6after 5 days of incubation. Fluorescence resonance (A, Molecular Probes, Inc.) were used as received. Water was
(1) Cell Fusion; Sowers, A. E., Ed.; Plenum Press: New York, 1987. (2) Ohki, S.;Doyle, D.; Flanagan, D; Hui, S. W.; Mayhew, E. Molecular Mechanisms ofMembraneFusion; Plenum Press: New York, 1987. Papahadjopoulos, D.; Nir, s.;Duzghes, N. J.Bioenerg. Biomembr. 1990,22, 157. (3) Nir, S.; Wilshut, J.; Bentz, J. Biochim. Biophys. Acta 1982, 688, 275. (4) Dugunes, N.; Allen, T. M.; Fedor, J.; Paphadjopoulos, D. Biochemistry 1987, 26, 8435.
(5) Flom, S. R.; Fendler, J. H. J. Phys. Chem. 1988, 92, 5908. (6). values and distribution calculations were baaed on the general definition of the kth moment of a distribution about a point, do. kth = x,f&di - do)&with f i = n i / x n i ; mean diameter - DH,k = 1, and do = 0; variance, k = 2 and do = d ; skewness, k = 3 and do = DH;and xz = simultaneous measurements of the random deviations of the data about the fit and the systematic differences between the data and the assumed functional form. x? is the reduced x2 = xz divided by the degree of freedom for fitting. (7) Herrmann, U.; Fendler, J. H. Chem. Phys. Lett. 1979,64, 270.
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0743-7463/91/2407-0623$02.50/00 1991 American Chemical Society
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624 Langmuir, Vol. 7, No. 4, 1991
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Figure 1. Relative A-D emission intensity, measured as Z5anm M DODAB, 4.0 X - Z & L ~in, ,solutions ~, that contained 4.0 X M Na~S04,3.2 X 10" M A, and 3.2 x 10" M D at 20.0 "C as a function of time. XEXC = 470 nm. Points are experimental data. energy transfer was used to distinguish between sulfateion-mediated vesicle-vesicle adhesion and fusion.8 Experiments were carried out on 1:lmixtures of labeled (by 0.8 mol % D and 0.8 mol 7% A) and nonlabeled vesicles prepared from 4.0 X M DODAB, also at 25.0 "C. Injection of appropriate amounts of Na2S04 (to give 4.0 X M) resulted in a slow decrease of the A-D emission intensity (Figure l),indicating the redistribution of D and A in the larger area of surfactant bilayer produced upon the merging of DODAB vesicles. In the absence of Nazso4, the A-D emission, in 1:l mixtures of labeled (by 0.8 mol 90 D and 0.8 mol 75 A) and non-labeled vesicles prepared from 4.0 X lo-* M DODAB, remained constant (within the experimental error) over the same period of time. Thus, the slow increase of D H following Na2S04 addition corresponds to fusion rather than to nonproductive adhesion. It should be emphasized that the time window of DODAB vesicle fusion is orders of magnitude longer than that customarily observed for vesicle and membrane fusions.'S2 Greater insight was obtained upon using DPH as a fluorescence probe sensitive to its microenvironment. Changes in the apparent relaxation time, T R values, of DPH in DODAB vesicles undergoing fusion at 20.0 O C also occur on along time scale (Figure 2). A possible mechanism of one cycle of vesicle fusion is illustrated in Figure 3. One can see, in the simplified overall picture, that the process starts with two small vesicles and ends with a large one. From start to finish, the curvature of the membrane and, accordingly, the average headgroup area of the amphiphile are decreased, while the packing is i n c r e a ~ e d . ~ The T R of DPH in membranes reflects the rate of rotational diffusion and the orientation order.1° Increasing the relative amount of amphiphiles in a given area hinders the motion of DPH and increases TR. The probability of quenching DPH emission by water molecules also decreases with tighter surfactant packing and, as a result, T F also increases. Similar considerations lead us to conclude that the observed (not shown) steady-state anisotropy, F, and residual anisotropy, rm,follow the same trend as T R , while the "average" angle of distribution, ( O ) , changes in the opposite direction. (8) Struck, D. K.; Hoekstra, D.; Pagano, E. Biochemistry 1981, 20, 4093. (9)Israelachvilli, J. M.; Marcelja, S.;Horn, R. G. Q.Reu. Biophys. 1980, 13, 2. (10)Kinosita, K.; Ikegami, A.; Kawato, S. Biophys. J. 1982,37, 461. Kinosita, K.; Kawato, S.; Ikegami, A. Biophys. J . 1977,20, 289. Lentz, B. R. Chem. Phys. Lipids 1989,50, 171.
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Figure 2. Apparent relaxation times ( T R values) of DPH in DODAB vesicles (XEXC = 300 nm and XEM = 450 nm) as a function of time after injection of NazS04 (the first point at time = 0 represents a T R value prior to the injection of Na2S04)at 20.0 "C. The fluorescence decay curves,Zll(t), were deconvolutedand fitted with a two-exponential function, ZI( t )- Ae-'lti + Be-t/r2,where T~ >. T Z . The values of T R and T F (huorescence lifetime) shown in Figure 4 were claculated as follows: T R = ( ~ 2 - l - ~ l - l ) - land T F = T ~ The . goodness ~ ~ of the fittings was within the range 1.0 < xu2 < 1.5. Determination of a data point look 3-6 min. All of the data points in this figure (as well as in Figure 4)have been passed through a smoothing process which did not change the trends.
Figure 3. A grossly oversimplified schematic diagram of one fusion cycle.
A closer inspection of the data in Figure 2 shows unexpectedly large fluctuations of the T R values of DPH, recorded subsequent to the addition of Na2S04 to DODAB vesicles. Expanding these fluctuations revealed previously unrecognized behavior. In the 19-26 and 45-50 h time windows, for example, T F , TR, F, F, and (8) underwent sets of systematic decreases and increases (see curves in Figure 4). It is important to note that these parameters were calculated by using several different and independent approaches. Such systematic fluctuations of the five experimental parameters were reproduced in several NazSO4-induced fusions of DODAB vesicles. In contrast, no systematic fluctuations were observed in two separate measurements of the fluorescence parameters of DPH in DODAB vesicles in the absence of added NazS04; 24 separate measurements of each sample over a 6-h period resulted in T F = 9.23 f 0.01 ns, in T R = 3.04 f 0.1 ns, and in random variations of successively determined T F and TR.
Combined experimental and theoretical analysis led to the prediction that spherical vesicles necessarily flatten
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Figure 4. Changes in the experimental parameters of DPH in DODAB vesicles in the 19-26 h and 45-50 h windows at 20.0 O C : (A) fluorescence lifetime (TF values) and apparent relaxation time (TB values); (B) steady-state anisotropy (F) calculated from F = [xmGZil(t)- C " Z l ( t ) ] / [ x " G Z , , ( t+ ) 2x;ZL(t)] (C = normalization factor) and residual anisotropy determined by plotting r ( t ) = [ C t l ( t ) - ZL(t)]/~CZll(t) + 2PL(t)]15and measuring the anisotropy value at the tail of r ( t ) ,according to r ( t ) = r , + (ro - r-)e-+R; (C) average angle of distribution, ( e ) , of the distribution of DPH in DODAB vesicular membranes calculated from r-/ro = 3 / 2 (cos2 0 ) - l/2.17 The anisotropy at time equal zero (ro) was determined to be 0.362 for DPH.lS
against each other (step iii in Figure 3) prior to adhesion which destabilizes the bilayer." Vesicle flattening is likely to be caused by the formation of a trans complex between the fusogenic agent (Sod2-,for example) and the headgroups of lipids in the two apposed bilayers.12 Flattening events decrease the curvature and dehydrate the headgroups of the adjacent bilayers13 with resultant increases in the "average" T F , TR, f , and rmvalues of DPH and corresponding decreases of ( e ) . Subsequent bilayer tension and deformation (steps iv-v in Figure 3) result in the destabilization of vesicles and the likely formation of lipidic intramembranous particles.'* Exposure of DPH to water molecules in steps iv-v (Figure 3) increases dramatically since the lipidic particles (reversed micelles and (11) Rand, R. P.; Parsegian, V. A. Annu. Rev. Physiol. 1986,48,201. (12) Rupert, L. A. M.; Engberts, J. B. F. M.; Hoekstra, D.J. Am. Chem. SOC.1986, 108, 3920-3925. (13) Wilshut, J.; Hoekstra, D. Trends Biochem. Sci. 1984, 9,479. (14) Verkleij, A. R. Biochim. Biophys. Acta 1984, 779,43.
related structures) formed represent equilibrium states which may give rise to fusion by the intermingling of the two aqueous compartments of both vesicles or, alternatively, which may re-form the antecedent structures. The overall consequence of lipidic structure formation is increased surfactant hydration and, hence, observable decreases in the "average" T F , TR, P, and r , values of DPH, but increases in ( e ) . Finally, merging of adjacent bilayers through an ellipsoid-like shape to enlarged spherical structures (steps vi-viii in Figure 3) changes the bilayer curvature and the relative amount of surfactant molecules per unit area in a way that, once again, increases the "average" TF, 7R, P, and r m values of DPH but decreases ( e ) . The whole process (steps i-viii in Figure 3) is then repeated until fusion terminates. Indeed, the overall increase of D H for DODAB vesicles from 80 to 740 nm indicates the occurrence of several complex fusion cycles. Individual data points (in Figures 1, 2, and 4) cannot be related, of course, to particular events depicted for one
626 Langmuir, Vol. 7, No. 4, 1991
fusion cycle in Figure 3. The observed decrease in the A-D emission intensity requires approximately 30 h (Figure 1) and corresponds to monitoring fusion events until very few vesicles remain unlabeled by A and D. Fusion continues further, of course (asindicated by the progressive increase in DHvalues), but cannot be seen by the decrease in the A-D emission intensity. Using the environmentally sensitive DPH fluorescence probes has allowed, however, monitoring of fusion on a longer time scale and the capture of subtle changes (Figure 4), which have indicated additional consecutive and concurrent events in the large ensemble of vesicles undergoing sulfate-ion-induced slow fusion (Figure 3). The long time scale of observable fusion is likely to be the consequence of the relatively low concentration of sulfate ions used and the high degree of stability in DODAB vesicles, which are present in their gel state under the experimental conditions used here. Fluctuations in microviscosity are not unique to vesicle fusion. In the neuroblastoma cell cycle, microviscosity is at a maximum in mitosis and decreases markedly in the
Letters G phase.15 Capturing subtle microviscosity changes which accompany vesicle fusion by carefully selected conditions and methods is the most significant result of the present communication. It provides an experimental approach for the in-depth examination of the mechanism of surfactant vesicle fusion. Acknowledgment. Support of this work by a grant from the National Science Foundation is gratefully acknowledged. David Yogev is the holder of a Dr. Chaim Weizmann Postdoctoral Fellowship for Scientific Research. We thank Mr. Michael Brandt for his competent technical assistance. (15) Delaat, S. W.; Van der Saag, P. T.; Shinitzky, M. Proc. Natl. Acad. Sei. U.S.A. 1977, 74, 4458. (16) Cross, A. J.; Fleming, G. R. Biophys. J . 1984, 46, 45. (17) Dale, R. E. In Time-Resolued Fluorescence Spectroscopy in Biochemistry and Biology; Cundall, R. B., Dale, R. E.,Eds.; Plenum Press: New York, 1983. (18)Shinitzky, M.; Barenholz, Y. J . B i d . Chem. 1974, 242, 2652.
