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Biochimica et Biophysica Acta, 4 6 5 ( 1 9 7 7 ) 6 6 1 - - 6 6 6 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA Report BBA 7 1 2 9 0

INTRACELLULAR DISTRIBUTION OF LIPOPHILIC FLUORESCENT PROBES IN MAMMALIAN CELLS

RICHARD

E. P A G A N O a ,

KEIKO

OZATOb

and J E A N - M A R I E

RUYSSCHAERT

c

Carnegie Institution of Washington, Department of Embryology, 115 West University Parkway, Baltimore, Md. 21210 (U.S.A.) (Received December 13th, 1976)

Summary The intracellular distribution of several hydrophobic fluorescent probes (1,6-diphenyl-l,3,5-hexatriene (DPH), perylene, and 2-p-toluidinyl-6-naphthalene sulfonate (TNS)) in mouse lymphocytes and a fibroblast cell line was examined using radiolabeled fluorescent probes and the technique of high resolution EM autoradiography. Following a short term incubation, DPH and perylene were found largely internalized in cells, while TNS was localized predominantly at the cell surface. These findings suggest that fluorescence polarization studies using such probes with intact cells do not necessarily monitor only the cell surface membrane and must be interpreted with caution.

The use of small lipophilic fluorescent molecules for probing the physical state of lipid membranes has been a subject of recent intensive investigation by membranologists. Such probes partition to varying degrees into the hydrophobic phase of the lipid bilayer and can be used to detect, by fluorescence, subtle changes in the molecular dynamics and physical state of the a To w h o m correspondenceshould be addressed. b P r e s e n t Address: G o o d S a m a r i t a n H o s p i t a l , O'Neill Memorial Laboratory, Baltimore, Md. 21239 (U.S.A.). c P e r m a n e n t Address: L a b o r a t o i r e de Chimie Physique des M a c r o m o l ~ c u l e s a u x I n t e r f a c e s , Campus Plaine, Universit~ Libre de Bruxelles, Bruxelles, 1000 (Belgium). A b b r e v i a t i o n s : DPH, 1,6-diphenyl-l,3,5-hexatriene; TNS, 2-p-toluidinyl-6-naphthalene s u l f o n a t e .

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lipid molecules in the vicinity of the probe. This approach has been particularly fruitful using model lipid dispersions or liposome systems [1--8]. Recently these probes have also been used with intact cells, including erythrocytes, cultured fibroblasts, lymphocytes, and plant cells [7, 9--16]. It is frequently assumed in such studies that the fluorescent molecules do not penetrate the intact cell, b u t are localized at the cell surface, and thus report on the "fluidity" or "microviscosity" of the plasma membrane lipid bilayer. Since a partitioning of such probes into internal compartments of the cell would greatly complicate the interpretation of these fluorescence measurements, it is essential that this assumption be rigorously tested. In the present study we examine the location of several lipophilic fluorescent probes in whole cells using EM autoradiographic methods. 1,6-Diphenyl-l,3,5-hexatriene (DPH), perylene, and 2-p-toluidinyl6-naphthalene sulfonate (TNS) were obtained from commercial sources, and radiolabeled by exposure to pure tritium gas in an ionizing electric field [17]. The tritiated samples were purified by crystallization (4 times) from the appropriate organic solvents and had specific activities of approximately 50 #Ci/mg (DPH), 600 ~Ci/mg (perylene) and 100 uCi/mg (TNS). Fluorescence spectra of the radiolabeled and unlabeled probe species were indistinguishable. Mouse t h y m o c y t e s were prepared using 4--8-week-old CBA male mice (Jackson Laboratory, Bar Harbor, Maine) as described [18] ; a b o u t 1% of the cells in the isolated t h y m o c y t e population were erythrocytes. The latter were also analyzed and served as an internal control (see below). Chinese hamster V79 fibroblasts were maintained in culture [19,20] and dissociated into single cell suspensions using a Ca2+and Mg 2+ free saline containing 1 mM EDTA. One volume of cell suspension ( 0 . 5 . 1 0 7 2.107 cells/ml balanced salt solution) was added to one volume of a 2 . 1 0 -6 M solution of 3H-labeled fluorescent probe in 0.15 M KCI, and the mixture incubated at 25°C for 30 min. These incubation conditions did n o t impair the viability of the treated cells as assessed by trypan blue staining. The labeled cells were then washed twice in a balanced salt solution, fixed in glutaraldehyde, postfixed in OsO4, and processed for EM autoradiography using Ilford L-4 emulsion as previously described [20,21]. In separate grids containing no sectioned material, background grains were random and less than 5 grains per grid square (200 mesh). Fig. l a shows a typical autoradiogram obtained for mouse t h y m o c y t e s treated with [3HI DPH. A large fraction of the silver grains appear to be localized in both cytoplasmic and nuclear regions of the cell. A similar grain distribution was seen using [3H] perylene. In contrast, t h y m o c y t e s treated with [3HI TNS (Fig. l b ) showed heavy labeling of the cell surface with relatively little internalized probe. These observations were quantitated as follows. Micrographs of t h y m o c y t e s treated with the radioactive fluorescent probes were taken at 14 000 ×, and the outline of individual cells and location of grains traced on paper, as indicated in Fig. lc. Each traced cell was then divided into t w o compartments and the number of silver grains in each c o m p a r t m e n t determined. A cell surface c o m p a r t m e n t was defined by a zone 0.3 pm wide and centered over the cell surface membrane contour. The remaining portion of the cell defined the internal compartment. Grain

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densities were obtained by dividing the number of grains in each compartm e n t by the area of t h a t compartment. The ratio of surface grain density to interior grain density was then calculated, and the average + S.E.M. calculated. The results of such measurements are summarized in Table I. It is readily seen, both from the percentage of silver grains found in the surface c o m p a r t m e n t , as well as the ratio of surface to internal c o m p a r t m e n t grain densities, that DPH and perylene are largely internalized in the treated cells, while TNS is confined mostly to the cell surface. Although our quantitative analysis of the intracellular distribution of fluorescent probes is limited to mouse t h y m o c y t e s , qualitatively similar distributions were seen using Chinese hamster V79 fibroblasts. A typical EM autoradiogram of a V79 cell treated with [3H] DPH is shown in Fig. 2.

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TABLE I DISTRIBUTION OF AUTORADIOGRAPHIC GRAINS OVER MOUSE THYMOCYTES TREATED WITH 3H-LABELED FLUORESCENT PROBES Probe

Total Grains Counted

Grains in Surface Compartment (%)

(Surface Grai n Density)/ (Interior Grain D e n s i t y ) ± S.E.M.

[3H]TNS [3H] DPH [3H] Perylene

302 990 918

69 16 27

11.4 ± 1.3 2 . 0 +- 0 . 3 2.4 ± 0.1

Fig. 2, E l e c t r o n m i c r o s c o p e a u t o r a d i o g r a m o f a Chinese h a m s t e r V 7 9 fibroblast i n c u b a t e d w i t h [SH] D P H . Bar is 5 Din.

As for t h y m o c y t e s , most of the radiolabeled probe was found internalized in the cell. Analysis of autoradiograms obtained for mouse erythrocytes treated with tritium labeled DPH, perylene, or TNS demonstrated t h a t the radioactive probes were symmetrically distributed about the red cell membrane, with approximately 50% of the silver grains found within about +- 0.15 ~m of the membrane surface (Fig. 3). This distribution demonstrates that, within the resolution of the EM autoradiographic technique (~ 0.15 ~m; ref. 22), the radiolabeled fluorescent probes are localized in the erythrocyte surface membrane. Furthermore, these data argue against a significant nonrandom redistribution of the probes during fixation and embedding for EM, since this would likely result in a skewed distribution of silver grains, as well as a high background outside the cell. Our autoradiographic findings show t h a t the more hydrophobic probes, DPH and perylene, when incubated with intact l y m p h o c y t e s or fibroblasts, partition not only into the cell surface membrane but also into cytoplasmic and nuclear regions of the cell. Presumably this partitioning

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Fig. 3. D i s t r i b u t i o n o f silver grains in autoradiograrns o f m o u s e e r y t h r o c y t e s t r e a t e d with [3HI DPH, N u m b e r o f silver grains is p l o t t e d vs. t h e i r d i s t a n c e f r o m t h e p l a s m a m e m b r a n e (+, o u t s i d e ; --, inside). Q u a n t i t a t i v e l y similar d i s t r i b u t i o n s w e r e o b t a i n e d using [3HI T N S and [ 3HI p e r y l e n e .

represents a rapid equilibration of the probes between the cell surface and hydrophobic elements within the cell, e.g. intracellular membranes. Such an equilibration is readily observed with DPH when t w o populations of artificial membranes are mixed with one another [ 6 ] . In contrast, TNS was found to be localized predominantly at the cell surface. This finding is consistent with the studies of model lipid dispersions using TNS, which suggest that this probe resides at the interface between the lipid polar head groups and the aqueous phase [ 2 3 ] . Our findings raise serious questions about the validity of using probes such as DPH with intact cells to estimate the "microviscosity" or "fluidity" of the cell surface membrane. Since large quantities of the fluorescent probe are internalized by treated cells, it seems quite likely that fluorescence polarization measurements represent some complicated average of fluorescence signals from various regions of the cell rather than from the cell surface alone. Thus, a rigorous interpretation of such data must await a determination of the relative amounts (and degrees of quenching) of a given probe in each of these regions. We gratefully acknowledge the expert technical assistance of Mr. W. Duncan and thank Drs. A. Sandra and B.J. Litman for critically reading this manuscript. This work was supported by National Institutes of Health Research Grant GM 2 2 9 4 2 to R.E.P., and by NATO Research Grant No. 771. References 1 Shinitzky, M., Dianoux, A.C., Gitler, C. and Weber, G. (1971) Biochemistry I 0 , 2 1 0 6 - - 2 1 1 3 2 Shinitzky, M. and B a r e n h o l z , Y. (1974) J. Biol. C h e m . 249, 2 6 5 2 - - 2 6 5 7 3 Papahadjopoulos, D., Jacobson, K., Poste, G. and S h e p h e r d , G. (1975) B i o c h i m . B i o p h y s . A c t a 394, 504--519 4 Andrich, M.P. and V a n d e r k o o i , J.M. (1976) Biochemistry 15, 1257--1261

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