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Ciliation of bovine aortic endothelial cells in culture
ATHEROSCLEROSIS ELSEVIER SCIENCE IRFLAND
Atherosclerosis
106 (1994) 75-8 I
Ciliation of bovine aortic endothelial cells in culture P. Briffeuilt,
R. Thibaut-Vercruyssen*,
M.-F. Ronveaux-Dupal
(Received 24 July 1992: revision received 13 October 1993: accepted 30 November 1993)
Abstract Solitary cilia were observed by electron microscopy in senescent bovine aortic endothelial cells in culture. Such single cilia have previously been seen in several tissues of various species, but as far as we know they have not been identified in cultured endothelial cells. The analysis of ultrathin sections enabled us to show that the cilia originated in one of the two centrioles. Vacuolar structures located at one end of the centrioles were also observed and might occur during the lengthening of the cilium. Moreover, the surface replication technique allowed us to show that the cilia could extend out of the cell. As senescent endothelial cells enter a quiescent stage, they could build up such a cilium as was observed for some strains of cultured fibroblasts. Key
words:
Solitary
cilia; Endothelial
cells; Ultrastructural
1. Introduction
The ciliation of centrioles is a phenomenon that has been observed over a long time both in vivo [l-6] and in cultured cells [7]. Solitary cilia have been observed in several tissues that were indexed by Odor and Blandau [8]. More recently Kojimahara [9] and Warfvinge and Elofsson [lo] reported for the first time the presence of solitary cilia, respectively in the endothelium of rat blood vessels and in the mammalian vaginal epithelium. In 1966, Stubblefield and Brinkley [7], using the mitotic inhibitor colcemid, were the first to de-
study; Freeze etching
scribe the formation of a cilium in cultured tibroblasts. They confirmed the relationship between cilia and mitotic activity originally proposed by Henneguy and Lenhossch [l 11. Since that time, such an inverse relationship has been proposed by several authors. Among them, Rash et al. [4] have shown that cilia are found during cardiac differentiation in the embryonic chick and suggested that the abrupt transformation from mitotic replicative tissue to non-mitotic differentiated tissue is correlated with the disappearance of centrioles and the formation of cilia. Other studies have confirmed the presence of cilia in numerous
* Corresponding author. Tel.: 3281 7243 14. tPresent address: DCpartement de Physiologie et Physiopathologie Humaine, Facultt de Midecine, FUNDP, 5000 Namur. Belgium. 002l-9150/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0021-9 150(93)05190-G
76
cultured libroblastic strains [ 12-151, in epithelial cells [ 161and in smooth muscle cells and endothelial cells surrounding or constitutive of atherosclerotic lesions of rabbit aortas [3]. Chaldakov [l] confirmed the results of Haust [3] and moreover described ciliated smooth muscle cells in normal rabbit aorta, aorta of colchicinetreated rabbits and the uterus of oestradiol-treated rats. He underlined the presence of ciliated endothelial cells in normal rabbit aorta and in the microvasculature of a leiomyoma of the uterus. In the course of a morphological analysis of bovine aortic endothelial cells in culture throughout their lifespan, we found and reported the first results concerning the presence of cilia in cultured endothelial cells [17]. In view of the results published by Haust [18] on endothelial cells of human aorta, we decided to illustrate fully the structural details of the cilia we observed in senescent bovine aortic endothelial cells. The purpose of this article is to illustrate the structural details of the observed cilia.
P. Briffeuil et al. /Atherosclerosis
centuation sionally.
of structure
106 (1994)
was used only
75-81
occa-
3. Results Following a casual observation of ciliated centrioles on cells which had undergone 7 passages (14 population doublings), we investigated thoroughly the presence of solitary cilia up to the end of the culture (14 passages-28 population doublings). Many cilia were observed from the beginning of the culture to its non-proliferating end at nearly every time of passage. 3.1. Morphology of the cilium On transmission electron microscopic examination of thin sections (Figs. l-4), each cilium consists of two distinguishable parts: a basal body
2. Methods Bovine aortic endothelial cells were cultured on gelatine-coated dishes in medium M 199 supplemented with 20% of newborn bovine serum. Culture techniques were adapted from the procedure of Booyse et al. [ 191. At the time of passage, cells were harvested after incubation in a buffered solution of trypsine/EDTA (0.25%/0.02%). At various stages of their lifespan, cells were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, at 4°C for 2 h, postfixed in 2% 0~0~ in the same buffer and used for conventional embedding in Epon 812 for thin section preparation. Serial cross-sections were performed only as required. Some cultures were fixed as described above and prepared for surface replication using the critical point drying technique. Replicas were made in a Balzers freeze etching apparatus (Balzers AG, Lichtenstein) by shadowing the cell surface with platinum and carbon at an angle of 38”. The cells were examined under a Philips EM 301 electron microscope. The technique described by Markham et al. (201 for enhancement of image details and ac-
Fig. I. Ciliated centriole. Note the ciliated centriole as the orthogonally located second centriole in a senescent thelial cell (20 population doublings). c, centriole; s, shaft. Lateral foot processes are clearly visible (arrow) as extending microtubules (MT).
well as endociliary as well
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106 (1994)
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Fig. 3. Section through
a basal body and a growing
cilium in
an endothelial cell of the first passage. The vesicle (v) expands to form the ciliary shaft, probably by addition of small vesicles (arrow).
Fig. 2. Serial cross-sections
through
the base of the cilium and
the top of the basal body. The light ring (arrowhead) in (A) and (B) represents the extracellular space surrounding the ciliary shaft. The extracellular space is still visible (arrowhead) near the obliquely sectioned basal body(C). (D) and (E) are first sections of the top of the basal body.
associated with the second centriole (c), and the ciliary shaft (s) which prolongs the basal body. Lateral foot processes are clearly visible as well as extending microtubules (MT). The second centriole is perpendicularly located relative to the basal body and both are found in the vicinity of the nucleus near the Golgi region. Tubular structures parallel to the longitudinal axis can be visualized at the periphery on either side of the shaft. However, central tubular structures can sometimes be seen in some sections. The tubules are associated in pairs and they usually appeared to be in continuity with similar structures of the basal body. The ciliary shaft seems to be enclosed within the cytoplasm and is flanked by two clear spaces. The tip of the cilium could not be visualized because the cilium usually bent or ran out of the plane of the section. In some cells, simple centrioles were observed and usually conveyed the
P. Briffeuil et al. /Atherosclerosis
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Fig. 5. Surface replica of endothelial cell (4 population doubling@. The cilium (arrowhead) as well as the shadow are clearly visible in the vicinity of the nucleus (N).
Fig. 4. Section through lial cell (16 population doublets can be seen generally situated close
the ciliary shaft of a senescent endothedoublings). The tubules associated in longitudinaly (arrow). The cilium is to the nucleus (N) and the Golgi apparatus (G).
image of a hollow cylinder, sometimes presenting one or two central vesicles. On the other hand, one of the parent centrioles could become associated with a larger vesicle which flattened across the end of the centriole. These vacuoles could probably play a leading part in the lengthening of the cilium. Figs. 1 and 3 show such a structure where the tubules are slightly elongated, the vesicle being more curved. The vesicle expands to form the ciliary shaft, probably by the addition of small vesicles. Some of them appeared coated (Fig. 3). As shown in Fig. 2, the solitary cilia could emerge from the cell but no sectioning enabled us to show it unequivocally. We thus decided to
employ the surface replication technique. This experiment has been carried out with endothelial cells in their growing phase after 2 passages. Indeed, some cells showed a ciliary-shaft structure emerging in the nuclear region. Moreover, those structures were of the same size as those measured on ultrathin sections, 0.2 pm in diameter and 2-3 pm long (Fig. 5). Nine fibers are protruding from the basal body near the point where it becomes a cilium (Figs. 2D and 2E). Cross-sections of the centrioles as well as of the ciliary shaft are not always sharply resolved, so we decided to use the technique of the enhancement of image details and accentuation of structures described by Markham et al. [20], which proved to be very useful (Fig. 6). 4. Discussion Following the observation of Stubblefield Brinkley [7], who showed cilia arising from trioles of cultured fibroblasts treated with cemid, a mitosis inhibitor, other studies
and cencolhave
P. Briffeuil et al. /Atherosclerosis
106 (1994)
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Fig. 6. (A) Cross-section of a centriole as shown by a conventional electron micrograph. (B) Rotational photo prepared by the method of Markham et al. [20]. Note the enhancement of the central structure as well as the incomplete triplets.
shown such cilia on several fibroblastic strains [12-151. The cilia we observed did not differ from those mentioned above, examined both in vivo and in cultured cells. The central vesicles in the centriole and sometimes in the basal body (not shown), also observed by Rash et al. [4] and by Reese [22], are a common constituent of normal centrioles in endothelial cells studied. Longitudinal sections gave us a great deal of information about the ciliary structure, whereas the rare cross-sectioned cilia did not permit us to determine ciliary axoneme for more than one cilium. The cilium contained 9 doublets of tubules in an irregular arrangement; this seems to be in keeping with Barnes’s [23] view that solitary cilia lack the two central tibres of motile cilia, and with the observations of Allen [24] which suggest that the 9 doublets of solitary cilia tend toward a random arrangement. Wheatley [14,15] and Fonte et al. [2] have shown that doublets could be arranged symmetrically in culture as well as in vivo. Nevertheless, biciliated cells were not observed
in our cultures even though Wheatley [14], Albrecht-Buehler [25] and Tucker et al. [ 131 have shown them at least in some cells of different libroblastic strains. The ciliary shaft originated in one of the centrioles of the centrosome. The major problem was the limited view of the cilia we had with ultrathin sections. Indeed, we could not show easily either an eventual relation of the cilium with the plasma membrane or that cilia emerged from the cell. The surface replication technique has been very helpful in this regard. In this case, we could show cilia emerging from some cells only in the vicinity of the nucleus. This experiment was carried out on cultured endothelial cells which had undergone 4 population doublings (2 passages) and were in an exponential growing phase. It would thus be of interest to realize the same experiment on senescent cells, where we found numerous ciliated centrioles. When solitary cilia were examined in mammalian cells, the suggestion was made that the formation of a cilium forced the cells to become quiescent by removing the centriole from the mitotic cycle [4].
80
The control of the cell cycle only by the centriole became less tenable when rapidly growing cells were found to have ciliated centrioles [2]. These conflicting results have been reconciled with the data obtained by Wheatley [15], who showed that cilium formation could occur in rapidly dividing mammalian cells, that ciliogenesis could begin with the completion of mitosis and that cilia may be present for almost the entire interphase period. In the same way, Wheatley [ 151noted that solitary cilia are expressed in primary fibroblastic cell cultures from embryonic mice in about the same frequency as in established murine cell lines. However, they are missing or very poorly expressed in old established L-929 cells. These results were confirmed by Tucker et al. [ 131, who found that ciliation of the centriole occurred in quiescent cells as well as in late Gl period of exponentially growing cells. As suggested by the results of Wheatley [15] and Tucker et al. [13], it seems reasonable to suppose that endothelial cells which reach the stationary phase, such as during senescence [26] or at confluence, will have a greater opportunity to develop ciliary appendages than cells passing rapidly through their division cycle. In ultrathin sections as well as on surface replication, the cilia seemed to be predominantly orientated parallel to the substrate. This observation is in agreement with the findings on 3T3 cells of Albrecht-Buehler [25] who, like Osborn and Weber [27], examined the solitary cilia by indirect immunofluorescence using antitubulin as first antibody. As seen in our study, several stages can happen between simple centriole and elongated ciliary shaft. In comparison with several authors [6,7,12], all stages in apparent ciliary development were seen. The earliest recognizable stage was the basal body in which tubules could be identified extending from one end of the centriole to an adjacent vesicle. This vesicle could be a larger vesicular component of the Golgi apparatus, as proposed earlier by Archer and Wheatley [ 121. It was suggested that thereafter this vesicle became more curved surrounding the elongated tubules. As shown in Ref. 12, the double membrane sheet surrounding the shaft is also enlarged, apparently by addition of vesicular elements. The function and significance of the solitary
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106 (1994)
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cilia are unknown. Since the two central tubules are lacking according to the majority of authors, it is now supposed that the cilia are not motile. There are suggestions that cilia could have a chemoreceptor [28] or a sensory function [23]. Boquist [29] suggested that the cilium resorption during the Sphase of the mitotic cycle could furnish a large amount of material for the elaboration of the mitotic spindle. Albrecht-Buehler [25] postulated a control of the migration. More recently, Haust [3] also found ciliated smooth muscle cells in aortic atherosclerotic lesions in rabbit. These observations were in contradiction to those of Chaldakov [1], who found ciliated smooth muscle and endothelial cells even in normal rabbit aortas. Although a large number of hypotheses have emerged from those results, the role of the solitary cilium remains unclear. Initially, we were surprised by the presence of cilia in cultured endothelial cells since, as far as we know, they have never been observed in these cells despite the fact that they have been studied by many laboratories for a long time all over the world. The only explanation we can give is that the ciliation could be due either to the cell strain used or to the culture condition. Indeed, Wheatley [ 151 has found variations of ciliary shaft occurrence in 3T6 tibroblasts cultured by himself from those of other laboratories, and has postulated subline differences in solitary cilium expression although the two fibroblastic strains appeared identical structurally. 5. Acknowledgments We wish to thank Mrs. Noelle Paulus-Ninane, and Chantal Devignon for Nicole F&et-Henry valuable technical work as well as Mr. Jacques for the Collet and Mr. Yves Houbion photographical work. 6. References I 2
Chaldakov, G.N., The ciliated smooth muscle and endothelial cell, Atherosclerosis, 56 (1985) 25 I. Fonte, V.G., Searls. R.L. and Hilfer, S.R., The relationship of cilia with cell division and differentiation, J. Cell. Biol.. 49 (1971) 226.
P. Brqfeeuil et al. /Atherosclerosis
3
4 5 6
I
8
9
IO
II
12
I3
14
I5
I6 I7
Haust,
M.D..
Ciliated
106 (1994)
smooth
muscle
75-81
cells
81
bovine
in aortic
atherosclerotic lesions of rabbit, Atherosclerosis. 50 (1984) 283. Rash, J.E.. Shay, J.W. and Biesele, J.J., Cilia in cardiac differentiation. J. Ultrastruct. Res.. 29 (1969) 470. Scherft. J.P. and Daems, W.Th., Single cilia in chondrocytes, J. Ultrastruct. Res., I9 (1967) 546. Smith, J.W., Christie, K.N. and Frame, J.. Desmosomes, cilia and acanthosomes associated with keratinocytes, J. Anat., 105 (1969) 383. Stubbletield. E. and Brinkley, B.R., Cilia formation in Chinese hamster tibroblasts in vitro as a response to colcemid treatment, J. Cell. Biol.. 30 (1966) 645. Odor, D.L. and Blandau, R.J., Observations on the solitary cilium of rabbit oviductal epithelium its motility and ultrastructure. Am. J. Anat.. 174 (1985) 437. Kojimahara, M., Endothelial cilia in rat mesenteric arteries and intramyocardial capillaries. Z. Mikrosk. Anat. Forsch. (Leipz.), I04 (1990) 412. Warfvinge. K. and Elofsson, R., Single modified cilia displayed by cells of human internal stratified epithelial (oral cavity, vagina), Cell Tissue Res.. 251 (1988) 237. Henneguy. L.F. and Lenhossch, Sur le rapport des cils vibratiles avec les centrosomes. Arch. Anat. Microsc. Morphol. Exp., I (1898) 481. Archer, F.L. and Wheatley. D.N., Cilia in cell-cultured fibroblasts. II. Incidence in mitotic and post-mitotic BHK 21iCl3 libroblasts. J. Anat.. 109 (1971) 277. Tucker, R.W., Pardee, A.B. and Fujiwara. K.. Centriole ciliation is related to quiescence and DNA synthesis in 3T3 cells, Cell, 17 (1979) 527. Wheatley, D.N.. Cilia in cell-cultured tibroblasts. I. On their occurrence and relative frequencies in primary cultures and established cell lines, J. Anat., I05 (1969) 35 I. Wheatley, D.N.. Cilia in cell-cultured libroblasts. III. Relationship between mitotic activity and cilium frequency in mouse 3T6 tibroblasts, J. Anat., I IO (1971) 367. Vorobjev, I.A. and Chentsov, Y.S., Centrioles in the cell cycle. I. Epithelial cells, J. Cell. Biol., 93 (1982) 938. Briffeuil, P. and Ronveaux-Dupal, M.F., Ciliation of
I8 I9
20
21
22 23
24
25
26
aortic
endothelial
cells in culture.
Arch.
Biol.
(Brux.). 97 (1986) 360. M.D.. Endothelial cilia in human aortic Haust, atherosclerotic lesions. Virchows Arch. A. 410 (1987) 317. Booyse. F.M., Sedlak. B.J. and Rapelson. M.E.. Culture of arterial endothelial cells: characterization and growth of bovine aortic cells. Thromb. Diath. Haemorrh., 34 (1975) 825. Markham, E., Frey, S. and Hills, G.. Methods for the enhancement of image detail and accentuation of structure in electron microscopy. Virology. 20 (1963) 88. Vorobjev. I.A. and Chentsov, Y.S.. The ultrastructure of centriole in mammalian tissue culture cells. Cell. Biol. Int. Rep.. 4 (1980) 1037. Reese. T.S.. Olfactory cilia in the frog. J. Cell. Biol.. 25 (1965) 209. Barnes. B.C.. Ciliated secretory cells in the pars distalis of the mouse adenohypophysis. J. Ultrastruct. Res.. 5 (1961) 453. Allen, R.A., Isolated cilia in inner retinal neurons and in retinal pigment epithelium, J. Ultrastruct. Res.. I2 (1965) 730. Albrecht-Buehler. G., Phagokinetic tracks of 3T3 cells: parallels between the orientation of track segments and of cellular structures which contain actin or tubulin, Cell. I2 (1977) 333. Rosen, E.M., Mueller, S.N.. Noveral. J.P. and Levine, E.M., Proliferative characteristics of clonal endothelial cell strains, J. Cell. Physiol., 107 (1981) 123.
27
Osborn. M. and Weber. K.. Cytoplasmic microtubules in tissue culture cells appear to grow from an organizing structure towards the plasma membrane, Proc. Natl. Acad. Sci. USA. 73 (1976) 867.
28
Munger, B.L., A light and electron microscopic study of cellular differentiation in the pancreatic islets of the mouse, Am. J. Anat., 103 (1958) 275. Boquist, L., Cilia in normal and regenerating islet tissue. An ultrastructural study in the Chinese hamster with par-
29
ticular reference to the P-cells and the ductular epitheliurn, Z. Zellforsch. Mikrosk. Anat.. 89 (1968) 519.
Atherosclerosis
106 (1994) 75-8 I
Ciliation of bovine aortic endothelial cells in culture P. Briffeuilt,
R. Thibaut-Vercruyssen*,
M.-F. Ronveaux-Dupal
(Received 24 July 1992: revision received 13 October 1993: accepted 30 November 1993)
Abstract Solitary cilia were observed by electron microscopy in senescent bovine aortic endothelial cells in culture. Such single cilia have previously been seen in several tissues of various species, but as far as we know they have not been identified in cultured endothelial cells. The analysis of ultrathin sections enabled us to show that the cilia originated in one of the two centrioles. Vacuolar structures located at one end of the centrioles were also observed and might occur during the lengthening of the cilium. Moreover, the surface replication technique allowed us to show that the cilia could extend out of the cell. As senescent endothelial cells enter a quiescent stage, they could build up such a cilium as was observed for some strains of cultured fibroblasts. Key
words:
Solitary
cilia; Endothelial
cells; Ultrastructural
1. Introduction
The ciliation of centrioles is a phenomenon that has been observed over a long time both in vivo [l-6] and in cultured cells [7]. Solitary cilia have been observed in several tissues that were indexed by Odor and Blandau [8]. More recently Kojimahara [9] and Warfvinge and Elofsson [lo] reported for the first time the presence of solitary cilia, respectively in the endothelium of rat blood vessels and in the mammalian vaginal epithelium. In 1966, Stubblefield and Brinkley [7], using the mitotic inhibitor colcemid, were the first to de-
study; Freeze etching
scribe the formation of a cilium in cultured tibroblasts. They confirmed the relationship between cilia and mitotic activity originally proposed by Henneguy and Lenhossch [l 11. Since that time, such an inverse relationship has been proposed by several authors. Among them, Rash et al. [4] have shown that cilia are found during cardiac differentiation in the embryonic chick and suggested that the abrupt transformation from mitotic replicative tissue to non-mitotic differentiated tissue is correlated with the disappearance of centrioles and the formation of cilia. Other studies have confirmed the presence of cilia in numerous
* Corresponding author. Tel.: 3281 7243 14. tPresent address: DCpartement de Physiologie et Physiopathologie Humaine, Facultt de Midecine, FUNDP, 5000 Namur. Belgium. 002l-9150/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0021-9 150(93)05190-G
76
cultured libroblastic strains [ 12-151, in epithelial cells [ 161and in smooth muscle cells and endothelial cells surrounding or constitutive of atherosclerotic lesions of rabbit aortas [3]. Chaldakov [l] confirmed the results of Haust [3] and moreover described ciliated smooth muscle cells in normal rabbit aorta, aorta of colchicinetreated rabbits and the uterus of oestradiol-treated rats. He underlined the presence of ciliated endothelial cells in normal rabbit aorta and in the microvasculature of a leiomyoma of the uterus. In the course of a morphological analysis of bovine aortic endothelial cells in culture throughout their lifespan, we found and reported the first results concerning the presence of cilia in cultured endothelial cells [17]. In view of the results published by Haust [18] on endothelial cells of human aorta, we decided to illustrate fully the structural details of the cilia we observed in senescent bovine aortic endothelial cells. The purpose of this article is to illustrate the structural details of the observed cilia.
P. Briffeuil et al. /Atherosclerosis
centuation sionally.
of structure
106 (1994)
was used only
75-81
occa-
3. Results Following a casual observation of ciliated centrioles on cells which had undergone 7 passages (14 population doublings), we investigated thoroughly the presence of solitary cilia up to the end of the culture (14 passages-28 population doublings). Many cilia were observed from the beginning of the culture to its non-proliferating end at nearly every time of passage. 3.1. Morphology of the cilium On transmission electron microscopic examination of thin sections (Figs. l-4), each cilium consists of two distinguishable parts: a basal body
2. Methods Bovine aortic endothelial cells were cultured on gelatine-coated dishes in medium M 199 supplemented with 20% of newborn bovine serum. Culture techniques were adapted from the procedure of Booyse et al. [ 191. At the time of passage, cells were harvested after incubation in a buffered solution of trypsine/EDTA (0.25%/0.02%). At various stages of their lifespan, cells were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, at 4°C for 2 h, postfixed in 2% 0~0~ in the same buffer and used for conventional embedding in Epon 812 for thin section preparation. Serial cross-sections were performed only as required. Some cultures were fixed as described above and prepared for surface replication using the critical point drying technique. Replicas were made in a Balzers freeze etching apparatus (Balzers AG, Lichtenstein) by shadowing the cell surface with platinum and carbon at an angle of 38”. The cells were examined under a Philips EM 301 electron microscope. The technique described by Markham et al. (201 for enhancement of image details and ac-
Fig. I. Ciliated centriole. Note the ciliated centriole as the orthogonally located second centriole in a senescent thelial cell (20 population doublings). c, centriole; s, shaft. Lateral foot processes are clearly visible (arrow) as extending microtubules (MT).
well as endociliary as well
P. Briffeuil et al. /Atherosclerosis
106 (1994)
75-81
Fig. 3. Section through
a basal body and a growing
cilium in
an endothelial cell of the first passage. The vesicle (v) expands to form the ciliary shaft, probably by addition of small vesicles (arrow).
Fig. 2. Serial cross-sections
through
the base of the cilium and
the top of the basal body. The light ring (arrowhead) in (A) and (B) represents the extracellular space surrounding the ciliary shaft. The extracellular space is still visible (arrowhead) near the obliquely sectioned basal body(C). (D) and (E) are first sections of the top of the basal body.
associated with the second centriole (c), and the ciliary shaft (s) which prolongs the basal body. Lateral foot processes are clearly visible as well as extending microtubules (MT). The second centriole is perpendicularly located relative to the basal body and both are found in the vicinity of the nucleus near the Golgi region. Tubular structures parallel to the longitudinal axis can be visualized at the periphery on either side of the shaft. However, central tubular structures can sometimes be seen in some sections. The tubules are associated in pairs and they usually appeared to be in continuity with similar structures of the basal body. The ciliary shaft seems to be enclosed within the cytoplasm and is flanked by two clear spaces. The tip of the cilium could not be visualized because the cilium usually bent or ran out of the plane of the section. In some cells, simple centrioles were observed and usually conveyed the
P. Briffeuil et al. /Atherosclerosis
106 (1994)
75-81
Fig. 5. Surface replica of endothelial cell (4 population doubling@. The cilium (arrowhead) as well as the shadow are clearly visible in the vicinity of the nucleus (N).
Fig. 4. Section through lial cell (16 population doublets can be seen generally situated close
the ciliary shaft of a senescent endothedoublings). The tubules associated in longitudinaly (arrow). The cilium is to the nucleus (N) and the Golgi apparatus (G).
image of a hollow cylinder, sometimes presenting one or two central vesicles. On the other hand, one of the parent centrioles could become associated with a larger vesicle which flattened across the end of the centriole. These vacuoles could probably play a leading part in the lengthening of the cilium. Figs. 1 and 3 show such a structure where the tubules are slightly elongated, the vesicle being more curved. The vesicle expands to form the ciliary shaft, probably by the addition of small vesicles. Some of them appeared coated (Fig. 3). As shown in Fig. 2, the solitary cilia could emerge from the cell but no sectioning enabled us to show it unequivocally. We thus decided to
employ the surface replication technique. This experiment has been carried out with endothelial cells in their growing phase after 2 passages. Indeed, some cells showed a ciliary-shaft structure emerging in the nuclear region. Moreover, those structures were of the same size as those measured on ultrathin sections, 0.2 pm in diameter and 2-3 pm long (Fig. 5). Nine fibers are protruding from the basal body near the point where it becomes a cilium (Figs. 2D and 2E). Cross-sections of the centrioles as well as of the ciliary shaft are not always sharply resolved, so we decided to use the technique of the enhancement of image details and accentuation of structures described by Markham et al. [20], which proved to be very useful (Fig. 6). 4. Discussion Following the observation of Stubblefield Brinkley [7], who showed cilia arising from trioles of cultured fibroblasts treated with cemid, a mitosis inhibitor, other studies
and cencolhave
P. Briffeuil et al. /Atherosclerosis
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75-81
Fig. 6. (A) Cross-section of a centriole as shown by a conventional electron micrograph. (B) Rotational photo prepared by the method of Markham et al. [20]. Note the enhancement of the central structure as well as the incomplete triplets.
shown such cilia on several fibroblastic strains [12-151. The cilia we observed did not differ from those mentioned above, examined both in vivo and in cultured cells. The central vesicles in the centriole and sometimes in the basal body (not shown), also observed by Rash et al. [4] and by Reese [22], are a common constituent of normal centrioles in endothelial cells studied. Longitudinal sections gave us a great deal of information about the ciliary structure, whereas the rare cross-sectioned cilia did not permit us to determine ciliary axoneme for more than one cilium. The cilium contained 9 doublets of tubules in an irregular arrangement; this seems to be in keeping with Barnes’s [23] view that solitary cilia lack the two central tibres of motile cilia, and with the observations of Allen [24] which suggest that the 9 doublets of solitary cilia tend toward a random arrangement. Wheatley [14,15] and Fonte et al. [2] have shown that doublets could be arranged symmetrically in culture as well as in vivo. Nevertheless, biciliated cells were not observed
in our cultures even though Wheatley [14], Albrecht-Buehler [25] and Tucker et al. [ 131 have shown them at least in some cells of different libroblastic strains. The ciliary shaft originated in one of the centrioles of the centrosome. The major problem was the limited view of the cilia we had with ultrathin sections. Indeed, we could not show easily either an eventual relation of the cilium with the plasma membrane or that cilia emerged from the cell. The surface replication technique has been very helpful in this regard. In this case, we could show cilia emerging from some cells only in the vicinity of the nucleus. This experiment was carried out on cultured endothelial cells which had undergone 4 population doublings (2 passages) and were in an exponential growing phase. It would thus be of interest to realize the same experiment on senescent cells, where we found numerous ciliated centrioles. When solitary cilia were examined in mammalian cells, the suggestion was made that the formation of a cilium forced the cells to become quiescent by removing the centriole from the mitotic cycle [4].
80
The control of the cell cycle only by the centriole became less tenable when rapidly growing cells were found to have ciliated centrioles [2]. These conflicting results have been reconciled with the data obtained by Wheatley [15], who showed that cilium formation could occur in rapidly dividing mammalian cells, that ciliogenesis could begin with the completion of mitosis and that cilia may be present for almost the entire interphase period. In the same way, Wheatley [ 151noted that solitary cilia are expressed in primary fibroblastic cell cultures from embryonic mice in about the same frequency as in established murine cell lines. However, they are missing or very poorly expressed in old established L-929 cells. These results were confirmed by Tucker et al. [ 131, who found that ciliation of the centriole occurred in quiescent cells as well as in late Gl period of exponentially growing cells. As suggested by the results of Wheatley [15] and Tucker et al. [13], it seems reasonable to suppose that endothelial cells which reach the stationary phase, such as during senescence [26] or at confluence, will have a greater opportunity to develop ciliary appendages than cells passing rapidly through their division cycle. In ultrathin sections as well as on surface replication, the cilia seemed to be predominantly orientated parallel to the substrate. This observation is in agreement with the findings on 3T3 cells of Albrecht-Buehler [25] who, like Osborn and Weber [27], examined the solitary cilia by indirect immunofluorescence using antitubulin as first antibody. As seen in our study, several stages can happen between simple centriole and elongated ciliary shaft. In comparison with several authors [6,7,12], all stages in apparent ciliary development were seen. The earliest recognizable stage was the basal body in which tubules could be identified extending from one end of the centriole to an adjacent vesicle. This vesicle could be a larger vesicular component of the Golgi apparatus, as proposed earlier by Archer and Wheatley [ 121. It was suggested that thereafter this vesicle became more curved surrounding the elongated tubules. As shown in Ref. 12, the double membrane sheet surrounding the shaft is also enlarged, apparently by addition of vesicular elements. The function and significance of the solitary
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cilia are unknown. Since the two central tubules are lacking according to the majority of authors, it is now supposed that the cilia are not motile. There are suggestions that cilia could have a chemoreceptor [28] or a sensory function [23]. Boquist [29] suggested that the cilium resorption during the Sphase of the mitotic cycle could furnish a large amount of material for the elaboration of the mitotic spindle. Albrecht-Buehler [25] postulated a control of the migration. More recently, Haust [3] also found ciliated smooth muscle cells in aortic atherosclerotic lesions in rabbit. These observations were in contradiction to those of Chaldakov [1], who found ciliated smooth muscle and endothelial cells even in normal rabbit aortas. Although a large number of hypotheses have emerged from those results, the role of the solitary cilium remains unclear. Initially, we were surprised by the presence of cilia in cultured endothelial cells since, as far as we know, they have never been observed in these cells despite the fact that they have been studied by many laboratories for a long time all over the world. The only explanation we can give is that the ciliation could be due either to the cell strain used or to the culture condition. Indeed, Wheatley [ 151 has found variations of ciliary shaft occurrence in 3T6 tibroblasts cultured by himself from those of other laboratories, and has postulated subline differences in solitary cilium expression although the two fibroblastic strains appeared identical structurally. 5. Acknowledgments We wish to thank Mrs. Noelle Paulus-Ninane, and Chantal Devignon for Nicole F&et-Henry valuable technical work as well as Mr. Jacques for the Collet and Mr. Yves Houbion photographical work. 6. References I 2
Chaldakov, G.N., The ciliated smooth muscle and endothelial cell, Atherosclerosis, 56 (1985) 25 I. Fonte, V.G., Searls. R.L. and Hilfer, S.R., The relationship of cilia with cell division and differentiation, J. Cell. Biol.. 49 (1971) 226.
P. Brqfeeuil et al. /Atherosclerosis
3
4 5 6
I
8
9
IO
II
12
I3
14
I5
I6 I7
Haust,
M.D..
Ciliated
106 (1994)
smooth
muscle
75-81
cells
81
bovine
in aortic
atherosclerotic lesions of rabbit, Atherosclerosis. 50 (1984) 283. Rash, J.E.. Shay, J.W. and Biesele, J.J., Cilia in cardiac differentiation. J. Ultrastruct. Res.. 29 (1969) 470. Scherft. J.P. and Daems, W.Th., Single cilia in chondrocytes, J. Ultrastruct. Res., I9 (1967) 546. Smith, J.W., Christie, K.N. and Frame, J.. Desmosomes, cilia and acanthosomes associated with keratinocytes, J. Anat., 105 (1969) 383. Stubbletield. E. and Brinkley, B.R., Cilia formation in Chinese hamster tibroblasts in vitro as a response to colcemid treatment, J. Cell. Biol.. 30 (1966) 645. Odor, D.L. and Blandau, R.J., Observations on the solitary cilium of rabbit oviductal epithelium its motility and ultrastructure. Am. J. Anat.. 174 (1985) 437. Kojimahara, M., Endothelial cilia in rat mesenteric arteries and intramyocardial capillaries. Z. Mikrosk. Anat. Forsch. (Leipz.), I04 (1990) 412. Warfvinge. K. and Elofsson, R., Single modified cilia displayed by cells of human internal stratified epithelial (oral cavity, vagina), Cell Tissue Res.. 251 (1988) 237. Henneguy. L.F. and Lenhossch, Sur le rapport des cils vibratiles avec les centrosomes. Arch. Anat. Microsc. Morphol. Exp., I (1898) 481. Archer, F.L. and Wheatley. D.N., Cilia in cell-cultured fibroblasts. II. Incidence in mitotic and post-mitotic BHK 21iCl3 libroblasts. J. Anat.. 109 (1971) 277. Tucker, R.W., Pardee, A.B. and Fujiwara. K.. Centriole ciliation is related to quiescence and DNA synthesis in 3T3 cells, Cell, 17 (1979) 527. Wheatley, D.N.. Cilia in cell-cultured tibroblasts. I. On their occurrence and relative frequencies in primary cultures and established cell lines, J. Anat., I05 (1969) 35 I. Wheatley, D.N.. Cilia in cell-cultured libroblasts. III. Relationship between mitotic activity and cilium frequency in mouse 3T6 tibroblasts, J. Anat., I IO (1971) 367. Vorobjev, I.A. and Chentsov, Y.S., Centrioles in the cell cycle. I. Epithelial cells, J. Cell. Biol., 93 (1982) 938. Briffeuil, P. and Ronveaux-Dupal, M.F., Ciliation of
I8 I9
20
21
22 23
24
25
26
aortic
endothelial
cells in culture.
Arch.
Biol.
(Brux.). 97 (1986) 360. M.D.. Endothelial cilia in human aortic Haust, atherosclerotic lesions. Virchows Arch. A. 410 (1987) 317. Booyse. F.M., Sedlak. B.J. and Rapelson. M.E.. Culture of arterial endothelial cells: characterization and growth of bovine aortic cells. Thromb. Diath. Haemorrh., 34 (1975) 825. Markham, E., Frey, S. and Hills, G.. Methods for the enhancement of image detail and accentuation of structure in electron microscopy. Virology. 20 (1963) 88. Vorobjev. I.A. and Chentsov, Y.S.. The ultrastructure of centriole in mammalian tissue culture cells. Cell. Biol. Int. Rep.. 4 (1980) 1037. Reese. T.S.. Olfactory cilia in the frog. J. Cell. Biol.. 25 (1965) 209. Barnes. B.C.. Ciliated secretory cells in the pars distalis of the mouse adenohypophysis. J. Ultrastruct. Res.. 5 (1961) 453. Allen, R.A., Isolated cilia in inner retinal neurons and in retinal pigment epithelium, J. Ultrastruct. Res.. I2 (1965) 730. Albrecht-Buehler. G., Phagokinetic tracks of 3T3 cells: parallels between the orientation of track segments and of cellular structures which contain actin or tubulin, Cell. I2 (1977) 333. Rosen, E.M., Mueller, S.N.. Noveral. J.P. and Levine, E.M., Proliferative characteristics of clonal endothelial cell strains, J. Cell. Physiol., 107 (1981) 123.
27
Osborn. M. and Weber. K.. Cytoplasmic microtubules in tissue culture cells appear to grow from an organizing structure towards the plasma membrane, Proc. Natl. Acad. Sci. USA. 73 (1976) 867.
28
Munger, B.L., A light and electron microscopic study of cellular differentiation in the pancreatic islets of the mouse, Am. J. Anat., 103 (1958) 275. Boquist, L., Cilia in normal and regenerating islet tissue. An ultrastructural study in the Chinese hamster with par-
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ticular reference to the P-cells and the ductular epitheliurn, Z. Zellforsch. Mikrosk. Anat.. 89 (1968) 519.