- Email: [email protected]
Carbocyanine dyes stain the sarcoplasmic reticulum of beating heart cells
5 14
Preliminary
notes
and 28% from the unifilarly substituted DNAs. The autoradiographs of second metaphases did not in most cases show this differential loss of label in all chromosomes (fig. la). Some chromosomes could be found in the cell where the loss of label from the pale chromatid was complete (fig. 1 b), while often in the same cell chromosomes can be found where the label was concentrated over the outer edges of the pale chromatid (fig. 1 c) suggesting that it is in the process of being leached from the chromosome. These effects are therefore likely to give an underestimate of the loss of label from the pale chromatids. These data therefore indicate that differential harlequin staining is a result of UV photolysis and differential loss of BUdRsubstituted DNA and that this represents the essential mechanism of the FFG staining technique in this material. BUdR-substituted DNA in chromatin has also been shown to be eluted at a higher pH than unsubstituted DNA [5] and also to denature at a higher temperature at pH 6.8 than unsubstituted DNA [6]. These factors may contribute to a differential loss of either substituted or unsubstituted DNA in other methods of harlequin or reverse harlequin staining, where variations in pH and temperature are used without necessarily involving exposure to UV and photolysis of BUdR-containing DNA as the major cause.
6. David, J, Gordon, J S & Ritter, W J, Proc natl acad sci 71 (1974) 2808. Received September 18, 1979 Revised version received November 5, 1979 Accepted November 8, 1979 Printed in Sweden Copyright @ 1980 by Academic Press, Inc. All rights of reproduction in any form reserved oOl4-4827/80/0205l4-05~2.00/0
Carbocyanine dyes stain the sarcoplasmic reticulum of beating heart cells J. HABICHT
and K. BRUNE, Department
macology, Biozentrum, Base/, Switzerland
University
of Basel,
of PharCH4056
Summary. In search of a fluorescent dye suitable for monitoring membrane potentials of beating heart cells, we noticed that the carbocvanine dves. CC, and CC,, show a unique pattern of httracellular distribution in vital and glutaraldehyde-fixed cardiomyoblasts. This distribution is clearly different from that observed in fibroblasts. In heart cells, it parallels the localization of actin-myosin containing my&laments as visualized by fluorescent antibody staining but it does not correspond to the localization of actin filaments or the microtubules. In tibroblasts these dyes stain only fine filaments and granules in the perinuclear space which correspond to the endoplasmic reticulum. This observation is evidence in support of the hypothesis that carbocyanine dyes accumulate selectively in the sarcoplasmic reticulum. It indicates that certain carbocyanine dyes may be useful tools to differentiate between muscle cells and connective tissue cells in cell cultures.
Recently, cultures of beating heart cells in culture have been employed for pharmacological experiments, e.g. for the assessment of anti-at-rhythmic activity [l]. Such cell cultures were derived from embryonic hearts and consequently contained heart cells at various stages of differentiation as The author is grateful to Mrs K. A. Hewitt for tech- well as connective tissue cells. Experimennical assistance. This work was supported by grants from the Medical Research Council, the Cancer Re- tation with such cultures has been found to search Campaign and the Leukaemia Research Fund. be unsatisfactory in two respects. In the first place, reliable monitoring of changes References in the membrane potential of beating heart 1. Burkholder, G D, EXD cell res 121 (1979) 209. cells can only be achieved by invasive 2. Regan, J D, Setlow, RB & Ley, R D, Proc natl acad methods, i.e. the insertion of microelecsci 68 (1971) 708. 3. Goto, K, Akematsu, T, Shimazu, H & Sugiyama, trodes which may considerably change the T, Chromosoma 53 (1975) 223. performance of the cell under investigation. 4. Perry, P&Wolff, S, Nature 251 (1974) 156. 5. Lapeyre, J N & Bekhor, I, J mol biol89 (1974) 137. Secondly, there is no defined structural Exp CdRes
125(1980)
Preliminary marker which allows for discrimination of heart cells from connective tissue cells by vital microscopy. We have now employed fluorescent cyanine dyes to achieve both, non-invasive measurement of membrane potentials of heart cells and clear discrimination of heart muscle cells from connective tissue cells. Cyanine dyes have recently been used as indicators of the membrane potential of cells, cell organelles and microorganisms in suspension (for review see [2]). In addition, there is biophysical and biochemical evidence that certain fluorescent dyes accumulate selectively in the T system and the sarcoplasmic reticulum of muscle cells [3,4,5]. In this paper we report morphological evidence that some carbocyanine dyes accumulate selectively in the sarcoplasmic reticulum of heart cells, thus allowing identification of heart cells in culture by vital staining. Materials
and Methods
Single beating heart cells for vital staining were obtained and cultured essentially according to the method of Goshima [6]. In brief: hearts of approximately twelve 15-16 day-old Sprague-Dawley rat embryos were isolated and placed in a Petri dish containing icecold incomplete (Ca-Mg-free) HBSS (Hank’s balanced salt solution). Excess tissue was trimmed off and the whole hearts were washed twice in cold HBSS. Digestion in three steps with warmed trypsin (5 ml 0.06%, containing EDTA and Phenol Red) was performed at 3PC in a 25 ml Erlenmeyer flask under agitation in a gyratory water bath. The first supematant obtained after 4 min of digestion was discarded since it contained mainly blood cells, non-viable cells and cell debris. The second and third fraction was obtained after 6 min of digestion followed by gentle dissociation of the tissue by repeated suction through a glass pipette. To each fraction 8 ml of fetal calf serum (FCS) supplemented DMEM (Dulbecco’s modified Eagle’s medium) was added to stop trypsin digestion and the resulting cell suspensions were filtered through gauze. Pellets obtained after centrifugation (7 min at 200 g) were resuspended in 2.0-2.5 ml of DMEM containing 1.Og/l sodium bicarbonate, 1% penicillin-streptomycin and 10% FCS. Cells (approx. 10s) were then seeded onto the round glass coverslios with which the uolvstyrene chambers were Iitted {as described by C&kshank et al. [7]). After incubation at 37”C/5% CO, for 24 h the cultures were washed and reincubated with fresh DMEM (10% FCS or horse serum). Vital staining with cyanine dyes and photography was usually performed when the cultures were 2 days old.
notes
5 15
Heart cells for immunofluorescence were prepared as for vital staining. The final cell suspension was distributed onto 10x20 mm glass coverslips placed in plastic Petri dishes. Fixation took place after 2 days of culture and was performed according to procedure 2 as described by Weber et al. [Sl. 3T3-Fibroblasts from our own stock were obtained-by detachment from a Petri dish with trypsin and 2~10~ cells were seeded into chambers as described by Cruickshank [l] and cultured in FCS-supplemented DMEM at 3PC and 5 % CO, for 2 days prior to staining and photography. Some cultures of heart cells and 3T3-fibroblasts on glass coverslips were fixed with glutaraldehyde (1% in phosphate buffered saline, vide infra) for 1 h before stainina, others were fixed and graduallv extracted with a&tone/PBS (25, 50, 75% f& 1 h each, 100% acetone for 4 h and in reverse 75.50.25 % fort h each) in an attempt to remove membrane lipids fromthe cells before staining (CCC, 1O-BM in PBS) and photography. All media and sera were purchased from North American Biologicals Inc. (Nabi). Tissue culture materials were obtained from Div. Becton Dickinson & Co. (Falcon) and Greiner Labortechnik fur Medizin und Forschung (Cruickshank-Chambers). The composition of Ca-Mg-free phosphate buffered saline (PBS) used in the cell fixation procedure for immunofluorescence was as follows (in g/l): NaCl, 8.0; KCl, 0.2; Na2HP0,* 7 H,O, 2.16; KH*IQ, 0.2. The dyes, 3,3’-dipentyloxacarbocyanine and 3,3’-dihexyloxacarbocyanine (hereafter termed CC, and CC,) were synthesized according to the procedure of Cohen et al. [9]. They were stored at 4°C as stock solutions in ethanol (1.5 mglml). Monovalent anti-chicken gizzardactin and anti-chicken oectoral muscle-mvosin. as well as FITC-labelled swine anti-rabbit-I-GG ‘(Sevac, Prague) were gifts from Professor R. M. Franklin, Biocenter, Basel. Anti-tubulin was prepared in our laboratory bv H. Kalin accordinr to the method of Fuller et al. [io]. Photography was performed on vital cells immediately after staining with 1O-6 M CC, or CC, in DMEM with standard equipment for fluorescence micrography (Leitz). The Ilford HP 5 high speed blackand-white film used was developed with Microphen (Ilford) as a 1600 ASA film consequently reducing the exposure times to 10-30 set for vital staining and 30-60 set for immunofluorescence. Phase contrast photography was done in parallel.
Results When heart cell cultures were exposed to the two carbocyanine dyes (CC, and CC,) two types of cells could be observed by direct fluorescence microscopy immediately after the addition of the dyes. One cell type (fig. 1) consisted of multiangular thick cells containing bundles of brightly fluorescent vesicles and tubules which extended straight across the cells from one Exp Cell
Res 125 (1980)
5 16
Preliminary
notes
Fig.
I. Cardiomyoblasts from embryonic rats were cultured for 2 days and then stained vitally with CC, or CC& ((b) beating heart cell, CC,; (d) quiescent heart cell, CC,) or fixed with glutaraldehyde followed by
either acetone extraction and staining with a cyanine dye (f) or fluorescent double antibody staining ((a) anti-tubulin; (c) anti-actin; (e) anti-myosin). Details in the text. Bar, 30 pm.
outer angle to another. Some of these cells were beating (fig. lb), others were resting (fig. 1 d) while under microscopic examination. In the beating cells the fluorescent structures were aligned in parallel to the
contraction vector. The other cell type consisted of large, thin cells (fig. 2a, c) showing only faint fluorescent staining of a perinuclear network of small granules and thin lamellae. None of these cells were observed
ExpC~llRrs
125(1980}
Preliminary
notes
5 17
Fig. 2. Fibroblast-like cells as found in primary heart cell cultures (LI, c) show a staining pattern similar to 3T3-mouse fibroblasts (h) when stained vitally with
CC, or CC6 (a, b) or fixed with glutaraldehyde and extracted with acetone before staining (c). Bar, 30 pm.
to beat. In light microscopic appearence and CC&C, fluorescence they resemble (fig. 2b) mouse fibroblasts (3T3 cells). The specific distribution of fluorescence remained almost unchanged in both cell types when the dyes were added after fixation of the cells with glutaraldehyde or even after fixation and subsequent delipidation by acetone extraction (fig. If and 2~). The localisation and appearance of structures stained by CC, and CC, were also compared with the structures made visible by indirect immunofluorescent staining of microtubules (fig. 1a), actin-containing filaments (fig. 1c) and myosin-containing filaments (fig. le). We found that the distribution and appearence of the tubulin-containing structures in heart cells and fibroblasts, as well as the actin filaments in fibroblasts (not shown) were clearly different from the distribution of CC,/CC, fluorescence in these two cell types. In contrast, the contractile actin-myosin bundles (myofibrils) of heart cells made visible by either anti-actin (fig. Ic) or anti-myosin (fig. le)
run parallel to the structures stained by CCJCC,. However, the periodicity and the high density of the actin-myosin bundles is not paralleled by similar morphological details in the CC&X,-stained heart cells.
3J-7YIX17
Discussion From our observations two major questions arise. The first concerns the identification of the intracellular structures which are stained selectively with the carbocyanines. Our results show that these structures are neither the microtubules nor the microfilaments since both structures show different arrangements in fibroblasts and heart cells. As there is a close similarity between the appearence and localization of the contractile myofilament system and the tubularvesicular bundles staining with CC, and CC, dyes, it could be argued that these dyes attach directly to actin-myosin bundles. However, this interpretation would not explain why cyanine staining of perinuclear granules is found in fibroblasts. Also, the fine structure of the actin-myosin fibrils is
518
Preliminary
notes
different, showing e.g. Z-lines. Moreover, it appears as if the space covered by these bundles is much larger than the space occupied by the structures stained by cyanine dyes. Since recent immunofluorescence studies of the membrane components of the ER and mitochondria [ 11, 121 show great resemblance to our vital staining, we cannot exclude the mitochondria as a locus of carbocyanine dye accumulation. The arrangement and density of the structures stained by cyanine dyes makes the endoplasmic reticulum and especially the sarcoplasmic reticulum (a specialized descendent of the ER, [ 13, 141) a more likely candidate for cyanine staining. Indeed, quantitative and qualitative differences between dye distribution in heart muscle cells and fibroblasts correlate well with difference in size and distribution of smooth endoplasmic and sarcoplasmic reticulum in these cell types. This interpretation is supported by evidence obtained from biophysical and biochemical methods [3, 4, 51. The other obvious question is why these cyanine dyes accumulate in structures which are probably the sarcoplasmic (endoplasmic) reticulum. Several explanations should be considered. One could (a) postulate concentration of these dyes in the sarcoplasmic reticulum by a pump system. Although this contention would be very attractive, the fact that the same general pattern of distribution occurs even after glutaraldehyde fixation and delipidation argues against it. The same argument can also be applied against the idea that these basic, cationic dyes are trapped as ions along an electrogenic (6), or a pH gradient (c). It is also possible to surmise that these relatively lipophilic dyes are concentrated in the sarcoplasmic (endoplasmic) reticulum due to the abundance of lipoid structures in this compartment (d). Again the almost un-
changed pattern of accumulation of CC, and CC, after acetone extraction is not compatible with this explanation. What remains is the interpretation that these carbocyanine dyes bind to sites on structures which are especially rich in the sarco- (endo-)plasmic reticulum and which are functionally not impaired by glutaraldehyde fixation and acetone extraction, The chemical nature of these structures has yet to be elucidated. For the present these observations may be useful in cell biology to discriminate different types of cells and to estimate the extent to which the sarcoplasmic reticulum is developed in cultured cardiomyoblasts by a simple vital staining method. This work was supported by the Swiss National Science Foundation (Grant No. 3.588-0.775 and a Scholarship to J. H.). The optical equipment was a donation of the Freiwillige Akademische Gesellschaft, Base].
References 1. Goshima, K, J mol cellular cardiology 8 (1976) 217. 2. Waaeoner, A S. J membrane bio127 (1976) 317. 3. Getrker, H, Baylor, S M & Chandler, W K, Nature 257 (1975) 693. 4. Easton, T G, Valinsky, J E & Reich, E, Cell 13 (1978) 475. 5. Russ&, T J, Beeler, T & Martonosi, A, J biol them 254 (1979) 2040. 6. Goshima, K, Exp cell res 92 (1975) 339. 7. Cruickshank, C N D, Cooper, J R & Conran, M B, Exp cell res 16 (1959) 695. 8. Weber, K, Rathke, P C & Osborn, M, Proc natl acad sci US 75 (1978) 1820. 9. Cohen, L B, Salzberg, B M, Davila, H V, Ross, W N, Landowne, D, Waggoner, A S &Wang, C H, J membrane biol 19 (1974) 1. 10. Fuller, G M, Brinkley, B R & Boughter, J M, Science 187 (1975) 948. 11. Franke, W W, Fink, A & Schmid, E, Cell biol intern reports 2 (1978) 465. 12. Krohne, G, Franke, W W, Ely, S, D’Arcy, A & Jost, E, Cytobiologie 18 (1978) 22. 13. Bloom, W & Fawcett, D W, A textbook of histology, 10th edn, p. 303. W B Saunders Co. Philadelphia, London, Toronto (1975). 14. Ezerman, E B & Ishikawa, H, J cell bio135 (1967) 405. Received September 25, 1979 Accepted October 1, 1979
Preliminary
notes
and 28% from the unifilarly substituted DNAs. The autoradiographs of second metaphases did not in most cases show this differential loss of label in all chromosomes (fig. la). Some chromosomes could be found in the cell where the loss of label from the pale chromatid was complete (fig. 1 b), while often in the same cell chromosomes can be found where the label was concentrated over the outer edges of the pale chromatid (fig. 1 c) suggesting that it is in the process of being leached from the chromosome. These effects are therefore likely to give an underestimate of the loss of label from the pale chromatids. These data therefore indicate that differential harlequin staining is a result of UV photolysis and differential loss of BUdRsubstituted DNA and that this represents the essential mechanism of the FFG staining technique in this material. BUdR-substituted DNA in chromatin has also been shown to be eluted at a higher pH than unsubstituted DNA [5] and also to denature at a higher temperature at pH 6.8 than unsubstituted DNA [6]. These factors may contribute to a differential loss of either substituted or unsubstituted DNA in other methods of harlequin or reverse harlequin staining, where variations in pH and temperature are used without necessarily involving exposure to UV and photolysis of BUdR-containing DNA as the major cause.
6. David, J, Gordon, J S & Ritter, W J, Proc natl acad sci 71 (1974) 2808. Received September 18, 1979 Revised version received November 5, 1979 Accepted November 8, 1979 Printed in Sweden Copyright @ 1980 by Academic Press, Inc. All rights of reproduction in any form reserved oOl4-4827/80/0205l4-05~2.00/0
Carbocyanine dyes stain the sarcoplasmic reticulum of beating heart cells J. HABICHT
and K. BRUNE, Department
macology, Biozentrum, Base/, Switzerland
University
of Basel,
of PharCH4056
Summary. In search of a fluorescent dye suitable for monitoring membrane potentials of beating heart cells, we noticed that the carbocvanine dves. CC, and CC,, show a unique pattern of httracellular distribution in vital and glutaraldehyde-fixed cardiomyoblasts. This distribution is clearly different from that observed in fibroblasts. In heart cells, it parallels the localization of actin-myosin containing my&laments as visualized by fluorescent antibody staining but it does not correspond to the localization of actin filaments or the microtubules. In tibroblasts these dyes stain only fine filaments and granules in the perinuclear space which correspond to the endoplasmic reticulum. This observation is evidence in support of the hypothesis that carbocyanine dyes accumulate selectively in the sarcoplasmic reticulum. It indicates that certain carbocyanine dyes may be useful tools to differentiate between muscle cells and connective tissue cells in cell cultures.
Recently, cultures of beating heart cells in culture have been employed for pharmacological experiments, e.g. for the assessment of anti-at-rhythmic activity [l]. Such cell cultures were derived from embryonic hearts and consequently contained heart cells at various stages of differentiation as The author is grateful to Mrs K. A. Hewitt for tech- well as connective tissue cells. Experimennical assistance. This work was supported by grants from the Medical Research Council, the Cancer Re- tation with such cultures has been found to search Campaign and the Leukaemia Research Fund. be unsatisfactory in two respects. In the first place, reliable monitoring of changes References in the membrane potential of beating heart 1. Burkholder, G D, EXD cell res 121 (1979) 209. cells can only be achieved by invasive 2. Regan, J D, Setlow, RB & Ley, R D, Proc natl acad methods, i.e. the insertion of microelecsci 68 (1971) 708. 3. Goto, K, Akematsu, T, Shimazu, H & Sugiyama, trodes which may considerably change the T, Chromosoma 53 (1975) 223. performance of the cell under investigation. 4. Perry, P&Wolff, S, Nature 251 (1974) 156. 5. Lapeyre, J N & Bekhor, I, J mol biol89 (1974) 137. Secondly, there is no defined structural Exp CdRes
125(1980)
Preliminary marker which allows for discrimination of heart cells from connective tissue cells by vital microscopy. We have now employed fluorescent cyanine dyes to achieve both, non-invasive measurement of membrane potentials of heart cells and clear discrimination of heart muscle cells from connective tissue cells. Cyanine dyes have recently been used as indicators of the membrane potential of cells, cell organelles and microorganisms in suspension (for review see [2]). In addition, there is biophysical and biochemical evidence that certain fluorescent dyes accumulate selectively in the T system and the sarcoplasmic reticulum of muscle cells [3,4,5]. In this paper we report morphological evidence that some carbocyanine dyes accumulate selectively in the sarcoplasmic reticulum of heart cells, thus allowing identification of heart cells in culture by vital staining. Materials
and Methods
Single beating heart cells for vital staining were obtained and cultured essentially according to the method of Goshima [6]. In brief: hearts of approximately twelve 15-16 day-old Sprague-Dawley rat embryos were isolated and placed in a Petri dish containing icecold incomplete (Ca-Mg-free) HBSS (Hank’s balanced salt solution). Excess tissue was trimmed off and the whole hearts were washed twice in cold HBSS. Digestion in three steps with warmed trypsin (5 ml 0.06%, containing EDTA and Phenol Red) was performed at 3PC in a 25 ml Erlenmeyer flask under agitation in a gyratory water bath. The first supematant obtained after 4 min of digestion was discarded since it contained mainly blood cells, non-viable cells and cell debris. The second and third fraction was obtained after 6 min of digestion followed by gentle dissociation of the tissue by repeated suction through a glass pipette. To each fraction 8 ml of fetal calf serum (FCS) supplemented DMEM (Dulbecco’s modified Eagle’s medium) was added to stop trypsin digestion and the resulting cell suspensions were filtered through gauze. Pellets obtained after centrifugation (7 min at 200 g) were resuspended in 2.0-2.5 ml of DMEM containing 1.Og/l sodium bicarbonate, 1% penicillin-streptomycin and 10% FCS. Cells (approx. 10s) were then seeded onto the round glass coverslios with which the uolvstyrene chambers were Iitted {as described by C&kshank et al. [7]). After incubation at 37”C/5% CO, for 24 h the cultures were washed and reincubated with fresh DMEM (10% FCS or horse serum). Vital staining with cyanine dyes and photography was usually performed when the cultures were 2 days old.
notes
5 15
Heart cells for immunofluorescence were prepared as for vital staining. The final cell suspension was distributed onto 10x20 mm glass coverslips placed in plastic Petri dishes. Fixation took place after 2 days of culture and was performed according to procedure 2 as described by Weber et al. [Sl. 3T3-Fibroblasts from our own stock were obtained-by detachment from a Petri dish with trypsin and 2~10~ cells were seeded into chambers as described by Cruickshank [l] and cultured in FCS-supplemented DMEM at 3PC and 5 % CO, for 2 days prior to staining and photography. Some cultures of heart cells and 3T3-fibroblasts on glass coverslips were fixed with glutaraldehyde (1% in phosphate buffered saline, vide infra) for 1 h before stainina, others were fixed and graduallv extracted with a&tone/PBS (25, 50, 75% f& 1 h each, 100% acetone for 4 h and in reverse 75.50.25 % fort h each) in an attempt to remove membrane lipids fromthe cells before staining (CCC, 1O-BM in PBS) and photography. All media and sera were purchased from North American Biologicals Inc. (Nabi). Tissue culture materials were obtained from Div. Becton Dickinson & Co. (Falcon) and Greiner Labortechnik fur Medizin und Forschung (Cruickshank-Chambers). The composition of Ca-Mg-free phosphate buffered saline (PBS) used in the cell fixation procedure for immunofluorescence was as follows (in g/l): NaCl, 8.0; KCl, 0.2; Na2HP0,* 7 H,O, 2.16; KH*IQ, 0.2. The dyes, 3,3’-dipentyloxacarbocyanine and 3,3’-dihexyloxacarbocyanine (hereafter termed CC, and CC,) were synthesized according to the procedure of Cohen et al. [9]. They were stored at 4°C as stock solutions in ethanol (1.5 mglml). Monovalent anti-chicken gizzardactin and anti-chicken oectoral muscle-mvosin. as well as FITC-labelled swine anti-rabbit-I-GG ‘(Sevac, Prague) were gifts from Professor R. M. Franklin, Biocenter, Basel. Anti-tubulin was prepared in our laboratory bv H. Kalin accordinr to the method of Fuller et al. [io]. Photography was performed on vital cells immediately after staining with 1O-6 M CC, or CC, in DMEM with standard equipment for fluorescence micrography (Leitz). The Ilford HP 5 high speed blackand-white film used was developed with Microphen (Ilford) as a 1600 ASA film consequently reducing the exposure times to 10-30 set for vital staining and 30-60 set for immunofluorescence. Phase contrast photography was done in parallel.
Results When heart cell cultures were exposed to the two carbocyanine dyes (CC, and CC,) two types of cells could be observed by direct fluorescence microscopy immediately after the addition of the dyes. One cell type (fig. 1) consisted of multiangular thick cells containing bundles of brightly fluorescent vesicles and tubules which extended straight across the cells from one Exp Cell
Res 125 (1980)
5 16
Preliminary
notes
Fig.
I. Cardiomyoblasts from embryonic rats were cultured for 2 days and then stained vitally with CC, or CC& ((b) beating heart cell, CC,; (d) quiescent heart cell, CC,) or fixed with glutaraldehyde followed by
either acetone extraction and staining with a cyanine dye (f) or fluorescent double antibody staining ((a) anti-tubulin; (c) anti-actin; (e) anti-myosin). Details in the text. Bar, 30 pm.
outer angle to another. Some of these cells were beating (fig. lb), others were resting (fig. 1 d) while under microscopic examination. In the beating cells the fluorescent structures were aligned in parallel to the
contraction vector. The other cell type consisted of large, thin cells (fig. 2a, c) showing only faint fluorescent staining of a perinuclear network of small granules and thin lamellae. None of these cells were observed
ExpC~llRrs
125(1980}
Preliminary
notes
5 17
Fig. 2. Fibroblast-like cells as found in primary heart cell cultures (LI, c) show a staining pattern similar to 3T3-mouse fibroblasts (h) when stained vitally with
CC, or CC6 (a, b) or fixed with glutaraldehyde and extracted with acetone before staining (c). Bar, 30 pm.
to beat. In light microscopic appearence and CC&C, fluorescence they resemble (fig. 2b) mouse fibroblasts (3T3 cells). The specific distribution of fluorescence remained almost unchanged in both cell types when the dyes were added after fixation of the cells with glutaraldehyde or even after fixation and subsequent delipidation by acetone extraction (fig. If and 2~). The localisation and appearance of structures stained by CC, and CC, were also compared with the structures made visible by indirect immunofluorescent staining of microtubules (fig. 1a), actin-containing filaments (fig. 1c) and myosin-containing filaments (fig. le). We found that the distribution and appearence of the tubulin-containing structures in heart cells and fibroblasts, as well as the actin filaments in fibroblasts (not shown) were clearly different from the distribution of CC,/CC, fluorescence in these two cell types. In contrast, the contractile actin-myosin bundles (myofibrils) of heart cells made visible by either anti-actin (fig. Ic) or anti-myosin (fig. le)
run parallel to the structures stained by CCJCC,. However, the periodicity and the high density of the actin-myosin bundles is not paralleled by similar morphological details in the CC&X,-stained heart cells.
3J-7YIX17
Discussion From our observations two major questions arise. The first concerns the identification of the intracellular structures which are stained selectively with the carbocyanines. Our results show that these structures are neither the microtubules nor the microfilaments since both structures show different arrangements in fibroblasts and heart cells. As there is a close similarity between the appearence and localization of the contractile myofilament system and the tubularvesicular bundles staining with CC, and CC, dyes, it could be argued that these dyes attach directly to actin-myosin bundles. However, this interpretation would not explain why cyanine staining of perinuclear granules is found in fibroblasts. Also, the fine structure of the actin-myosin fibrils is
518
Preliminary
notes
different, showing e.g. Z-lines. Moreover, it appears as if the space covered by these bundles is much larger than the space occupied by the structures stained by cyanine dyes. Since recent immunofluorescence studies of the membrane components of the ER and mitochondria [ 11, 121 show great resemblance to our vital staining, we cannot exclude the mitochondria as a locus of carbocyanine dye accumulation. The arrangement and density of the structures stained by cyanine dyes makes the endoplasmic reticulum and especially the sarcoplasmic reticulum (a specialized descendent of the ER, [ 13, 141) a more likely candidate for cyanine staining. Indeed, quantitative and qualitative differences between dye distribution in heart muscle cells and fibroblasts correlate well with difference in size and distribution of smooth endoplasmic and sarcoplasmic reticulum in these cell types. This interpretation is supported by evidence obtained from biophysical and biochemical methods [3, 4, 51. The other obvious question is why these cyanine dyes accumulate in structures which are probably the sarcoplasmic (endoplasmic) reticulum. Several explanations should be considered. One could (a) postulate concentration of these dyes in the sarcoplasmic reticulum by a pump system. Although this contention would be very attractive, the fact that the same general pattern of distribution occurs even after glutaraldehyde fixation and delipidation argues against it. The same argument can also be applied against the idea that these basic, cationic dyes are trapped as ions along an electrogenic (6), or a pH gradient (c). It is also possible to surmise that these relatively lipophilic dyes are concentrated in the sarcoplasmic (endoplasmic) reticulum due to the abundance of lipoid structures in this compartment (d). Again the almost un-
changed pattern of accumulation of CC, and CC, after acetone extraction is not compatible with this explanation. What remains is the interpretation that these carbocyanine dyes bind to sites on structures which are especially rich in the sarco- (endo-)plasmic reticulum and which are functionally not impaired by glutaraldehyde fixation and acetone extraction, The chemical nature of these structures has yet to be elucidated. For the present these observations may be useful in cell biology to discriminate different types of cells and to estimate the extent to which the sarcoplasmic reticulum is developed in cultured cardiomyoblasts by a simple vital staining method. This work was supported by the Swiss National Science Foundation (Grant No. 3.588-0.775 and a Scholarship to J. H.). The optical equipment was a donation of the Freiwillige Akademische Gesellschaft, Base].
References 1. Goshima, K, J mol cellular cardiology 8 (1976) 217. 2. Waaeoner, A S. J membrane bio127 (1976) 317. 3. Getrker, H, Baylor, S M & Chandler, W K, Nature 257 (1975) 693. 4. Easton, T G, Valinsky, J E & Reich, E, Cell 13 (1978) 475. 5. Russ&, T J, Beeler, T & Martonosi, A, J biol them 254 (1979) 2040. 6. Goshima, K, Exp cell res 92 (1975) 339. 7. Cruickshank, C N D, Cooper, J R & Conran, M B, Exp cell res 16 (1959) 695. 8. Weber, K, Rathke, P C & Osborn, M, Proc natl acad sci US 75 (1978) 1820. 9. Cohen, L B, Salzberg, B M, Davila, H V, Ross, W N, Landowne, D, Waggoner, A S &Wang, C H, J membrane biol 19 (1974) 1. 10. Fuller, G M, Brinkley, B R & Boughter, J M, Science 187 (1975) 948. 11. Franke, W W, Fink, A & Schmid, E, Cell biol intern reports 2 (1978) 465. 12. Krohne, G, Franke, W W, Ely, S, D’Arcy, A & Jost, E, Cytobiologie 18 (1978) 22. 13. Bloom, W & Fawcett, D W, A textbook of histology, 10th edn, p. 303. W B Saunders Co. Philadelphia, London, Toronto (1975). 14. Ezerman, E B & Ishikawa, H, J cell bio135 (1967) 405. Received September 25, 1979 Accepted October 1, 1979