Cell and Tissue. Research. 9 Springer-Verlag 1981. A Phase-Contrast and Immunofluorescence Study of Adrenal Medullary Chromaffin Cells in Culture:.
Cell Tissue Res (1981) 218:331-343
Cell and Tissue Research 9 Springer-Verlag 1981
A Phase-Contrast and Immunofluorescence Study of Adrenal Medullary Chromaffin Cells in Culture: Neurite Formation, Actin and Chromaffin Granule Distribution John E. Hesketh*, Jaroslava Ciesielski-Treska**, and Dominique Aunis** * The Rowett Research Institute, Bucksburn, Aberdeen, U.K. ; ** Unite INSERM U-44, Centre de Neurochimie du CNRS, 5, Rue Blaise Pascal, Strasbourg, France
Summary. Immunofluorescence studies of bovine chromaffin cells in culture with specific antibodies against dopamine-/~-hydroxylase gave a distinct punctate pattern of labelling, reflecting the distribution of chromaffin granules. There was strong staining of cell extensions and growth cones. Linear arrays of fluorescent dots were observed, suggesting an association of granules with a filamentous cytoskeleton. Labelling of neuritic processes was periodic, perhaps indicative of a packaging of secretory granules. Chromaffin cells stained strongly with specific anti-actin antisera. Fine filament bundles were observed, and also diffuse staining, some punctate labelling and staining of the plasma membrane or sub-membranous cytoplasm. Growth cones and non-terminal cytoplasmic varicosities contained significant amounts of actin. Colchicine (5 x 10-SM) caused retraction of neuritic extensions and formation of lateral growth cones. Cytochalasin (10gg/ml) caused ballooning of terminal growth cones and non-terminal cytoplasmic varicosities. Phalloidin (10-4 M) stimulated microspike formation. The results are discussed in terms of the role of the cytoskeleton in growth cone formation, cell-substratum contacts and the transport of chromaffin granules. Key words: Actin - Dopamine-/~-hydroxylase - Chromaffin cell - Growth cone - Immunofluorescence
The chromaffin cells of the adrenal medulla are of neuronal origin, the adrenal medulla being a modified sympathetic ganglion, and are highly specialised to synthesize, store and secrete large amounts of catecholamines (Winkler 1977). Secretion of catecholamines from adrenal medulla has long been considered a model for neurotransmitter release from neurones. Although catecholamines are known to be stored in vesicular storage organelles (chromaffin granules) and are thought to be released from these granules by exocytosis, little is known about how Send offprint requests to: Dr. J.E. Hesketh, Rowett Research Institute, Bucksburn, Aberdeen, AB 2 9 SB, U.K.
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the granules are transported in the cell and how they are brought into contact with the plasma membrane. It has been suggested that both microtubular and microfilament (actin) systems are involved in such intracelltdar transport and also in other aspects of the secretory process (Douglas 1975; Poisner and Cooke 1975; Trifaro 1977), but there is little evidence on these points. Actin and myosin are known to occur in adrenal medulla, in chromaffin cells (Trifaro 1977; Hesketh et al. 1978; Aunis et al. 1980a, b), and in neurones (Bray 1977; Kuczmarski and Rosenbaum 1979; Aunis et al. 1980a, b); however, their function(s) are still speculative (Bray 1977; Trifaro 1977) but could be related to axonal growth and neurosecretion. In the present experiments we have studied the growth of chromaffin cells in culture, their normal disposition of actin and their reaction to cytoskeleton-disrupting agents in order to investigate the possible association of actin with secretory granules and to study its role in the formation and growth of neuritic cell processes. Furthermore, since dopamine-/~-hydroxylase (D/?H) is exclusively localised in the chromaffin granules (Winkler 1976), immunocytochemical studies with anti-D/~H antiserum have allowed us to follow the distribution of these organelles during periods of neuritic formation and growth. Materials and Methods Cyanogen bromide-activated Sepharose 4 B was obtained from Pharmacia France SA. Collagenase from Clostridium histolyticum (EC 3.4.24.3) was purchased from Boehringer Mannheim. Fluorescein isothiocyanate-conjugated sheep anti-rabbit immunoglobulins were prepared by Institut Pasteur Production (29.4 mg protein per ml; fluorochrome/protein molar ratio of 6.1). Non-immunised sheep antisera were from Miles Laboratories. Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum were purchased from Gibco. Nerve growth factor (NGF) was from Wellcome Laboratories.
Preparation of Actin from Bovine Skeletal Muscle 100,&_filaments were extracted from bovine skeletal muscles using the procedure described by Lazarides and Hubbard (1976). After successive extraction with KC1 and KI buffers, the insoluble material was enriched in desmin but still contained a considerable amount of actin. Actin and desmin were further extracted in the presence of 8 M urea and separated by preparative electrophoresis on 4 - 2 0 ~ polyacrylamide slab gels (8 • 8 • 0.27 cm) in the presence of 2 ~ sodium dodecylsulphate.
Preparation of Antibodies to Act&; Isolation of Anti-Actin Antibodies by Antigen Affinity Chromatography On day 1 rabbits received 1.5 mg protein emulsified in complete Freund's adjuvant in 20-30 intradermal injections distributed along one side of the back, corresponding to I ml of the emulsion. At a separate site near the neck, 1 ml of anti-whooping cough vaccine was injected intradermally. Fifteen days later each rabbit received 2mg of actin emulsified with incomplete Freund's adjuvant in 20-30 intradermal injections along the other side of the back. Booster injections of 1 mg protein adsorbed with KAI(SO4)2 were administered intravenously every three weeks. Animals were bled from the ear veins at intervals of three weeks. Ammonium sulphate-precipitated immunoglobulins were chromatographed on an actin-Sepharose 4 B column. Cyanogen bromide-activated Sepharose 4 B (1.5 g) was washed with 1 m M HC1 and mixed with 20 mg of pure actin in 5 ml of0A M NaHCO 3 containing 0.5 M NaC1 at final pH 8.5. The coupling reaction was performed at room temperature for 2 h. The mixture was filtered, washed with 150 ml of coupling buffer and resuspended in 5 ml of 1 M ethanolamine in a 1 M NaHCO3 buffer containing 0.5 M NaCI.
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Incubation was performed for one hour at room temperature and unbound material was removed by four cycles of washing, each cycle consisting of a wash at pH 8.5 with 250ml of 0.1 M borate buffer containing 0.5 M NaC1 followed by a wash at pH 4 with 200 ml of 0.1 M acetate buffer containing 0.5 M NaCI. The gel was then equilibrated with phosphate-buffered saline (PBS). To the gel suspended in 15ml of PBS was added 3ml of ammonium sulphate-fractionated gammaglobulin fraction in PBS (35 mg protein per ml) and incubated for 3 h at room temperature. The mixture was then packed into a Pharmacia K 9/15 column and washed extensively with PBS. Elution of anti-actin molecules was performed with 0.2 M glycine-HCl at pH 2.75 (Fuller et al. 1975). Fractions were read at 280 nm, pooled and immediately neutralized by addition of 1 M NaOH dropwise before dialyzing against PBS. The mixture was then concentrated in a 3 ml Diaflo cell through a PM-30 membrane to a concentration of 1 mg protein per ml, divided, into 0.1 ml aliquots under N 2 atmosphere and stored at - 2 0 ~C. Anti-actin antibodies were shown to be specific to actin by immunodiffusion, immunoelectrophoresis, staining of the I-band of bovine or rat myofibrils (Aunis et al. 1980b) and staining of fibroblast stress fibres.
Indirect Immunofluorescence The indirect immunofluorescence technique was a modification of that described by Lazarides (1976). Cells were cultured on coverslips, washed briefly in PBS and then fixed in 4 ~ formaldehyde in PBS for 20 min at room temperature. Cells were extracted for 10 min at room temperature with 0.2 ~ Triton X 100 in 3.6 ~ p-formaldehyde in PBS, and the coverslips were then rinsed with PBS for 10 min and incubated at 4 ~C with successively 50 ~ acetone in water (v/v) for 3 min, 100 ~ acetone for 5 min, and 50 ~ acetone in water for 3 min. Coverslips were washed with PBS for 10 min, covered with 100 ~tl nonimmunized sheep immunoglobulins (2mg protein per ml, in PBS) for 15 min at room temperature, and washed with PBS for 5 min. Coverslips were then covered with purified anti-actin antibodies (50 ~tl at a concentration of 50 lxg per ml) or with anti-dopamine-~-hydroxylase antiserum (dilution 1 : 200 with PBS), and incubated for 30-35 min in a humid atmosphere at room temperature. Coverslips were washed with PBS (20 min, changed every 5 min) and then incubated for 30 min with fluorescein isothiocyanatelabelled sheep anti-rabbit immunoglobulins (0.5mg protein per ml PBS) at room temperature. Coverslips were then washed with PBS and mounted on a drop of 50 ~ glycerol - 50 ~o PBS (v/v). Samples were examined under a Leitz microscope with oil-immersion objectives. Control experiments were performed using the crude gamma globulin fractions which were not retained on the actin-Sepharose column (at a concentration of 1 mg protein per ml in PBS). For antidopamine-~-hydroxylase labelling experiments, controls were run using ammonium sulphateprecipitated immunoglobulin fraction previously incubated with excess chromaffin-granule membranes. Both controls gave only very low staining of the cell nucleus.
Culture of Chromaffin Cells from Bovine Adrenal Medulla Chromaffin cells were isolated in Ca ++ free Krebs solution containing 0.05 ~ collagenase and passed through a nylon sieve of 120 jam pore size as described by Aunis et al. (1980b). Cells were cultured (Aunis et al. 1980b) in Dulbecco's modified Eagle's medium supplemented with 20 ~ fetal calf serum containing 50 units per ml of penicillin, 50 ~tg per ml of streptomycin and 10 units per ml of Nerve Growth Factor. Cells were seeded on collagen-coated coverslips (40 x 40 nm) which were placed into Falcon Petri dishes (60nm diameter). Cell concentrations were 105 cells per ml. Living cultures were examined with an inverted, phase contrast, light microscope.
Antibodies to Dopamine-~-Hydroxylase Chromaffin granules were purified from bovine adrenal medulla using discontinuous sucrose gradients (Smith and Winkler 1967) as reported elsewhere (Aunis et al. 1977). Dopamine-fl-hydroxylase was purified from the soluble matrix by affinity chromatography on Concanavalin A-Sepharose 4 B (Aunis and Miras-Portugal 1976). Antibodies were prepared and characterized as described elsewhere (Aunis et al. 1975, 1980a).
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Results
Dopamine-~-Hydroxylase Immunofluorescence Chromaffin cells in culture stained strongly with antisera raised against bovine adrenal dopamine-/3-hydroxylase, and in all cases the fluorescence was seen as a punctate pattern, presumably reflecting the distribution of chromaffin granules. Cells cultured for 24-48 h showed a mainly bipolar or multipolar shape, with few fine, neuritic processes. In such cases fluorescent labelling with anti-D/3H serum was particularly strong in the "poles" of the cell, suggesting an accumulation of granules in these regions of the cytoplasm (Fig. 1). There was no staining of the plasma membrane or the submembranous cytoplasm. In certain cells it was evident that the fluorescent dots were arranged in linear arrays (Figs. 2, 3) suggesting some specific organisation of granules. Cells cultured for periods over 72 h developed very fine neuritic processes with bulb-shaped endings similar to the processes and growth cones observed in cultured neurones (Yamada et al. 1971). Approximately 70 % of cells formed neuritic extensions. Anti-D/?H antiserum showed strong staining of the growth cones at the ends of such processes (Figs. 2, 3, 5, 6) but not the leading edge, membrane or microspikes of the growth cone. Linear arrangements of fluorescent dots were observed in the poles and extensions of many cells (Figs. 2, 4, 5). Anti-D/~H antiserum also stained certain parts of the "axonal" region (to be referred to as the trunk) of such fine cellular extensions. Not all the trunk was stained; fluorescence was restricted to discrete regions which occurred periodically along the "axon" (Fig. 3) and also to non-terminal cytoplasmic varicosities (Fig. 6). Cells cultured for long periods (100 h) formed complicated networks of branching neuritic processes, each with a terminal growth cone. These networks were similar to those observed in cultures of sympathetic neurones (Bray 1973). In such cells, the growth cones and the areas of cytoplasm at the junctions of the branching axons stained strongly with anti-D/~H antiserum (Fig. 3).
Actin Immunofluorescence Anti-actin antiserum stained chromaffin cells to show a characteristic pattern of labelling, quite distinct from that observed in fibroblasts. Figures 7, 12 and 14 show typical chromaffin cells; fluorescent labelling of fine fibres or filament bundles was evident but there were no fluorescently labelled large stress fibres as observed in fibroblasts (Fig. 8). The fine fibres were observed throughout the cell, though they were more evident towards the cell periphery and in the"poles" of the cell (Figs. 7, 9, 11). Parallel arrays of such actin fibres were observed. Anti-actin staining was also seen as a diffuse staining, as a punctate pattern and was also associated with the plasma membrane of the chromaffin cell (Figs. 7, 12, 13, 14). In cells which had formed fine neuritic processes, the growth cones of such processes were strongly fluorescent following labelling with anti-actin antiserum (Figs. 13, 14); in addition to diffuse staining there were many labelled fine filaments, which were sometimes seen to be arranged in parallel arrays. Anti-actin antiserum gave distinct labelling of the membrane and microspikes of the growth cone whereas such areas were unlabelled by anti-D/?H antibodies. Cytoplasm at the junction between branching
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Figs. 1-3. Immunochemical staining o f chromaffin cells with specific anti-dopamine-fl-hydroxylase antibodies, x 820. Note the strong staining of"poles '~ of the cell in Figs. 1, 2 and the growth cones in Figs. 2, 3. Periodic staining of neuritic extensions is indicated by a r r o w in Fig. 3
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Figs. 4--6. Immunochemical staining of chromaffin cells with specific anti-dopamine-fl-hydroxylase antibodies, x 820. Note the linear arrangement of staining in Figs. 4, 5 (arrows). Non-terminal cytoplasmic varicosities are labelled in Fig. 6
" a x o n s " was labelled strongly by anti-actin a n t i b o d i e s (Fig. 10) whereas labelling o f the t r u n k region o f the processes was weak.
Effects of Cytochalasin, Colchicine and Phalloidin on Chromaffin Cells T r e a t m e n t o f c h r o m a f f i n cells with c y t o c h a l a s i n caused the b a l l o o n i n g o f b o t h t e r m i n a l g r o w t h cones a n d n o n - t e r m i n a l varicosities a l o n g the fine cell processes (Fig. 15). Such varicosities were stable, r e m a i n i n g as long as 24 h after r e t u r n to n o r m a l m e d i u m . There was s o m e r e t r a c t i o n o f the extensions b u t this was n o t as n o t i c e a b l e as t h a t which o c c u r r e d following t r e a t m e n t with colchicine (Fig. 15). Cell extensions b e c a m e t h i n n e r after 5-15 m i n o f colchicine treatment, then they b e g a n to r e t r a c t a n d b e c o m e m o r e r o u n d e d (30 min). In some cases there was evidence o f c y t o p l a s m i c varicosities, similar to those seen in c o n t r o l cells a n d similar to lateral
Figs. 7-10. Immunochemical staining of chromaffin cells (Figs. 7, 9, 10) and fibroblasts (Fig. 8) with specific anti-actin antibodies, x 820. Note the fine fibres (arrows) stained in Figs. 7, 9, compared with the prominent stress fibres in the fibroblast (Fig. 8). Membrane (submembranous) staining is evident in Fig. 7 (double arrow). Note the diffuse staining and the apparent network of labelling in Fig. 7. In Fig. 10 the cell has formed a complex network of neuritic extensions. The growth cones, non-terminal varicosities (arrows) and cytoplasmic junctions are strongly stained
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Figs. 11-14. Immunochemical staining of chromaffin cells with specific anti-actin antibodies, x 820. Fig. 11 shows fine fibres stained with anti-actin antibodies. Note the punctate pattern of labelling (Fig. 12, arrow), the strong membrane labelling both in the cell body and the growth cone (Figs. 12-14) and the apparent network of labelling. Fig. 14 shows strong labelling of the growth cone
Fig. 15. Phase-contrast micrographs showing the effects ofcytochalasin (10 lxg/ml) (a-f) and 5 x 10- s M colchicine (g-l) on chromaffin cells in culture, x410. (a) Zerotime, (b) 10min, (e) 35 min, (d) 90min, (e) 150 min, and (f) 210 min after addition of cytochalasin. Cytochalasin was present in the medium for 40 min after which time the cells were washed and resuspended in normal medium without the drug. Note the non-terminal varicosities (arrows)before treatment (a) and the ballooning of both these and the terminal growth cone after treatment with cytochalasin, g-j Sequential photographs of the same cell at zero time (g) and 15 min (h), 30 min (i), and 24 h (j) after addition of colchicine. 75 min after addition of colchicine cells were washed and resuspended in normal medium without drug. k and i A second cell before and after colchicine treatment (15 min). Note the accumulations of cytoplasm (arrows) along the trunk of the cell processes during process retraction (L)
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Fig. 16. Immunochemicalstaining of a chromaffin cell with specificanti-actin antibodies, x 820. The cell had been treated with phalloidin (10- 4 M) for 1 h. Note the many microspikescoveringthe wholecell surface growth cones, along the trunk of the cell processes during process retraction (Fig. 15). These may represent lateral growth cones formed in response to colchicine, as observed in cultured sympathetic neurones (Bray et al. 1979). After 75min there was almost total retraction of the fine processes. Addition of phalloidin to cultures of chromaffin cells led to an increase in microspike formation over the whole cell surface (Fig. 16).
Discussion Growth Cone Formation After 1-2 days in culture bovine chromaffin cells were mostly bipolar or multipolar but when cultured for longer periods (3 days) in the presence of nerve growth factor the cells were found to form neuritic processes and terminal growth cones in a similar fashion to neurones (Yamada et al. 197l). This is evidence of their inherent and latent neuronal characteristics. This behaviour is similar to that of rat chromaffin cells (Unsicker et al. 1978) but contrasts with the reported failure of nerve growth factor to stimulate neuritic formation in bovine chromaffin cells (cf. Unsicker et al. 1980). In addition the fine processes and growth cones formed by chromaffin cells generally reacted to addition of cytochalasin or colchicine in a similar manner to these structures formed by neurones in culture (Yamada et al. 1971; Spooner 1978). The retraction of axons following colchicine treatment suggests that chromaffin cells, like neurones, form cellular extensions which depend for their maintenance on the presence of an intact tubulin-microtubule system. Many chromaffin cells formed intermittent varicose swellings along the trunk of the fine cell processes and these stained strongly with both antiactin and anti-DflH antisera; they may represent lateral growth cones. Although such varicosities were observed in control cells they were more evident, larger and "ballooned" in cells treated with cytochalasin. Cytochalasin also caused ballooning of terminal growth
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cones, as found in neurones (Yamada et al. 1971; Spooner 1978). One interpretation of this ballooning is that it is due to the destruction of contacts between the growth cone and the substratum as a result ofmicrofilament disruption. Following such a hypothesis, the "ballooning" of non-terminal varicosities would be due to disruption of similar contacts formed by certain areas of cytoplasm along the trunk of the cell process. The results suggest therefore that certain cytoplasmic regions represented by non-terminal varicosities are important areas of contact between the neuritic process and the substratum; furthermore they could be points for the formation of lateral growth cones. It would seen reasonable for lateral growth cones to be associated with stable areas of cytoplasm attached to the substratum. Actin Distribution and Function
Actin was observed throughout the chromaffin cell as a fine cytoplasmic network composed of punctate labelling, diffuse labelling (presumably due to unpolymerised actin) and fine actin fibres, and also observed in association with the plasma membrane or the submembranous zone of the cytoplasm both in the cell body and the growth cone. Different cells showed their own pattern of fluorescence, with a different balance of fibre, punctate and diffuse staining. The punctate pattern of labelling (Aunis et al. 1980b) might represent a focal point of filaments (Lazarides and Weber 1974) or an association of actin with chromaffin granules. It is important to investigate the latter possibility since it might provide the basis for a mechanism of granule movement. Actin was present in particularly significant amounts in the growth cone both in association with the plasma membrane and as a fine punctate and fibrillar network; it was also associated with the varicose structures along the trunk of the cell processes whereas the trunk of the neuritic process was only weakly labelled. No actin-containing stress fibres were seen in chromaffin cells, but the cytoplasm contained many fine actin fibres, probably bundles of microfilaments. The observed lack of stress fibres, as previously noted for neurones (Yamada et al. 1971; Bunge 1973; Kuczmarski and Rosenbaum 1979), may reflect less close adhesions from these cells to the substratum than the focal contacts associated with the characteristic stress fibres found in fibroblasts (Abercrombie 1980). Obviously the form of the actin assemblies differ in chromaffin cells and neurones compared with stationary fibroblasts. Although there was often no apparent order in the arrangement of these fine actin fibres, both the broad extensions of multipolar cells and the growth cones of fine processes exhibited filaments arranged in parallel arrays, the arrays themselves being roughly parallel to the direction of cell growth. Actin-containing filaments were particularly evident at the cell periphery in cellular expansions and growth cones. The distribution of actin and the results of experiments with cytochalasin emphasise one possible function of actin-filaments in chromaffin cells, namely the formation and maintenance of cell-substratum contacts by terminal and nonterminal axonal growth cones and the importance of actin in axon structure and growth. Other functions of actin in chromaffin cells remain to be elucidated, in particular the role of plasma membrane-associated/submembranous actin and whether it is involved in the exocytosis. Treatment of cells with phalloidin greatly
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increased microspike formation over the whole cell surface. Since phalloidin penetrates cells very poorly (Wehland et al. 1979) it would seem likely that this phenomenon is due to an induction of plasma membrane-associated actin polymerization which stimulates microspike formation. Such interactions of phalloidin with membrane or submembranous actin may be related to its observed inhibition of synaptosomal neurotransmitter release (Babitch et al. 1979) and the reported increased microspike formation by chromaffin cells in response to secretagogues such as acetylcholine (Englert 1980).
Distribution of Chromaffin Granules Since chromaffin cells are rich in storage granules containing dopamine-flhydroxylase they present a potentially useful system with which to study granule disposition by immunocytochemical means. In culture, chromaffin cells were stained very strongly by anti-DflH antiserum showing that in culture, these cells retain a high content of dopamine-fl-hydroxylase. Both the observed punctate pattern of fluorescence and the labelling of chromaffin granules in sections of adrenal medulla (Aunis et al. 1980a) strongly suggest that the pattern of fluorescence obtained with anti-D/~H antiserum reflects the distribution of chromaffin granules. In the early stages of culture, chromaffin granules, as shown by labelling with anti-DflH antiserum, accumulated in the periphery of the cell cytoplasm, particularly in the broad extensions or "poles" of bipolar and multipolar cells. Granules were also seen in the perinuclear region but their accumulation in the "poles" of the cell and in the regions destined to form neuritic extensions, was quite characteristic. Such a distribution of granules could be due to some specific mechanism or to non-specific protoplasmic flow. As the cells formed fine processes and growth cones, granules were labelled in both these structures, there being particularly large accumulations in the growth cone cytoplasm. Labelling of the trunk regions was intermittent, suggesting in packaging of granules so that they travel down the process in pulses, and in addition anti-DflH fluorescence was observed in the non-terminal cytoplasmic varicosities seen along the fine processes of chromaffin cells. The results show that chromaffin cells, in culture, transport chromaffin granules containing dopamine-fl-hydroxylase from the perinuclear region of the cytoplasm to the cell periphery; there is a particular accumulation in regions about to form neuritic extensions. Granules are subsequently transported along such cell processes to the terminal growth cone. Some granules may remain associated with varicosities along the trunk of the process. However, the results shed no light on the mechanism of such transport. Most interestingly, however, many cells labelled with anti-DflH antiserum showed fluorescent dots in linear arrays suggesting some organisation of the chromaffin granules in the cell cytoplasm and growth cone. The observed linear arrangement is suggestive of an association with some kind of filamentous cytoskeleton, probably either microtubules or microfilaments.
Acknowledgements.J.E.H. acknowledgesthe support ofa FEBSfellowship.The work was supportedby grants from the French DGRST (79-7-1058) and INSERM (CRL 80-6-017).The authors thank B. Guerold for help in preparinganti-actinantibody and D. Thierseand G. Ulrichfor technicalassistance.
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References Abercrombie M (1980) The crawling movement of metazoan cells. Proc R Soc Lond B 207:129-147 Aunis DA, Miras-Portugal MT (1976) Isolation ofdopamine-]?-hydroxylase. In: Bittiger H, Schebli HO (eds) Concanavalin A as a tool. Wiley, London, pp 349-354 Aunis D, Miras-Portugal MT, Mandel P (1975) Bovine adrenal medullary dopamine-]i-hydroxylase: studies on the interaction with concanavalin A. J Neurochem 24:425431 Aunis D, Bouclier M, Pescheloche M, Mandel P (1977) Properties of membrane bound dopamine-//hydroxylase in chromaffin granules from bovine adrenal medulla. J Neurochem 29:439-447 Aunis D, Hesketh JE, Devilliers G (1980a) Immunohistological and immunocytochemical localization of myosin, chromogranin A and dopamine-/~-hydroxylase in nerve cells in culture and in adrenal glands. J Neurocytol 9:255-274 Aunis D, Guerold B, Bader MF, Ciesielski-Treska J (1980h) Immunocytochemical and biochemical demonstration of contractile proteins in chromaffin cells in culture. Neuroscience 5:2261-2277 Babitch JA, Gage FH, Valdes JJ (1979) Effects of phalloidin on K + dependant, Ca ++ independant neurotransmitter effiux and Ca dependant neurotransmitter release. Life Sci 24:117-124 Bray D (1973) Branching patterns of individual sympathetic neurones in culture. J Cell Bio156: 702-712 Bray D (1977) Actin and myosin in neurones: a first review. Biochimie 59:1-6 Bray D, Thomas C, Shaw G (1979) Growth cone formation in cultures of sensory neurones. Proc Nail Acad Sci USA 75:5226-9 Bunge MB (1973) Fine structure of nerve fibres and growth cones of isolated sympathetic neurones in culture. J Cell Biol 56:713-735 Douglas WW (1975) Secretomotor control of adrenal medullary secretion: synaptic membrane and ionic events in stimulus-secretion coupling. In: Blaschko H, Sayers G, Smith AD (eds) Handbook of Physiology. Endocrinology 6:367-388 Englert DF (1980) An optical study of isolated rat adrenal chromaffin cells. Exp Cell Res 125: 369-376 Fuller GM, Brinkley BK, Boughter JM (1975) Immunofluorescence of mitotic spindles by using monospecific antibody against bovine brain tubulin. Science 187:948-951 Hesketh JE, Aunis D, Devilliers G, Mandel P (1978) Biochemical and morphological studies of bovine adrenal medullary myosin. Biol Cell 33:199-208 Kuczmarski ER, Rosenbaum JL (1979) Studies on the organization and localization of actin and myosin in neurones. J Cell Biol 80:356-371 Lazarides E (1976) Actin, ~-actinin and tropomyosin interaction in the structural organization of actin filaments in non-muscle cells. J Cell Biol 68:202-219 Lazarides E, Weber K (1974) Actin antibody: the specific visualization of actin filaments in non-muscle cells. Proc Natl Acad Sci USA 71 : 2268-2272 Lazarides E, Hubbard BD (1976) Immunological characterization of the subunit of the 100/~ filaments from muscle cells. Proc Natl Acad Sci USA 73:4334-4338 Poisner AM, Cooke P (1975) Microtuhules and the adrenal medulla. Ann N Y Acad Sci 253:653-669 Smith AD, Winkler H (1967) A simple method for the isolation of adrenal chromaffin granules on a large scale. Biochem J 103:480-2 Spooner BS (1978) In Cytochalasins: Biochemical and cell biological aspects. Tanenbaum SW (ed). North Holland Trifaro JM (1977) Contractile proteins in tissues originating from the neural crest. Neurosci 3:1-24 Unsicker U, Krisch B, Otten U, Thoenen H (1978) Nerve growth factor-induced fiber outgrowth from isolated rat adrenal chromaffin cells: impairment by glucocorticoids. Proc Natl Acad Sci USA 75: 3498-3502 Unsicker U, Griesser G-H, Lindmar R, Loffelholz K, Wolf U (1980) Establishment, characterization and fibre outgrowth of isolated bovine adrenal medullary cells in long-term cultures. Neuroscience 5:1445-1460 Wehland J, Osborn M, Weber K (1977) Phalloidin-induced actin polymerization in the cytoplasm of cultured cells interferes with cell locomotion and growth. Proc Natl Acad Sci USA 74:5613-7 Winkler H (1976) The composition of adrenal chromaffin granules: an assessment of controversial results. Neuroscience 1 : 65-80 Winkler H (1977) The biogenesis of adrenal chromaffin granules. Neuroscience 2:657-683 Yamada KM, Spooner B S, Wessels NK (1971) Ultrastructure and function of growth cones and axons of cultured nerve cells. J Cell Biol 49:614-635 Accepted February 10, 1981
